Showing papers in "Current Pharmaceutical Biotechnology in 2009"

Lantibiotics: Mode of Action, Biosynthesis and Bioengineering

Abstract: Lantibiotics are gene-encoded peptides that contain intramolecular ring structures, introduced through the thioether containing lanthionine and methyllanthionine residues The overwhelming majority of the lantibiotics shows antibacterial activity Some lantibiotics, eg nisin, are characterized by a dual mode of action These peptides form a complex with the ultimate cell wall precursor lipid II, thereby inhibiting cell wall biosynthesis The complexes then aggregate, incorporate further peptides and form a pore in the bacterial membrane Recent results show that complexing of lipid II is widespread among lantibiotics; however, pore formation depends on the overall length of the peptide and the lipid composition of the test strain membrane In the two-component system of lacticin 3147, the two functions are performed by the two different peptides The genetic information for production of lantibiotics is organized in gene clusters which contain a structural gene (lanA) for the lantibiotic prepeptide The modifications are introduced by one biosynthetic enzyme (LanM) or a combination of a dehydratase (LanB) and a cyclase (LanC) These enzymes have been in the focus of recent bioengineering studies: The structure of NisC has been resolved, the reaction mechanism of LctM was elucidated and the active site residues were characterized by mutagenesis studies In vitro modification systems have successfully been used to introduce thioether rings into other biologically active peptides Furthermore, variant lantibiotics with enhanced properties have been engineered and at least one promising new lantibiotic with strong activity against multiresistant pathogens has been described

Ganoderma lucidum: a potent pharmacological macrofungus.

Abstract: Ganoderma lucidum (Ling Zhi) is a basidiomycete white rot macrofungus which has been used extensively as "the mushroom of immortality" in China, Japan, Korea and other Asian countries for 2000 years. A great deal of work has been carried out on therapeutic potential of Ganoderma lucidum. The basidiocarp, mycelia and spores of Ganoderma lucidum contain approximately 400 different bioactive compounds, which mainly include triterpenoids, polysaccharides, nucleotides, sterols, steroids, fatty acids, proteins/peptides and trace elements which has been reported to have a number of pharmacological effects including immunomodulation, anti-atherosclerotic, anti-inflammatory, analgesic, chemo-preventive, antitumor, chemo and radio protective, sleep promoting, antibacterial, antiviral (including anti-HIV), hypolipidemic, anti-fibrotic, hepatoprotective, anti-diabetic, anti-androgenic, anti-angiogenic, anti-herpetic, antioxidative and radical-scavenging, anti-aging, hypoglycemic, estrogenic activity and anti-ulcer properties. Ganoderma lucidum has now become recognized as an alternative adjuvant in the treatment of leukemia, carcinoma, hepatitis and diabetes. The macrofungus is very rare in nature rather not sufficient for commercial exploitation for vital therapeutic emergencies, therefore, the cultivation on solid substrates, stationary liquid medium or by submerged cultivation has become an essential aspect to meet the driving force towards the increasing demands in the international market. Present review focuses on the pharmacological aspects, cultivation methods and bioactive metabolites playing a significant role in various therapeutic applications.

Mechanisms of protein aggregation.

Abstract: Aggregation or reversible self-association of protein therapeutics can arise through a number of different mechanisms. Five common aggregation mechanisms are described and their relations to manufacturing processes to suppress and remove aggregates are discussed.

Marine sponges: potential sources of new antimicrobial drugs.

Abstract: Sponges (phylum Porifera) are sessile marine filter feeders that have developed efficient defense mechanisms against foreign attackers such as viruses, bacteria, or eukaryotic organisms. Marine sponges are among the richest sources of pharmacologically-active chemicals from marine organisms. It is suggested that (at least) some of the bioactive secondary metabolites isolated from sponges are produced by functional enzyme clusters, which originated from the sponges and their associated microorganisms. More than 5,300 different products are known from sponges and their associated microorganisms, and more than 200 new metabolites from sponges are reported each year. As infectious microorganisms evolve and develop resistance to existing pharmaceuticals, the marine sponge provides novel leads against bacterial, viral, fungal and parasitic diseases. Many marine natural products have successfully advanced to the late stages of clinical trials, as for example ara-A (vidarabine), an anti-viral drug used against the herpes simplex encephalitis virus. This substance is in clinical use for many years. Moreover, a growing number of candidates have been selected as promising leads for extended preclinical assessment, including manzamine A (activity against malaria, tuberculosis, HIV, and others), lasonolides (antifungal activity) and psammaplin A (antibacterial activity). In this review we have surveyed the discoveries of products derived from marine sponges and associated bacteria that have shown in vivo efficacy or potent in vitro activity against infectious and parasitic diseases, including bacterial, viral, fungal and protozoan infections. Our objective was to highlight the substances that have the greatest potential to lead to clinically useful treatments.

Structure-function relationships of the non-lanthionine-containing peptide (class II) bacteriocins produced by gram-positive bacteria.

Abstract: This review focuses on the structure and mode-of-action of non-lanthionine-containing peptide bacteriocins produced by Gram-positive bacteria. These bacteriocins may be divided into four groups: (i) the anti-listerial one-peptide pediocin-like bacteriocins that have very similar amino acid sequences, (ii) the two-peptide bacteriocins that consist of two different peptides, (iii) the cyclic bacteriocins, and (iv) the linear non-pediocin-like one-peptide bacteriocins. These bacteriocins are largely cationic, contain 20 to 70 residues, and kill cells through membrane-permeabilization. The pediocin-like bacteriocins are the ones that are best characterized. Upon contact with target membranes, their cationic N-terminal half forms a beta-sheet-like structure that binds to the target cell surface, while their more hydrophobic helical-containing C-terminal half penetrates into the hydrophobic core of target-cell membranes and apparently binds to the mannose phosphotransferase permease in a manner that results in membrane leakage. Immunity proteins that protect cells from being killed by pediocin-like bacteriocins bind to the bacteriocin-permease complex and prevent bacteriocin-induced membrane-leakage. Recent structural analyses of two-peptide bacteriocins indicate that they form a helix-helix structure that penetrates into cell membranes. Also these bacteriocins may act by binding to integrated membrane proteins. It is proposed that many membrane-active peptide bacteriocins kill target-cells through basically the same mechanism; the common theme being that a membrane-penetrating part of bacteriocins bind to a membrane embedded region of an integrated membrane protein, thereby causing conformational alterations in the protein that in turn lead to membrane-leakage and cell death.

Effect of additives on protein aggregation.

Abstract: This paper overviews solution additives that affect protein stability and aggregation during refolding, heating, and freezing processes. Solution additives are mainly grouped into two classes, i.e., protein denaturants and stabilizers. The former includes guanidine, urea, strong ionic detergents, and certain chaotropic salts; the latter includes certain amino acids, sugars, polyhydric alcohols, osmolytes, and kosmotropic salts. However, there are solution additives that are not unambiguously placed into these two classes, including arginine, certain divalent cation salts (e.g., MgCl ) and certain polyhydric alcohols (e.g., ethylene glycol). Certain non-ionic or non-detergent surfactants, ionic liquids, amino acid derivatives, polyamines, and certain amphiphilic polymers may belong to this class. They have marginal effects on protein structure and stability, but are able to disrupt protein interactions. Information on additives that do not catalyze chemical reactions nor affect protein functions helps us to design protein solutions for increased stability or reduced aggregation.

A Critical Review of Methods for Size Characterization of Non-Particulate Protein Aggregates

Abstract: Although size exclusion chromatography (SEC) has been, and will continue to be, the primary analytical tool for characterization of the content and size distribution of non-particulate aggregates in protein pharmaceuticals, regulatory concerns are driving increased use of alternative and complementary methods such as analytical ultracentrifugation and light scattering techniques. This review will highlight and critically review the capabilities, advantages, and drawbacks of SEC, analytical ultracentrifugation, and light scattering methods for characterizing aggregates with sizes below about 0.3 microns. The physical principles of the biophysical methods are briefly described and examples of data for real samples and how that data is interpreted are given to help clarify capabilities and weaknesses.

Plant Extracts: Search for New Alternatives to Treat Microbial Diseases

Abstract: Medicinal plants constitute the base of health care systems in many societies. The recovery of the knowledge and practices associated with these plant resources are part of an important strategy linked to the conservation of biodiversity, discovery of new medicines, and the bettering of the quality of life of poor rural communities. Research in phytosciences, an emerging multidisciplinary science, is almost unlimited, with several aspects to be discussed. Therefore, the focus of the present review is mainly on the antimicrobial and antioxidant properties of bioactive phytocompounds resultant of our research with crude plant extracts and essential oils of medicinal plants belonging to different families, used in various infectious disorders. The results obtained in the last years warrant the present review, discussing not only the use of several medicinal plants against bacteria, yeast, filamentous fungi and protozoa, but also their mechanisms of action, interactions with macromolecules and potential for toxicity in mammalian cells. Problems related to the efficacy of the isolation techniques and stability of bioactive compounds are also commented on. In addition, this review aims to emphasize the greatest importance to investigate plant species that have not been the subject of pharmacological studies, although their popular uses have been reported.

A Critical Review of Analytical Methods for Subvisible and Visible Particles

Abstract: The subvisible and visible particles present in a solution are often classified based on size, and are quantified by the actual number of particles present rather than by weight or molar amounts. The analysis of these particles in protein therapeutics are governed by compendial methods and the regulatory agencies, and the methods available to measure them originally evolved focusing on potential safety issues, including capillary occlusion and immunogenicity, that might arise from their presence. Ultracentrifugation, size exclusion chromatography, etc., discussed in previous articles, can be used to analyze aggregates of less than 0.10 microns. This article will focus on methods for analyzing and quantitating sub visible particles (SbVP) of 2 microns or larger. At the present time there is no routine method for quantitating sub visible particles (SbVP) between 0.1 microns and 2 microns. The most common technique for quantitating the amount of subvisible particles between 2 and 100 microns is the light obscuration method. This technique can determine size and amount of particles, but cannot differentiate between the types of particles, such as protein particles, foreign material, micro bubbles or silicone oil droplets, that can be present in protein solutions. The difficulties in adapting this method, originally developed for small molecule drugs for IV administration, to protein therapeutics delivered subcutaneously is discussed. The flow imaging techniques can determine morphology and optical characteristics of the particles, but still not identify the chemical composition. Other methods that can also be used, but are applicable for characterization purposes only, are discussed. The primary method for quantitating visible particles is visual inspection, a method that can be subjective and relies on adequate training of the human inspectors. Automated methods for visible particle determination are being developed. Identification of the chemical composition of isolated particles greater than about 50 microns is possible using several micro-spectroscopic methods, and these will also be discussed.

Advances in peptide pharmaceuticals.

Abstract: Drug delivery strategies for peptide pharmaceuticals have incorporated a wide range of structure activity relationships, analog generation to impart protease resistance and increased bioavailability, novel formulations, and delivery systems to target optimal therapeutic dosing requirements. Advances in peptide pharmaceuticals have provided products for the treatment of diabetes, obesity, Crohn's disease, osteoporosis, cancer, cardiovascular disease, immunotherapy, acromegaly, enuresis, pain, and antimicrobials. Here we review these marketed peptides and new peptidomimetic therapies currently in clinical trials.

Staphylococcal antimicrobial peptides: relevant properties and potential biotechnological applications.

Abstract: Bacteriocins are bacterial antimicrobial peptides with bactericidal activity against other bacteria. Staphylococcins are bacteriocins produced by staphylococci, which are Gram-positive bacteria with medical and veterinary importance. Most bacteriocins produced by staphylococci are either lantibiotics (e.g., Pep5, epidermin, epilancin K7, epicidin 280, staphylococcin C55/BacR1, and nukacin ISK-1) or class II bacteriocins (e.g., aureocins A70 and 53). Only one staphylococcin belonging to class III, lysostaphin, has been described so far. Production of staphylococcins is a self-protection mechanism that helps staphylococci to survive in their natural habitats. However, since these substances generally have a broad spectrum of activity, inhibiting several human and animal pathogens, they have potential biotechnological applications either as food preservatives or therapeutic agents. Due to the increasing consumer awareness of the risks derived not only from food-borne pathogens, but also from the artificial chemical preservatives used to control them, the interest in the discovery of natural food preservatives has increased considerably. The emergence and dissemination of antibiotic resistance among human and animal pathogens and their association with the use of antibiotics constitute a serious problem worldwide requiring effective measures for controlling their spread. Staphylococcins may be used, solely or in combination with other chemical agents, to avoid food contamination or spoilage and to prevent or treat bacterial infectious diseases. The use of combinations of antimicrobials is common in the clinical setting and expands the spectrum of organisms that can be targeted, prevents the emergence of resistant organisms, decreases toxicity by allowing lower doses of both agents and can result in synergistic inhibition.

Suppression of protein aggregation by L-arginine.

Abstract: L-Arginine is one of the most commonly used and most generally applicable suppressors of protein aggregation Its effect as enhancer of in vitro protein refolding was serendipitously discovered two decades ago This article aims at giving a brief overview about the discovery of the arginine effect, the range of its applications that have been explored over the past two decades, and of the current state of the discussion regarding the mechanisms responsible for the action of L-arginine as suppressor of aggregation

Multiple Myeloma Bone Marrow Niche

Abstract: "Niche" is defined as a specialized regulatory microenvironment, consisting of components which control the fate specification of stem and progenitor cells, as well as maintaining their development by supplying the requisite factors. Bone marrow (BM) niche has a well-organized architecture and is composed of osteoblasts, osteoclasts, bone marrow endothelial cells, stromal cells, adipocytes and extracellular matrix proteins (ECM). These elements play an essential role in the survival, growth and differentiation of diverse lineages of blood cells, but also provide optimal growth environment for multiple hematological malignancies including multiple myeloma (MM). MM is a neoplastic plasma cell disorder which not only resides in BM but also converts it into specialized neoplastic niche. This niche aids the growth and spreading of tumor cells by a complex interplay of cytokines, chemokines, proteolytic enzymes and adhesion molecules. Moreover, the MM BM microenvironment was shown to confer survival and chemoresistance of MM cells to current therapies. However, our knowledge in this field is still in infancy and many details are unknown. Therefore, there is a strong need to further dissect the MM BM niche and understand the process of how the complex interactions with BM milieu influence MM growth, survival and development of resistance to chemotherapy. A better and more detailed understanding of neoplastic MM niche will provide a guiding model for identifying and validating novel targeted therapies directed against MM. Therefore, in the present review, we have focused principally on the basic features, physical structures, and functions of the BM niche and have highlighted its interaction with MM cells.

Antibacterial and Antitumorigenic Properties of Microcin E492, a Pore- Forming Bacteriocin

Abstract: Microcins are a family of low-molecular weight bacteriocins produced and secreted by Gram-negative bacteria. This review is focused on microcin E492, a pore-forming bacteriocin produced by Klebsiella pneumoniae RYC492 that exerts its antibacterial action on related strains. The steps necessary for the production of active microcin E492 involve post-translational modification with a catechol-type siderophore at the C-terminal and proteolytic processing during export to the extracellular space. This bacteriocin has a modular structure, with a toxic domain at the N-terminal and an uptake domain at the C-terminal of the mature protein. The mechanism by which the C-terminal of microcin E492 is recognized by catecholate siderophore receptors is called the "Trojan horse" strategy, because the C-terminal structure mimics essential bacterial elements, which are recognized by the respective receptors and translocated across the outer membrane to exert antibacterial action. The C-terminal uptake module can be exchanged and used with other toxic domains. Microcin E492 also has a cytotoxic effect on malignant human cell lines. The cytotoxic mechanism is through apoptosis, a desired mechanism for cancer therapy. The ability of microcin E492 to form amyloid-like fibrils constitutes a property that can be exploited in the formulation of this bacteriocin as an antitumoral agent, because these fibrils can behave as stable depots to ensure the sustained release of a biologically active molecule. Alternatively, live bacteria can be used as a continuous source of microcin E492 production in specific tumors.

Trafficking of Mature miRNA-122 into the Nucleus of Live Liver Cells

Abstract: The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA- 122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMBprobes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.

Momordica balsamina: a medicinal and neutraceutical plant for health care management.

Abstract: Momordica balsamina, African pumpkin (Cucurbitaceae), is a tendril-bearing, wild climber containing wide spectrum of medicinal and nutritional values and has been used as a traditional folk medicine in many countries. The leaves, fruits, seeds, and bark of the plant contains resins, alkaloids, flavonoids, glycosides, steroids, terpenes, cardiac glycoside, saponins having various medicinal importance viz. anti-HIV, anti-plasmodial, shigellocidal, anti-diarrheal, anti-septic, anti-bacterial, anti-viral, anti-inflammatory, anti-microbial, hypoglycemic, antioxidant, analgesic and hepatoprotective properties. The therapeutic agent 'Momordin' is capable of inhibiting the growth of HIV and other viruses. The leaves are also important source of nutrients having 17 amino acids with adequate mineral composition like potassium, magnesium, phosphorus, calcium, sodium, zinc, manganese and iron. It also helps to combat the problem of micronutrient deficiencies in soil and high value of protein and fat with low fibre content. High potassium content is a good source for the management of hypertension and other cardiovascular conditions. This plant is being promoted as a protein supplement for cereal-based diets in poor rural communities. The commercial exploitation of this plant for biopharmaceuticals and neutraceuticals are some of the prospective future potential of this wild herb. This review discusses the potential of medicinal and nutritional importance of this wild herb for health care management.

10 years of tension on chromatin: results from single molecule force spectroscopy.

Abstract: The compact, yet dynamic organization of chromatin plays an essential role in regulating gene expression. Although the static structure of chromatin fibers has been studied extensively, the controversy about the higher order folding remains. In the past ten years a number of studies have addressed chromatin folding with single molecule force spectroscopy. By manipulating chromatin fibers individually, the mechanical properties of the fibers were quantified with piconewton and nanometer accuracy. Here, we review the results of force induced chromatin unfolding and compare the differences between experimental conditions and single molecule manipulation techniques like force and position clamps. From these studies, five major features appeared upon forced extension of chromatin fibers: the elastic stretching of chromatin's higher order structure, the breaking of internucleosomal contacts, unwrapping of the first turn of DNA, unwrapping of the second turn of DNA, and the dissociation of histone octamers. These events occur sequentially at the increasing force. Resolving force induced structural changes of chromatin fibers at the single molecule level will help to provide a physical understanding of processes involving chromatin that occur in vivo and will reveal the mechanical constraints that are relevant for processing and maintenance of DNA in eukaryotes.

Pharmacology and chemistry of a potent hepatoprotective compound Picroliv isolated from the roots and rhizomes of Picrorhiza kurroa royle ex benth. (kutki).

Abstract: Natural products from plants are of major pharmaceutical and therapeutic importance, several of which are often obtained from the underground parts of the concerned plants. Deviation from standard rules in modern medicines, where instead of a single isolated fraction, a group of naturally occurring components exerts the desired therapeutic effect, was noted in case of Picroliv or Kutkin of Picrorhiza kurroa. "Picroliv" mainly a glucoside, is one such compound, normally obtained from 3 - 4 years old roots and rhizomes of an endangered medicinal plant - Picrorhiza kurroa (kutki) and constitute an important component of many Indian herbal preparations, used mainly for the treatment of a variety of liver ailments. It is an iridoid glycoside mixture containing 60% picroside I and kutkoside in the ratio of 1:1.5. Picroliv has shown efficacy comparable to silymarin in rodent models of galactosamine, paracetamol, thioacetamide and CCl(4) induced hepatic damage. Picroliv has also shown cholerectic effect in rats and anti-cholestatic effect in rats, guinea pigs and cats treated with paracetamol and ethinyl estradiol. It has also anti-viral and immune-stimulant activities and is devoid of any significant CNS and CVS, autonomic and other systemic activity. Because of its apparent ability as a strong hepato-protective and immune-modulatory compound, it is in high demand in both national and international markets. The review discusses the potential of Picrorhiza in various hepatic diseases as well as the chemistry and activity of individual compound of crude drug Picroliv.

Targeting the immune system in cancer.

Abstract: The concept of cancer immunotherapy provides a fresh perspective as it is not associated with many of the drawbacks of conventional therapies such as chemotherapy, radiotherapy and surgery. When fully activated the immune system has immense potential as is evident from mis-matched transplanted organs undergoing rapid immunological attack and rejection. However, the development of immune strategies for cancer therapy has been associated with challenges of their own. Early attempts at cancer vaccination were carried out in an empirical manner that did not always lead to reproducibility. This led to a search of tumor associated antigens with the belief that specific targeting of these antigens would lead to successful tumor elimination. Active vaccination with TAA peptides or passive vaccination with specific lymphocytes against these TAAs did not however demonstrate encouraging results in clinical trials. This was mainly because of the lack of an activating immune response which is required for continuous stimulation of lymphocytes and also because of the selection of tumor escape variants that did not express the particular TAA. On the positive side, attempts at characterizing TAAs illuminated the molecular changes that attribute a malignant phenotype to cancer cells. Attempts at cytokine therapy were also met with challenges of high systemic toxicity and a lack of specific lymphocyte activation. It was therefore realized that an ideal vaccinating agent should be able to combine the effects of both these therapeutic strategies, i.e., it should be able to induce an innate immune response which can be tailored to a tumor specific adaptive immune response. By this, the immunosuppressive tumor environment can be altered to become immune activating, thus facilitating the infiltration of myeloid and lymphoid cells that can act in concert leading to tumor regression. In this regard, immunotherapeutic approaches such as DNA vaccines, dendritic cell based vaccines, HSP based vaccines and gene transfer technology, are being developed and further refined to overcome their inherent limitations. Animal experiments with these therapeutic modalities have demonstrated exciting results, although their evaluation in clinical trials has not indicated exceptional tumor protection in a large percentage of the patients. These observations only further underscore the multivariate and dynamic nature of the immune system and the many ways in which tumor cells modulate themselves and their surroundings to escape immune surveillance. Assessment of successful therapeutic intervention will require periodic evaluations of the suppressive nature of the tumor microenvironment accompanied by qualitative and quantitative measurements of lymphocyte responses in patients. With the development of advanced genetic technologies and continuous identification of tumor antigens, the field of cancer immunotherapy is progressing at an exciting pace giving us hope for the advent of effective treatment modalities that will prolong tumor free survival and enhance the quality of life in patients with malignant disease.

The German Mouse Clinic: a platform for systemic phenotype analysis of mouse models

Abstract: The German Mouse Clinic (GMC) is a large scale phenotyping center where mouse mutant lines are analyzed in a standardized and comprehensive way. The result is an almost complete picture of the phenotype of a mouse mutant line - a systemic view. At the GMC, expert scientists from various fields of mouse research work in close cooperation with clinicians side by side at one location. The phenotype screens comprise the following areas: allergy, behavior, clinical chemistry, cardiovascular analyses, dysmorphology, bone and cartilage, energy metabolism, eye and vision, hostpathogen interactions, immunology, lung function, molecular phenotyping, neurology, nociception, steroid metabolism, and pathology. The German Mouse Clinic is an open access platform that offers a collaboration-based phenotyping to the scientific community (www.mouseclinic.de). More than 80 mutant lines have been analyzed in a primary screen for 320 parameters, and for 95% of the mutant lines we have found new or additional phenotypes that were not associated with the mouse line before. Our data contributed to the association of mutant mouse lines to the corresponding human disease. In addition, the systemic phenotype analysis accounts for pleiotropic gene functions and refines previous phenotypic characterizations. This is an important basis for the analysis of underlying disease mechanisms. We are currently setting up a platform that will include environmental challenge tests to decipher genome-environmental interactions in the areas nutrition, exercise, air, stress and infection with different standardized experiments. This will help us to identify genetic predispositions as susceptibility factors for environmental influences.

Regulation of cellular signal transduction pathways by the extracellular calcium-sensing receptor.

Abstract: The extracellular calcium-sensing receptor (CaR) is a class III G-protein coupled receptor that coordinates cellular responses to changes in extracellular free Ca2+ or amino acid concentrations as well as ionic strength and pH. It regulates signalling cascades via recruiting and controlling the activities of various heterotrimeric G-proteins, including Gq/11, Gi/0, and G12/13, even Gs in some “unusual” circumstances, thereby inducing changes in the metabolism of membrane lipids, the phosphorylation state of protein kinases and their targets, the activation state of monomeric G-proteins and the levels of intracellular second messengers including cAMP, Ca2+ ions, fatty acids and other small molecules. According to its site(s) of expression and available signalling pathways, the CaR modulates cell proliferation and survival, differentiation, peptide hormone secretion, ion and water transport and various other processes. In this article we consider the complex intracellular mechanisms by which the CaR elicits its cellular functions. We also consider some of the better understood CaR-regulated cell functions and the nature of the signalling mechanisms that support them.

Separation and characterization of protein aggregates and particles by field flow fractionation.

Abstract: Field flow fractionation (FFF) is a technique that holds great promise for the analysis and characterization of protein aggregates and particles, due to its wide dynamic range and matrix-free separation mechanism. FFF can be routinely used to achieve good monomer-oligomer separation and quantification for a variety of protein types, and is a reasonable choice for an orthogonal method for size exclusion chromatography and analytical ultracentrifugation. Quantifying sub-micrometer particles in protein therapeutics is a potential of the FFF technique that is yet to be realized, due to the lack of detection with sufficient sensitivity. In this article the effect of several important parameters on the optimization of FFF analyses are explored, and the strengths, weaknesses, and potential new applications of the technique are discussed.

Use of lactobacilli and their pheromone-based regulatory mechanism in gene expression and drug delivery.

Abstract: Lactobacilli are common microorganisms in diverse vegetables and meat products and several of these are also indigenous inhabitants in the gastro-intestinal (GI) tract of humans and animals where they are believed to have health promoting effects on the host. One of the highly appreciated probiotic effects is their ability to inhibit the growth of pathogens by producing antimicrobial peptides, so-called bacteriocins. Production of some bacteriocins has been shown to be strictly regulated through a quorum-sensing based mechanism mediated by a secreted peptide-pheromone (also called induction peptide; IP), a membrane-located sensor (histidine protein kinase; HPK) and a cytoplasmic response regulator (RR). The interaction between an IP and its sensor, which is highly specific, leads to activation of the cognate RR which in turn binds to regulated promoters and activates gene expression. The HPKs and RRs are built up by conserved modules, and the signalling between them within a network is efficient and directional, and can easily be activated by exogenously added synthetic IPs. Consequently, components from such regulatory networks have successfully been exploited in construction of a number of inducible gene expression systems. In this review, we discuss some well-characterised quorum sensing networks involved in bacteriocin production in lactobacilli, with special focus on the use of the regulatory components in gene expression and on lactobacilli as potential delivery vehicle for therapeutic and vaccine purposes.

The role of the calcium-sensing receptor in bone biology and pathophysiology.

Abstract: Bone cells, particularly osteoblasts and osteoclasts, exhibit functional responses to calcium (Ca(2+)). The identification of the calcium-sensing receptor (CaR) in parathyroid glands as the master regulator of parathyroid hormone (PTH) secretion proved that cells could specifically respond to changes in divalent cation concentration. Yet, after many years of study, it remains unclear whether this receptor, which has also been identified in bone, has functional import there. Various knockout and transgenic mouse models have been developed, but conclusions about skeletal phenotypes remain elusive. Complex endocrine feedback loops involving calcium, phosphorus, vitamin D, and PTH confound efforts to isolate the effects of a single mineral, hormone, or receptor and most models fail to account for other local factors such as parathyroid hormone related protein (PTHrP). We review the relevant mouse models and discuss the importance of CaR in chondrogenesis and osteogenesis. We present the evidence for a non-redundant role for CaR in skeletal mineralization, including our experience in patients with activating CaR mutations. Additionally, we review emerging research on the importance of the CaR to the regulation of serum calcium homeostasis independent of PTH, the role of the CaR in the hematopoietic stem cell niche with implications for bone marrow transplant, and early evidence that implies a role for the CaR as a factor in skeletal metastasis from breast and prostate cancer. We conclude with a discussion of drugs that target the CaR directly either as agonists (calcimimetics) or antagonists (calcilytics), and the consequences for bone physiology and pathology.

Targeting the tumor stroma in cancer therapy.

Abstract: Increasing evidence shows that the interaction between neoplastic cells and the surrounding stroma is a critical factor in solid tumor growth. The tumor stroma is made up of diverse cellular populations including macrophages, lymphocytes, vascular cells, and carcinoma-associated fibroblasts. The complex interactions between the stroma and neoplastic cells are largely unexplored. Initial therapies aimed at disrupting angiogenesis within the tumor microenvironment have met with success in a number of tumor types. An improved understanding of stromal signaling pathways is likely to identify additional novel therapeutic targets.

Preparation of polymeric carriers for drug delivery with different shape and size using an electric jet.

Abstract: A method for generating poly(lactic-co-glycolic acid) and polycaprolactone biodegradable particles of different size and shape using a jet generated in an electric field is elucidated. These particles are suitable for use as drug carriers and the method can be developed into a mass production route. The effect of different parameters such as applied voltage, collecting distance, flow rate and polymer concentration on inducing size and shape differences in these particles was studied. It was found that the flow rate, polymer concentration and collecting distance have a significant impact on the size of the generated particles and by changing the collecting distance a systematic reduction in the particle size by at least an order of magnitude (10microm-100nm) can be achieved. By using a high polymer concentration (30 wt. %) the shape and surface morphology of these particles can also be controlled from spherical to fibrous, and smooth to irregular, respectively, which presently is an interesting strategy and concept in drug delivery. This method is very useful as a one-step generator of different sizes of drug carriers with morphological variations.

IgG Aggregate Removal by Charged-Hydrophobic Mixed Mode Chromatography

Abstract: Charged-hydrophobic mixed mode chromatography methods have been applied to antibody purification for decades and have focused more recently on the specific task of aggregate removal. They exploit various combinations of alkyl and aromatic hydrophobic groups with positively and/or negatively charged residues. Charge and hydrophobicity remain relatively constant as function of pH for some ligands; one or both vary for others. All of these compound selectivities and their associated elution strategies are intended to achieve purification of native IgG through preferential retention of aggregates. This review focuses on the two members of this family that have shown the most promise for aggregate removal: MEP HyperCel() and Capto() adhere. It defines how they work, how they interact with various classes of biomolecules, how those interactions are controlled by different elution strategies, and how to determine which may be most effective for a particular antibody. Consideration is also given to their specific strengths and limitations from an industrial perspective.

Chlorophytum borivilianum: a white gold for biopharmaceuticals and neutraceuticals.

Abstract: Chlorophytum borivilianum Santapau & Fernandes (Liliaceae) also known as ‘Safed Musli’ is a traditional rare Indian medicinal herb which has many therapeutic applications in Ayurvedic, Unani, Homeopathic and Allopathic systems of medicine. Its roots (tubers) are widely used for various therapeutic applications. It is used to cure physical illness and weakness, as an aphrodisiac agent and revitalizer, as general sex tonic, remedy for diabetes, arthritis and increasing body immunity, curative for natal and postnatal problems, for rheumatism and joint pains, increase lactation in feeding mothers, as antimicrobial, anti-inflammatory, antitumor agent, also used in diarrhea, dysentery, gonorrhea, leucorrhea etc. It has spermatogenic property and is found useful in curing impotency, now it is considered as an alternative ‘Viagra’. Its root contains steroidal and triterpenoidal saponins, sapogenins and fructans which act as therapeutic agents and play vital role in many therapeutic applications. It is a rich source of over 25 alkaloids, vitamins, proteins, carbohydrates, steroids, saponins, potassium, calcium, magnesium, phenol, resins, mucilage, and polysaccharides and also contains high quantity of simple sugars, mainly sucrose, glucose, fructose, galactose, mannose and xylose. The commercial exploitation of this plant and their secondary metabolites, germplasm conservation and in vitro production of secondary metabolites for quality control are some of the major prospects of this rare medicinal herb. The focus of the present review is to galvanize the potential of therapeutic and nutritive values of this herb and production of their secondary metabolites. The in vitro tuber induction, extraction, purification and characterization of saponins are also discussed in the present review.

Tumor Initiating Cells

Abstract: Cancer Stem cells (CSC) are defined as a population of cells found within a tumor that have characteristics similar to normal stem cells. Like normal stem cells they have the potential to self renew and differentiate. The cellular origin of these cancer stem cells--whether they originate from stem cells that have lost the ability to regulate proliferation, or they arise from more differentiated population of progenitor cells that have acquired abilities to self-renew is still unclear. Investigators have reported isolation of cancer stem cells or tumor initiating cells using techniques developed for isolating hematopoietic stem cells and assays that identify a small subset of tumor initiating cells. The TICs are thought to play an important role in tumor development, progression as well response to therapy and relapse. Strategies that combine conventional therapies with newer approaches that target the TICs may be more effective in tumor cell kill are discussed.

Recent Advancement in Application of Hydrophobic Interaction Chromatography for Aggregate Removal in Industrial Purification Process

Abstract: Hydrophobic interaction chromatography (HIC) is a classic purification tool applied in protein and antibody, laboratory and industrial production process. It has been mainly used for the removal of both product-related impurities such as aggregates, as well as process contaminants such as host cell proteins. This review will focus on the recent development of HIC in its applications in the industrial purification processes. The process economy and requirements of high product purity and quality have driven much of the recent advancement in HIC chromatography in terms of increased throughput and enhanced selectivity or resolution. Meanwhile, high throughput screening (HTS), design of experiments (DoE) and platform approach for process development have been applied to shorten the development time. The throughput improvement has been achieved through new resins with increased binding capacity, using dual salts for load conditioning, and operating in the flow-through mode. In addition, hydrophobic interaction membrane filter chromatography technology reduces bed volumes and buffer usage and potentially improves process throughput by reducing cycle time. Selectivity and/or resolution enhancements have been achieved through optimization of operation parameters such as temperature and efforts such as application of solvent additives.