Showing papers in "BioMed Research International in 2001"

The Human Y Chromosome: The Biological Role of a "Functional Wasteland"

Abstract: "Functional wasteland," "Nonrecombining desert" and "Gene-poor chromosome" are only some examples of the different definitions given to the Y chromosome in the last decade. In comparison to the other chromosomes, the Y is poor in genes, being more than 50% of its sequence composed of repeated elements. Moreover, the Y genes are in continuous decay probably due to the lack of recombination of this chromosome. But the human Y chromosome, at the same time, plays a central role in human biology. The presence or absence of this chromosome determines gonadal sex. Thus, mammalian embryos with a Y chromosome develop testes, while those without it develop ovaries (Polani, 1981). What is responsible for the male phenotype is the testis-determining SRY gene (Sinclair, 1990) which remains the most distinguishing characteristic of this chromosome. In addition to SRY, the presence of other genes with important functions has been reported, including a region associated to Turner estigmata, a gene related to the development of gonadoblastoma and, most important, genes related to germ cell development and maintenance and then, related with male fertility (Lahn and Page, 1997). This paper reviews the structure and the biological functions of this peculiar chromosome.

High-Pressure Biotechnology in Medicine and Pharmaceutical Science.

Abstract: High-pressure (HP) biotechnology is an emerging technique initially applied for food processing and more recently in pharmaceutical and medical sciences. Pressure can stabilize enzymes and modulate both their activity and specificity. HP engineering of proteins may be used for enzyme-catalyzed synthesis of fine chemicals, pharmaceuticals, and production of modified proteins of medical or pharmaceutical interest. HP inactivation of biological agents is expected to be applicable to sterilization of fragile biopharmaceuticals, or medical compounds. The enhanced immunogenicity of some pressure-killed bacteria and viruses could be applied for making new vaccines. Finally, storage at subzero temperatures without freezing is another potential application of HP for cells, animal tissues, blood cells, organs for transplant, and so forth.

Identification of Stem Cell Units in the Terminal End Bud and Duct of the Mouse Mammary Gland.

Abstract: The mouse mammary gland may undergo cycles of proliferation, terminal differentiation, tissue remodeling, and more importantly malignant transformation. Mammary epithelial stem cells and their progeny participate in these processes. Mammary epithelial stem cells are multipotent, exhibit properties of self renewal (up to 7 divisions) and may exist either as long-lived nondividing cells or as proliferating-differentiating cells. The focus of this study was to locate stem cells by identifying them as long-lived, label-retaining mammary epithelial cells (LRCs) in growth active (developing) or growth static (aged) mammary ducts. Initially, primary epithelial cells were pulse labeled with either fluorescent tracker dye and/or BrdU. Cells were then transplanted into cleared juvenile syngeneic mammary fat pads and held for 5 weeks or 8 weeks. In this study, we demonstrate that LRCs are stem cells and their progeny (transitional cells) are arranged as transitional units (TUs). Additionally, TUs are located every 250 ± 75 µm in ducts or in the terminal end bud 200–600 µm in diameter. Molecules expressed in TUs were Zonula Occludens-1 and α-catenin proteins which were significantly detected in 75%–91% ( P< 0.001) of the LRCs cells that make up the TU. These data suggest that transitional units may be a group of label-retaining stem cells and maybe involved in the developmental or cancer process.

The Spectrum of β-Thalassemia Mutations in the Arab Populations

Abstract: The Arab countries encompass a wide region stretching from the Persian Gulf to the Atlantic Ocean. The Arab population is quite heterogeneous and has experienced various invasions and migrations throughout history. β-thalassemia is endemic in all countries of the Arab world. Our review of the molecular basis of β-thalassemia in various Arab countries reveals the presence of 52 mutations, which are mostly of Mediterranean and Asian origin. The distribution of mutations reflects the geographical and historical backgrounds of each region. However, no specific mutation is confined to the Arabs, although some Arab countries do have unique mutations.

Genomic Regions That Underlie Soybean Seed Isoflavone Content.

Abstract: Soy products contain isoflavones (genistein, daidzein, and glycitein)that display biological effects when ingested by humans and animals, these effects are species, dose and age dependent. Therefore, the content and quality of isoflavones in soybeans is a key to their biological effect. Our objective was to identify loci that underlie isoflavone content in soybean seeds. The study involved 100 recombinant inbred lines (RIL)from the cross of ‘Essex’ by ‘Forrest,’ two cultivars that contrast for isoflavone content. Isoflavone content of seeds from each RIL was determined by high performance liquid chromatography (HPLC). The distribution of isoflavone content was continuous and unimodal. The heritability estimates on a line mean basis were 79% for daidzein, 22% for genistein, and 88% for glycitein. Isoflavone content of soybean seeds was compared against 150 polymorphic DNA markers in a one-way analysis of variance. Four genomic regions were found to be significantly associated with the isoflavone content of soybean seeds across both locations and years. Molecular linkage group B1 contained a major QTL underlying glycitein content (P = 0.0001 ,R 2 = 50.2%), linkage group N contained a QTL for glycitein (P = 0.0033 ,R 2 = 11.1%)and a QTL for daidzein ( P = 0.0023 ,R 2 = 10.3%) and linkage group A1 contained a QTL for daidzein (P = 0.0081 ,R 2 = 9.6%). Selection for these chromosomal regions in a marker assisted selection program will allow for the manipulation of amounts and profiles of isoflavones (genistein, daidzein, and glycitein)content of soybean seeds. In addition, tightly linked markers can be used in map based cloning of genes associated with isoflavone content.

Sunlight and Skin Cancer.

Abstract: It is well known that chronic exposure to ultraviolet (UV) radiation present in sunlight is responsible for the induction of most nonmelanoma skin cancer (NMSC) in humans. Wavelengths in the UV-B (290–320 nm) region of the solar spectrum are absorbed into the skin, producing erythema, burns, and eventually skin cancer. NMSC is the most common type of human cancer. Recent surveys indicate that around one million new cases of skin cancer are diagnosed each year in the United States, about 70% of which result from repeated exposure of the skin to sunlight. Laboratory studies have shown that UV-B region of the solar spectrum is responsible for this effect. The first step in UV skin carcinogenesis involves the induction of DNA damage. Occasional mistakes during the repair of this damage leads to the incorporation of wrong bases into the genetic material. The DNA damage that is left unrepaired may also disrupt cellular processes by obstructing the DNA and RNA synthesizing machineries and introduce wrong bases into the DNA. These types of mistakes often result in mutation leading to loss or inappropriate expression of affected genes. Recent studies indicate that genetic alterations in the p53 tumor suppressor gene play an important role in the development of skin cancer. The p53 protein is also involved in programmed cell death (apoptosis), and it has been proposed that p53 serves as a “guardian of the genome” by aiding DNA repair or causing elimination of cells with excessive DNA damage. Unrepaired photoproducts in the p53 gene are transformed into mutations thereby initiating the process of carcinogenesis. Following repeated exposures to UV, keratinocytes carrying p53 mutations acquire a growth advantage by virtue of their increased resistance to apoptosis. Recent studies have shown that UV-B damaged keratinocytes (sunburn cells) are eliminated by Fas/Fas-ligand interaction and that this pathway is dysregulated during UV skin carcinogenesis resulting in accumulation of p53 mutations in DNA damaged keratinocytes. These results demonstrate a link between p53 pathway and Fas/Fas-ligand pathway and that dysregulation of both pathways can lead to the pathogenesis of UV-induced skin cancer. Several studies have shown that UV induces unique types of p53 mutations in skin cancers at a high frequency that are not commonly found in other types of human cancer. Analogous to human skin cancers, skin cancers induced in laboratory mice by UV radiation also display UV signature p53 mutations at a high frequency. More interestingly, p53 mutations are also present in sun-exposed skin and it can serve as an indicator of prior solar exposure in humans. It has been shown that p53 mutations in mouse skin arise as early as one week of chronic UV-irradiation and the frequency of p53 mutations reach a maximum at 4–8 week of UV exposure. These results suggest that p53 mutations arise well before skin cancer development and that they can serve as a surrogate early biological endpoint in skin cancer prevention studies. In fact, it has been shown that application of SPF-15 sunscreens to mouse skin before each UV-irradiation protect mice against induction of p53 mutations as well as skin cancer development. In addition to the p53 gene, another tumor suppressor gene known as patched (ptc) has been implicated in nevoid basal cell carcinoma syndrome (NBCCS). NBCCS, also called basal cell nevus syndrome or Gorlin's syndrome, is a rare autosomal dominant disorder characterized by multiple BCCs that appear at a young age on sun-exposed areas of the skin. Studies of NBCCS patients have shown that they have both germ-line and somatic mutations in the ptc gene. Somatic ptc mutations have been found in BCCs from otherwise normal individuals suggesting that genetic alterations in the ptc gene may also play a role in the development of BCC. In summary, recent advances have aided in the understanding of the mechanisms by which UV radiation induces skin cancer. Continued efforts should result in a greater understanding of the genetic mechanisms involved in the function of tumor suppressor and oncogenes that are now known, as well as genes yet to be discovered. The efforts of research in skin cancer may help to increase overall awareness of the harmful effects of UV exposure and result in better methods of skin cancer prevention and treatment.

Cellular Senescence: Ex Vivo p53-Dependent Asymmetric Cell Kinetics.

Abstract: Although senescence is a defining property of euploid mammalian cells, its physiologic basis remains obscure. Previously, cell kinetics properties of normal tissue cells have not been considered in models for senescence. We now provide evidence that senescence is in fact the natural consequence of normal in vivo somatic stem cell kinetics extended in culture. This concept of senescence is based on our discovery that cells engineered to conditionally express the well-recognized tumor suppressor protein and senescence factor, p53, exhibit asymmetric cell kinetics. In vivo, asymmetric cell kinetics are essential for maintenance of somatic stem cells; ex vivo, the same cell kinetics yield senescence as a simple kinetic endpoint. This new “asymmetric cell kinetics model” for senescence suggests novel strategies for the isolation and propagation of somatic tissue stem cells in culture.

The Thermal Stability of the Fusarium solani pisi Cutinase as a Function of pH

Abstract: We have investigated the thermal stability of the Fusarium solani pisi cutinase as a function of pH, in the range from pH 2–12. Its highest enzymatic activity coincides with the pH-range at which it displays its highest thermal stability. The unfolding of the enzyme as a function of pH was investigated by microcalorimetry. The ratio between the calorimetric enthalpy (� Hcal)and the van’t Hoff enthalpy (� Hv)obtained, is far from unity, indicating that cutinase does not exhibit a simple two state unfolding behaviour. The role of pH on the electrostatic contribution to the thermal stability was assessed using TITRA. We propose a molecular interpretation for the pH-variation in enzymatic activity.

Trypanosoma cruzi-Induced Host Immune System Dysfunction: A Rationale for Parasite Immunosuppressive Factor(s) Encoding Gene Targeting.

Abstract: An intense suppression of T cell proliferation to mitogens and to antigens is observed in a large number of parasitic infections. The impairment of T cell proliferation also occurred during the acute phase of Chagas’ disease, caused by the intracellular protozoan parasite Trypanosoma cruzi. A wealth of evidence has accumulated that illustrates the ability of T. cruzi released molecules to influence directly a variety of diverse immunological functions. In this paper, we review the data concerning the immunoregulatory effects of T. cruzi Tc24 (a B cell activator antigen)and Tc52 (an immunosuppressive protein)released molecules on the host immune system. The gene targeting approach developed to further explore the biological function(s)of Tc52 molecule, revealed interesting unexpected functional properties. Indeed, in addition to its immunusuppressive activity a direct or indirect involvement of Tc52 gene product alone or in combination with other cellular components in T. cruzi differentiation control mechanisms have been evidenced. Moreover, targeted Tc52 replacement allowed the obtention of parasite mutants exhibiting low virulence in vitro and in vivo. Thus, the generation of a complete deficiency state of virulence factors by gene targeting should provide a means to assess the importance of these factors in the pathophysiological processes and disease progression. It is hoped that such approaches might allow rational design of tools to control T. cruzi infections.

Journal of Biomedicine and Biotechnology

Abstract: Pourquoi un nouveau journal médical? Pourquoi spé-cialement ce nouveau journal? Quelles sont ses ambitions? Quel public peut-il toucher? Des spécialistes, des généralistes ou les deux à la fois? Il se veut être un lien entre la communauté scientifique internationale, plus particulièrement dans les domaines de la science du vivant et leur application technologique. Il y a incontestablement un espace à occuper, un rôle important à jouer pour unir d'avantage cette commu-nauté. Je me réjouis de l'initiative prise par ses promo-teurs, qui ont eu le courage d'aborder une concurrence bien établie et à se lancer dans une aventure intellectuelle exaltante. Pour ma part, je me permettrai d'aborder en quelques lignes quelques aspects qui me sont chers et qui sont suff-isamment généraux pour faire l'objet d'un éditorial. Où va en effet la médecine et que nous réserve la médecine de demain? Les progrès fantastiques dont nous avons été témoins, éblouis, entrent progressivement dans la pratique courante. La révolution à laquelle nous avons assisté se nomme, en particulier: la vaccination après Pasteur, qui a encore de beaux jours et de nouvelles perspectives (n'y-a-t'il pas de vaccin encore contre le paludisme ni contre le SIDA!), plus près de nous l'antibiothérapie, en plein essor mais qui commence à s'essouffler, à montrer ses limites du fait du développement rapide des résistances aux microorganismes. Ce n'est que depuis quelques décennies que la chirurgie im-munologique a permis la transplantation d'organes ou de moelle osseuse, transplantation vieux rêve de l'humanité en-fin réalisé et dont les succès eux-même entraînent, du fait de la pénurie des organes à transplanter, des déceptions parmi les très nombreux malades inscrits sur la liste d'attente. N'est-il pas admirable que, dans le monde entier, 6 millions d'individus sont prêts à donner leur moelle pour sauver des malades, en particulier leucémiques. A cette liste, devrait-on ajouter les nombreuses victoires en cancérologie dues à un meilleur monitoring des agents antitumoraux? Et enfin, ne pas oublier les méthodes d'investigation biologique ou d'images médicales, qui se sont profondément améliorées récemment. Ce panorama serait bien incomplet si je n'y ajoutais pas l'intrusion spectaculaire de la génétique médicale, qui per-met de localiser, d'isoler les gènes de nombreuses maladies monogéniques et bientôt la gamme des gènes agissant de concert dans les maladies polygéniques, qui sont le lot désolant de nos sociétés industrialisées. Muni de tout cet arsenal, le médecin, qu'il soit spécialiste ou généraliste, peut désormais proposer à ses malades des thérapeutiques de …

Breaching the Kinetic Barrier to In Vitro Somatic Stem Cell Propagation.

Abstract: Here we have reviewed the conventional definitions and fundamental characteristics of the two basic types of stem cells, embryonic stem cells (ESCs) and somatic stem cells (SSCs). By taking into account the often-overlooked asymmetric cell kinetics of SSCs, we consider the evidence that should SSCs retain these growth kinetics in vitro, a natural kinetic barrier to SSC propagation exists. Recent discoveries showing that the tumor suppressor gene p53 can act as a regulator of asymmetric cell kinetics provide a target pathway for in vitro SSC propagation strategies.

A model for UV-induction of skin cancer

Abstract: The cellular and molecular events that contribute to the development of UV-induced skin cancer is a complex process involving at least two distinct pathways that interact or converge to cause skin cancer (Figure ​(Figure1).1). One pathway involves the action of UV on target cells (keratinocytes) for neoplastic transformation, and the other involves the effects of UV on the host's immune system [1]. There is evidence to indicate that UV-induced DNA damage plays an important role in both pathways.

In Vitro Investigations on the Toxicity and Cell Death Induced by Tamoxifen on Two Non-Breast Cancer Cell Types

Abstract: Tamoxifen, a potent anticancer agent known to interrupt the enhanced estrogen activity of malignant mammary gland cells, was recently approved by the Food and Drug Administration (FDA) for the treatment of breast cancer. In this investigation, the toxic effects of tamoxifen were evaluated through cell multiplication, and cytological, surface ultrastructural, and biochemical studies on human cervical carcinoma cells (HeLa) and/or murine erythroleukemic (MEL) cells (BB-88). Tamoxifen treatment demonstrated an inhibitory effect on HeLa cell multiplication at lower concentrations and toxicity at higher concentrations and longer treatment durations. The drug also triggered morphological and biochemical changes as revealed by light microscopy, scanning electron microscopy (SEM), fluorescence microscopy, Nucleosome ELISA, and the DNA smear pattern. Cytological observations showed nuclear condensation, cell shrinkage, multinucleation, and apoptotic bodies. Surface ultrastructure of tamoxifen treated cells examined under the SEM revealed abnormalities such as membrane blebbing, holes, and cytoplasmic extrusions, all of which are characteristics of programmed cell death (apoptosis). Redistribution of the membrane phospholipid phosphatidylserine (PS) from the protoplasmic surface of the plasma membrane to the cell surface was identified using annexin V-enhanced green fluorescent protein (EGFP) in tamoxifen treated MEL BB-88 cells, a general feature of cells undergoing apoptosis. Tamoxifen treated cells demonstrated internucleosomal damages of the genomic DNA and DNA fragmentations, evidenced by an increase in free nucleosomes, and distinctive DNA smear patterns on the agarose gel.

Induction of Apoptosis in Rat Peripheral Blood Lymphocytes by the Anticancer Drug CI-994 (Acetyldinaline)

Abstract: CI-994 (acetyldinaline)is an investigational anticancer drug currently in clinical trials. In preclinical safety studies in rats and dogs, CI-994 resulted in significant toxicity to bone marrow and lymphoid tissue. To determine if apoptosis was involved in CI-994 toxicity, peripheral blood lymphocytes were isolated from untreated male Wistar rats and exposed to CI-994 (1, 3, 10, or 30 µM) in vitro for up to 24 hours. Morphological and biochemical features of apoptosis were evaluated using several techniques, and lactate dehydrogenase (LDH)release was measured as an indicator of cell necrosis. No evidence of apoptosis or necrosis was detected in lymphocytes exposed to CI-994 for 4 hours. After 24 hours, concentration-dependent increases in apoptosis characterized by DNA condensation, DNA fragmentation, and/or externalization of phosphatidyl serine were seen at CI-994 concentrations as low as 1 µM and were statistically significant beginning at 10 µM. Ultrastructural analysis confirmed the presence of DNA condensation, DNA fragmentation, cell shrinkage, and membrane blebbing in cells exposed to 30 µM CI-994. After 24 hours, the percent of maximum LDH release from lymphocytes treated with 10 and 30 µM CI-994 was 7% and 15%, respectively, compared with 0% in the controls. In comparison, morphological changes of apoptosis detected by fluorescent microscopy were observed in 79% of the lymphocytes at these two concentrations. Additionally, apoptosis was seen in more than 24% of lymphocytes exposed to 1 and 3 µM CI-994, whereas maximum LDH release was less than or equal to 1% at these concentrations. These results show that apoptosis is the primary mode of cell death in rat lymphocytes exposed to CI-994 in vitro.

Genetic Stabilization by p53 Involves Growth Regulatory and Repair Pathways

Abstract: p53 performs a plethora of activities, which are directed towards the maintenance of the genomic integrity and constitute its universal role as a tumor suppressor. 1000 to 10000 latent p53 molecules are permanently available in order to monitor DNA exchange processes in mitotically growing cells. After the introduction of major DNA injuries the levels of posttranslationally modified p53 proteins rise, which in turn transcriptionally signal transient cell cycle arrest or apoptotic cell death, depending on the extent of damage. Taken together, p53 inhibits the manifestation of genomic instabilities at different control levels both during naturally occurring metabolic processes and in response to genotoxic treatments.

Somatic embryogenesis, rhizogenesis, and morphinan alkaloids production in two species of opium poppy

Abstract: A study of somatic embryogenesis and rhizogenesis and their influence on production of morphinan alkaloids on two species of opium poppy is presented. We identified the ratios of auxin and cytokinin that caused somatic embryogenesis and rhizogenesis in hypocotyl and cotyledons of Papaver somniferum album and Papaver orientale splendidissimum. The hypocotyls and cotyledons both show somatic embryogenesis in Papaver somniferum album whereas only the cotyledons were embryogenic in Papaver orientale splendidissimum. For rhizogenesis, the most important response is on the cotyledons and leaves in these two species. Histology showed characteristic stages of somatic embryo: Globular, cotyledonous, and heart cotyledonary. High performance liquid chromatography analysis showed that the roots of both species synthesized codeine, thebaine, and papaverine. Morphine was only detected in aerial parts of Papaver somniferum album. Codeine and thebaine were detected in the rhizogenous but no embryonic callus. These results suggest that root organogenesis is causally related to alkaloid biosynthesis.

Two Novel Frame Shift, Recurrent and De Novo Mutations in the ITGB2 (CD18) Gene Causing Leukocyte Adhesion Deficiency in a Highly Inbred North African Population.

Abstract: We have identified four different mutations causing leukocyte adhesion Deficiency (LAD) in the ITGB2 gene of patients from a highly inbred population. Two were novel single-bp deletions (1497delG and 1920delG) causing frame shift and the two others were the missense mutations G284S and R593C. In our study, the G284S was a recurrent mutation while the R593C occurred de novo. We have also characterized a novel Xba1 polymorphic site located at the 5' end of the ITGB2 locus. Family studies showed that the 1497delG mutation segregated with this marker and the intragenic AvaII polymorphic marker, suggesting the presence of a founder effect. The observation of a heterogeneous spectrum including de novo and recurrent mutations causing LAD in a highly inbred population is rather unexpected. In view of the literature published on the molecular genetics of LAD and considering the ethnic origin of the patients studied, our findings confirm the heterogeneity of the mutations causing LAD and point out potential mutational hot spots in the ITGB2 gene.

The impact of genetic diseases on Jordanians:strategies towards prevention

Abstract: Genetic disorders are diseases in which genetic factorsplay an important role in their etiology. They are classi-fied into chromosomal abnormalities,monogenic and mul-tifactorialdisorders.Whilechromosomalabnormalitiesandmonogenic disorders are purely genetic in nature,multifac-torialdisordersareproducedbytheinteractionbetweenen-vironmentalandgeneticfactors.Althoughmostgeneticdis-easesareindividuallyraretherearemanyof them.Thevastmajority are serious, none is curable and relatively few areamenabletosatisfactorytreatment.Anumberofsurveyshaveindicatedthatatleastoneineveryfiftynewbornshasama-jor congenital anomaly, one in hundred has a monogenicdisorderandoneintwohundredhasachromosomalabnor-mality.However,theprevalenceof geneticdisordersisquitevariable among different ethnic groups and across differentagegroups.Thecontrolanddeclineofenvironmentalcausesofchild-hoodmortalityinwesterncountriesthrewthegeneticcausesintogreaterprominence.Althoughthepicturemaybediffer-ent in third world countries, improvement in living condi-tionsiscausingasimilarshift.Themagnitudeof theimpactofgeneticdisordersonallsocietiesisquitesignificantneces-sitating their control which can be principally achieved byprevention.

A C-Terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein.

Abstract: Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2 )o f HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressed in vitro or in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms. While N-terminal deletions up to 111 amino acids had no effect, the deletion of amino acids 585–610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585–610 of the ORF2 protein might be critical for capsid biogenesis.

Telomeres in cancer therapy

Abstract: Finding new targets to improve current cancer therapies is one of the areas of Biomedicine and Biotechnology that generates greater expectations. The telomeres, special protective structures at the end of eukaryotic chromosomes have been metaphorically proposed to be cancer's Achilles heel, since they are essential to stabilize linear chromosomes [1]. There is mounting evidence that loss of telomere function, either by altering telomere-binding proteins or by loss of telomeric sequences, is associated with loss of cell viability through induction of apoptosis [1]. Most of the attempts to impair telomere function have consisted in the inhibition of the enzyme telomerase, a ribonucleoprotein DNA polymerase that synthesizes telomeres, de novo [2]. The characterization of mice that lack the RNA component of telomerase showed that telomerase inhibition in a mammal leads to telomere shortening, increased chromosomal instability, and loss of viability [3]. Furthermore, inhibition of telomerase in various cancer cell lines, either using dominant versions of the enzyme or antisense oligonucleotides against the RNA component, also lead to telomere shortening and cell death or differentiation [4, 5, 6]. All these studies suggested that telomerase inhibition might compromise tumor growth by leading to an accelerated telomere shortening and cell death, hence, anticancer therapies based in telomerase inhibition could be a promising approach. Some data suggest, however, that the situation may be more complex. The first doubt thrown on the efficacy of an anticancer therapy based on telomerase inhibition came from the fact that telomeres can be maintained by means other than telomerase itself [7, 8]. Such telomere maintenance mechanisms seem to be selected when telomeres reach a critical short length, chromosomal abnormalities have occurred and cell viability has been compromised [9]. In other words, even though telomerase inhibition in a tumor might result in short telomeres and cell death, there is a possibility that resistant clones might arise that would be refractory to the treatment and would bear a higher chromosomal instability. This is supported by evidence from the mouse model without telomerase: even though these mice show loss of viability associated with telomere shortening, a fraction of telomerase-deficient mice appear to develop lymphomas at a higher frequency than the wild-type counterparts [10]. These tumors are possibly the consequence of loss of check-points associated with telomere loss, this allowing the growth of cells bearing high chromosomal instability and maintaining telomeres without telomerase [11]. An analogous situation could happen in human cancers where the proliferative pressure is very high. Such telomerase-independent telomere maintenance mechanisms should be targeted if we want to assure an efficient telomere-based therapy. We have learned from studies in yeast that these mechanisms might involve homologous recombination and DNA repair proteins [12, 13]. More recently, it has been proposed that a special structure at human telomeres, known as the telomeric loop, could also account for telomerase-independent telomere elongation [14]. It is possible, however, that different tissues have different sensitivities to telomere loss. In this regard, the skin of telomerase-deficient mice is resistant to chemical tumorigenesis [15]. This supporting that telomerase inhibition in skin tumors might cease their growth. A second possible problem of a tumor therapy based on telomerase inhibition is that tumor cells may divide without telomerase before reaching critically short telomeres. In other words, telomerase inhibition is not expected to have an immediate effect on tumor growth. In summary, there is strong evidence that telomeres may be a very good target for new anticancer therapies. To date, most of the efforts to compromise telomere function in cancer cells have involved telomerase inhibition. The fact that telomeres can also be maintained in a telomerase-independent way and that telomeres have to reach a critical length before effects on viability are seen, might result in a low efficiency of a treatment based on telomerase inhibition. This should encourage researchers to focus future studies not only on development of telomerase inhibitors but also on inhibitors of telomeric proteins, disruption of telomere structure, or disruption of alternative telomere maintenance pathways.

Response of Escherichia coli Containing Mycobacterial Carotene Genes to UV Radiation.

Abstract: The plasmid pC5, which encodes biogenesis of lycopene in Mycobacterium aurum A(+), was partially digested by restriction endonucleases and generated fragments were cloned. After transformation of Escherichia coli (colorless bacteria) with the plasmids so constructed, seven orange clones were detected and found to carry the same recombinant plasmid (pC51). E. coli cells containing this plasmid synthesize neurosporene and lycopene, and were more resistant to ultraviolet irradiation than non pigmented strain.

Interaction Between Mevalonate Pathway and Retinoic Acid-Induced Differentiation

Abstract: All trans retinoic acid (ATRA) is a potent inducer of differentiation of HL-60 cell line. The pretreatment of the cells by compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase, during 24 hours, enhances the ATRA-induced cell differentiation. At 50 nM, the percentage of cell differentiation is 34.9% ± 2 and 73% ± 2.96 in the control and compactin-treated cells, respectively. The removal of compactin boosts the level of HMG-CoA reductase and therefore the biosynthesis of sterol and nonsterol isoprenoid compounds. The participation of sterol and nonsterol pathway was then investigated. The supply of an excess of cholesterol (up to 80 μg/ml of LDL) leads to a significant decrease of cell differentiation by ATRA from 78% ±0.1 to 54% ±2.8. A concomitant decrease of cell growth (51% ± 6.4) was observed. The pretreatment of cells by the geranylgeranyltransferase inhibitor (GGTI-298) has no effect on the cell differentiation process. By contrast, the farnesyltransferase inhibitors (FTI-II and FTI-277) completely abolish theATRA-induced differentiation, thus confirming the involvement of farnesylated proteins in the differentiation mechanism.

Some Provocative Thoughts on Damage and Repair of DNA.

Abstract: The instability of nucleic acid bases, responsible for hereditary information storage is amazing. Is there a system in this madness? All the nitrogen bases may undergo different kinds of rapid, spontaneous, or induced modification. They may form adducts and photoproducts, may be deaminated, methylated, and oxidized, or may be lost spontaneously or in the course of enzymatic processes. The list of potentially mutagenic lesions in DNA is long (some of them are given in parentheses)and includes products of deaminations (hypoxanthine, uracil, 5-methyluracil (thymine)); of methylation (3-methyladenine, O-6-methylguanine); of oxidation (8-oxoguanine, thymine glycol, 5-hydroxycytosine, 5-hydroxyuracil, 5-formyluracil), and apurinic/apyrimidinic (AP)sites. The frequency of base oxidation much outweighs the remaining modifications. The most susceptible base to oxidation damage (and to depurination), both in nucleotide pools as well as in DNA, is guanine, with formation of the highly mutagenic base, 8-oxoguanine. It has been calculated that in a single cell per day as much as ∼ 100 000 modifica

The Role of Neuraminidase Inhibitors in the Treatment and Prevention of Influenza

Abstract: The causative agents of acute respiratory infections in children and adults are mostly thought to be viruses. Many types of viruses could cause similar symptoms of ARI. Among them, influenza viruses A and B and respiratory syncytial virus (RSV) are thought to be the most important because of the severity of illness after infection and their high communicability in the human population [1]. Type C influenza virus usually is not serious and typically not included in discussion about influenza infection [2]. In this article, discussion is limited to influenza A and B only. These viruses belong to the orthomyxoviridae family of viruses [3]. Influenza infects an estimated 120 million people in the United States, Europe, and Japan each year and is a major cause of morbidity and mortality. In periods of flu pandemics, infection rates could be even higher. In the United States, out of an estimated 20 million cases of flu each year, up to 40 000 Americans die, and more than 400 000 are hospitalized. The resulting economic burden has been estimated to be as high as $12 billion. In the last few years, new developments in the prevention, diagnosis, and treatment of influenza have been introduced that will change the clinical milieu in the new millennium. The traditional vaccine approaches to the prophylaxis have been problematic owing to the ability of these viruses to undergo antigenic shift by exchanging genomic segments or by undergoing antigenic drift, consisting of point mutations in the surface glycoproteins haemagglutinin (HA) and neuraminidase (NA) genes as a result of an error-prone viral polymerase. Antigenic shift is seen only in influenza A viruses and occurs when a novel HA or NA subtype is introduced from an animal influenza virus reservoir into the human population. Antigenic drift occurs in influenza A and B in and around the antibody binding site. Therefore, the annual update of influenza is necessary because of the annual occurrence of antigenic drifts. For example, for the 1999–2000 season, it was recommended that vaccines contain an A/Sydney/5/97 (H3N2)-like virus, an A/Beijing/262/95 (H1N1)-like virus, and a B/Beijing/184/93-like virus or B/shangdong/7/97-like virus. However, significant morbidity and mortality were clinically observed during this season, and indicated the obvious limitations of this method of prevention. As a result of these ongoing observations, other prevention and treatment modalities have been investigated and some have already been introduced to practice. The most recent breakthrough in that direction is the introduction of neuraminidase inhibitors for the treatment and prevention of influenza infection. The neuraminidase is a surface glycoprotein that is composed of eleven conserved residues [4] which catalyze the cleavage of sialic acid residues terminally linked to glycoproteins and glycolipids and plays an important role in the replication of the virus. A recent new study demonstrated that Tyr409 is the most critical residue for enzyme activity, and that Asp149, Arg223, Glu275, and Arg374 also play important roles in enzyme catalysis [2]. Neuraminidase function is critical for the spread of virus to new cells, and if the enzyme activity is inhibited, then virus infection is abrogated [2, 5]. Also, there is evidence that this enzyme plays some role in the introduction of apoptosis to the infected cells [6]. Currently, there are two neuraminidase inhibitors (NI) available for clinical use: zanamivir and oseltamivir. In phase II trials (NAIB2005 and NAIB2008), benefit was seen in early treatment (within 36 hours of onset of illness). Virological substudies showed mean reductions in virus shedding after 24 hours of treatment of 1.5 to 2.0 log [10] 50% tissue culture infective doses compared with a placebo, with no reemergence of virus after completion of the therapy [7, 8]. In phase III trials, median times to alleviation of major symptoms were reduced by up to 2.5 days after treatment. Benefit of treatment in terms of time to return to normal activities, reductions in consumption of acetaminophen, and the reductions in the level of interference of influenza with sleep, work, leisure, and recreational activities were reported [9]. In the prophylaxis trials, statistically significant reductions in the incidence of influenza A was reported with the use of inhaled zanamivir in a double blind study that included 1107 persons in two university communities [9, 10]. In this study, zanamivir was 67% efficacious in preventing laboratory-confirmed clinical influenza and 84% efficacious in preventing laboratory-confirmed illnesses with fever. Also, intravenous zanamvir (600 mg twice daily for 5 days) used 4 hours prior to intranasal inoculation with infectious doses of influenza A virus was successful in reducing the frequency of viral shedding (0% versus 100% in placebo), seroconversion (14% versus 100% in placebo), fever (14% versus 88% in placebo), upper respiratory tract illness (0% versus 100% in placebo), and total symptom score (1% versus 44% in placebo) [11]. Another trial showed that outbreak of influenza A and B were prevented in a nursing home community [9]. Oral administration of oseltamivir (GS4104) in two randomized trials (1559 subjects given 75 mg once or twice daily) [12] reduced the risk of infection during the influenza season from 4.8% in placebo to 1.2% and 1.3% in the treatment groups. The protective effects of the drug were 74% in all study sites combined and 84% in the areas where the rate of influenza was higher. Another smaller study reached similar conclusions [13]. The coadministration of NI with inactivated trivalent influenza vaccine was investigated in England. It was found that it does not adversely affect the production of antihaemagglutinin antibodies in the serum [14]. Finally, although fighting influenza seems like boxing a heavyweight champion and odds are you will end up flat on your back, but with proper precautions and the development of new drugs you may be able to avoid getting knocked out.

A Tragic Compromise on Stem Cell Derivation.

Abstract: It appears that the official policy of the US government for federally funded studies of human embryo stem (ES) cell research will exclude the derivation of ES cells (National Institute of Health Guidelines for Research Using Human Pluripotent Stem Cells, 65 FR 69951 (November 21, 2000)). In taking this decision, the National Institutes of Health (NIH) is not to be blamed, nor should we wring our hands about the political climate that has led to it. ES cell derivation and the destruction human embryos it entails are caught in the maelstrom of the culturally divisive abortion debate that continues to fester in the United States. At the very least, however, persons who support the promise of stem cell therapy should understand the implications of this limitation and raise their voices in protest against it. The significance of the derivation compromise has largely been lost in the popular press. Even scientifically knowledgeable journalists have not done the point justice. Yet it is known that ES cell properties depend on the methods used to derive them, particularly in their pluripotency. Worse, the multiple changes that can occur within the cells as they are cultured render their properties inconsistent and unreliable. The instability associated with their growth conditions means that ES cells cannot be simply produced centrally by a nonfederally funded source and then distributed to federally funded researchers (in order to insulate the latter from violation of regulations) without jeopardizing the cells' ability to contribute to tissue. Extended passage in culture seems to allow an accumulation of mutations that change gene expression. Perhaps the most direct lost opportunity presented by the antiderivation policy is that of studying the process of derivation itself. The immense decade-long effort to derive and grow mouse ES cells needs to be undertaken for other mammalian species. Although that work has already begun, the time required to achieve stable ES cells from other mammals could be greatly shortened by the application of the considerable intellectual, financial, and organizational resources of the US government medical research system. The ethical objections to public funding of stem cell derivation are almost shockingly weak. Consider one source, embryonic germ (EG) cells are derived from cadaveric fetal tissues from induced abortions. First, it has been alleged that the knowledge that an embryo stem cells could contribute to science might cause people to rationalize abortions and therefore increase their frequency. Yet there is no evidence that the frequency of abortion would be increased under these conditions, and various mechanisms have been proposed to insulate the abortion decision from the embryo donation decision, on analogy with policies for organ donation and harvesting. Second, it is argued the use of human materials remaining from abortions taints the work of medical scientists. Yet surely the surgeon involved in the transplant of an organ from a shooting victim is not thereby endorsing the murder, but rather trying to promote health and life. As for the use of “extra” embryos remaining following fertility treatments, there is deep disagreement among religious traditions about the moral status of the embryo. Although the official Roman Catholic doctrine that full moral status must be conferred to the human embryo at conception is well known, not even all Catholic theologians subscribe to this view. And it is clear that other Christian faiths, as well as Jewish and Islamic traditions, do not accord full moral status to the early embryo. Surely the embryo must be respected as a form of human life, but it does not follow that it must be treated on a moral par with persons. Finally, the argument that work should instead go forward using adult stem cells cannot exclude the importance of continuing on the ES cell track, nor have recent promising events demonstrated that adult stem cells possess the pluripotency of ES cells. Scientists can and must work within the constraints of policy decisions made in the political realm. In this case, however, as in the case of artificial reproduction techniques, the net result of the ban on ES cell derivation not only slows progress without good reason. It also leaves this area of science without the powerful imprimatur and guidance of public oversight, a circumstance that only compounds the tragic irony of US policy on stem cell derivation.

CD26 Immuno-Expression and Periodontal Disease Progression.

Abstract: Immunological mechanisms participate in the pathogenesis of human chronic inflammatory periodontal disease (CIPD). Human CD4+ lymphocytes express functionally heterogeneous profiles of cytokine production. CD26 is an integral membrane glycoprotein, that is, a marker of Th1-like cytokine development. The purpose of the present study was to compare the immuno-expression of CD26 receptor in periodontal sites with and without clinical attachment loss (CAL). Five patients with rapidly progressing periodontitis and one with juvenile periodontitis were investigated. Each patient presented at least one site with and without CAL. Ten sites with CAL and nine without any CAL were biopsied, followed by the immunohistochemical identification of the CD26 receptor using the MIB-DS2/7 antibody. The results demonstrated that the percentage of positive cells for this antigen in the periodontal sites with CAL was not significantly different from those without attachment loss. Therefore, Th1 cell impairment may not be directly involved with periodontal attachment loss.