Showing papers in "Cellular and Molecular Biology in 1993"

Generation of senescent cell antigen on old cells initiates IgG binding to a neoantigen

Abstract: An aging antigen, senescent cell antigen, resides on the 911 amino acid membrane protein band 3. It marks cells for removal by initiating specific IgG autoantibody binding. Band 3 is a ubiquitous membrane transport protein found in the plasma membrane of diverse cell types and tissues, and in nuclear, mitochondrial and Golgi membranes. Band 3 in tissues such as brain performs the same functions as it does in red cells. Senescent cell antigen is generated on brain membranes. Oxidation is a mechanism for generating senescent cell antigen. Neither cross-linking nor hemoglobin appear to play a role in generating senescent cell antigen. Although storage is the only in vitro model that mimics cellular aging in situ, we have discovered three alterations/mutations of band 3 that permit insight into aging in situ. One mutation with an addition to band 3 has normal or decelerated red cell aging. In contrast, another band 3 alteration with a suspected deletion or substitution that renders band 3 more susceptible to proteolysis, shows accelerated aging. The third alteration which is also more susceptible to proteolysis is associated with neurologic defects. Peptide technology was used to map the aging antigenic sites and anion transport sites on band 3 using a competitive inhibition assay and immunoblotting with IgG directed against the aging antigen on old cells. Results indicate that: a) aging antigenic sites reside on human band 3 residues 538-554, and 812-830; b) a putative ankyrin binding region peptide is not involved in senescent cell antigen activity and c) carbohydrate moieties are not required for the antigenicity or recognition of senescent cell antigen since synthetic peptides alone abolish binding of senescent cell IgG to erythrocytes. Peptide residues 588-594 (a 7 amino acid peptide), 822-839 and 869-883 were the most active inhibitors of anion transport (P < or = 0.001 compared to control without peptide). Localization of the active antigenic and transport sites on band 3 molecule facilitates the definition of molecular changes occurring during aging that initiate molecular as well as cellular degeneration. The role of senescent cell antigen and band 3 in brain aging and Alzheimer's disease is discussed.

Collagen cross-linking by pyridinoline occurs in non-reversible skin fibrosis.

Abstract: The extent of the covalent cross-linking of collagen molecules by pyridinoline was measured in skin lesions from patients with chromomycosis, a chronic fungal infection leading to an extensive and chronic dermal fibrosis. These data were compared to those collected from patients with a localized cutaneous leishmaniasis, an acute inflammatory process leading to an extensive and reversible remodelling of the extracellular matrix. The amount of the mature cross-linking amino acid pyridinoline increased in chromomycosis patients when compared to controls and was significantly higher than in leishmaniasis patients. These data confirm and extend our previous studies on liver fibrosis showing that a high level of pyridinoline is associated to irreversible fibrotic lesions. They also suggest that an increase in the mature collagen cross-linking by pyridinoline in the course of fibrosis is not restricted to liver, but might be a general feature of irreversible and chronic fibrosis.The extent of the covalent cross-linking of collagen molecules by pyridinoline was measured in skin lesions from patients with chromomycosis, a chronic fungal infection leading to an extensive and chronic dermal fibrosis. These data were compared to those collected from patients with a localized cutaneous leishmaniasis, an acute inflammatory process leading to an extensive and reversible remodelling of the extracellular matrix. The amount of the mature cross-linking amino acid pyridinoline increased in chromomycosis patients when compared to controls and was significantly higher than in leishmaniasis patients. These data confirm and extend our previous studies on liver fibrosis showing that a high level of pyridinoline is associated to irreversible fibrotic lesions. They also suggest that an increase in the mature collagen cross-linking by pyridinoline in the course of fibrosis is not restricted to liver, but might be a general feature of irreversible and chronic fibrosis.

Mercury, Zinc and Selenium Bioaccumulation in Tissues and Organs of Mediterranean Striped Dolphins Stenella-coeruleoalba Meyen Toxicological Result of Their Interaction

Abstract: Neutron activation analysis of 13 Mediterranean striped dolphins Stenella coeruleoalba showed high mercury and selenium contaminations of main tissues and organs of these cetaceans. The mercuric contents were excessive, particularly in liver (from 68 to 2272 mug/g dry wt. basis), then in kidney, lung, muscle, heart and brain. The selenium concentrations were also high in liver (from 45 to 1320 mug/g dry wt. basis), then in kidney, lung, muscle, skin and heart. The main way of contamination seems to be the food through trophic network, but skin and lung are also able to play a part which must be elucidated. The average Hg/Se ratios in liver and kidney were respectively 1.82 and 1.59. Linear relationship between mercury and selenium concentrations in tissues and organs, particularly in liver and kidney, were confirmed. The mercury and selenium interaction on a toxicological point of view was established by a statistical approach; in the same way, intervention of zinc, metallothioneins and glutathiones have been discussed.

Distribution and subcellular immunolocalization of 1,25-dihydroxyvitamin D3 receptors in rat epiphyseal cartilage.

Abstract: The distribution and subcellular localization of the 1,25-dihydroxyvitamin D3 receptor (VDR) in the epiphyseal cartilage of normal weaning rats were examined immunocytochemically at the light and electron microscope level using a monoclonal anti-VDR antibody (9A7 gamma). VDR immunoreactivity was detected in the nuclei of chondrocytes in all zones of the epiphyseal plate cartilage from the resting to calcifying chondrocytes, and at much lower concentrations, in the cytoplasms. Perichondrial mesenchymal cells contained no VDR immunoreactivity. VDR immunoreactivity developed in the nuclei of cells in the lateral margin area as they acquired the chondroblast phenotype. VDR immunoreactivity was also found over the nucleoli of chondrocytes in all cells zones of the epiphyseal plate and appeared in the nucleoli of the cells in the lateral margin area before immunostaining of the nuclei, as the mesenchymal cells differentiated into chondroblasts. Electron microscopy showed that the immunoreactivity for 1,25(OH)2D3 receptor, indicated by gold particles, was associated with scattered clumps of compact chromatin and small clumps of dispersed chromatin. But the nuclei immunostaining patterns before and after mitosis were different in proliferative chondrocytes. The heterochromatin along the nuclear envelope was immunonegative in interphase chondrocytes, but there was VDR immunostaining over the rim of the perinuclear chromatin just after mitosis. In the nucleoli, the dense fibrillar component was immunostained, but the fibrillar centers and the perinuclear chromatin were not. This distribution of VDR immunoreactivity suggests that the hormone is directly involved in differentiation, proliferation and maturation of cartilage cells, and also with extracellular calcification in epiphyseal cartilage. The presence of immunoreactive VDR receptors in nucleoli of chondrocytes, particularly the fibrillar component, suggests that 1,25(OH)2D3 may be involved in regulation of ribosomal genes.

Murine prosaposin: expression in the reproductive system of a gene implicated in human genetic diseases.

Abstract: Expression of the prosaposin gene, implicated in human genetic diseases, was studied in the mouse by Northern blot analysis and in situ hybridization Using a cloned mouse prosaposin cDNA as a probe, we observed high expression of the prosaposin gene in the adult and embryonic nervous and reproductive systems while lower levels of prosaposin mRNA were detected in the heart, kidneys, liver, adrenals and lymphoid organs Ubiquitous low level of prosaposin mRNA were detected by in situ hybridization in 125 days-old embryos However, high expression was restricted to the hindbrain, the dorsal ganglia and to the genital ridge, the primordium for both male and female gonads RNA transcription in the testis was detected in Sertoli cells, Leydig cells and peritubular cells but not in germ cells of adult mice Similar observations were obtained with the atrichosis mutant mouse known to lack normal germ cells, indicating that prosaposin gene expression was not dependent on the presence of germ cells in the testis Prosaposin was found to be expressed in the mature female gonads at various stages of the corpus luteum development These results strongly suggest that prosaposin may play an important role in the reproductive system as well as the nervous system

Effect of sodium butyrate on the expression of retinoblastoma (RB1) and P53 gene and phosphorylation of retinoblastoma protein in human colon tumor cell line HT29.

Abstract: Sodium butyrate is known to induce morphological and biochemical changes associated with cell differentiation in some colon tumor cell lines including HT29. In our present study we observed that sodium butyrate treatment caused a decrease in the level of expression of RB1 gene on day seven of butyrate treatment but a gradual six to sevenfold decrease in the level of expression of p53 gene. Western blot analysis revealed a decrease in the level of the phosphorylated form of Rb protein (pRb) and an increase in the level of underphosphorylated pRb as compared to the control cells. These changes in the phosphorylation level were observed from day three of sodium butyrate treatment. In addition, the flat foci forming large differentiated cells also began to appear after 3 days of sodium butyrate treatment. In this study, we are able to show that, besides induction of differentiation, sodium butyrate treatment can also cause a reversal in the phosphorylation status of the pRb in colon tumor cell line HT29. These results suggest that the phosphorylation level of pRb could be associated with the cell differentiation process in human colonic epithelium and as a consequence in its neoplastic development.

Alterations of retinoblastoma susceptible gene accompanied by c-myc amplification in human bone and soft tissue tumors.

Abstract: The present study was undertaken to examine oncogene abnormalities in human bone and soft tissue tumors. Twenty four tumor tissues and one human cell strain established from an osteosarcoma were examined by Southern blot analysis using a recessive oncogene (p0.9R and p3.8R derived from a cDNA of the Rb gene) and eight dominant oncogenes (c-myc, c-K-ras, c-fos, c-raf-1, c-fms, c-sis, N-myc, and c-erb B) as probes. Homozygous deletions or other alterations within the Rb locus were found in 3 of 6 osteosarcomas, 1 osteosarcoma cell line, 1 of 3 malignant fibrous histiocytomas and 1 of 2 Ewing's sarcomas. On the other hand, amplification of c-myc was found in 2 osteosarcomas and 1 osteosarcoma cell line. All cases with c-myc amplification had alterations in the structure of the Rb locus, and these patients showed rapid clinical malignancy progression and a probable tendency to bone metastases. Results of this study suggest that structural alterations of the Rb gene and amplification of c-myc might play an important role in the clinical course and pathogenesis of osteosarcoma.

Postnatal development and aging of esophageal epithelium in mouse: a light and electron microscopic radioautographic study.

Abstract: DNA synthesis and fine structure of the esophageal epithelium of ddY mouse in different age groups, from neonatal to senescent, were investigated by light and electron microscopic radioautography after 3H-thymidine labeling in vitro. At 1-3 days after birth, the esophagus was lined with two cell types, ciliated and nonciliated cells. From 1 week on, ciliated cells could not be found. At 2 weeks, the superficial cells still possessed nuclei and short microvilli on the apical plasma membranes. From 1 month, the superficial cells became keratinized and the microvilli on the apical plasma membrane disappeared. With aging of the mice, the epithelia became markedly thick. These results show that disappearance of ciliated cells, keratinization of superficial cells and increase of the thickness are the main changes in structure of mouse esophageal epithelium from neonatal to senescent ages. By light microscopic radioautography, labelled cells were almost confined in the basal layer, regardless of the aging stages. The highest labeling index was recorded at 1 day after birth, then decreased with age. By electron microscopic radioautography, silver grains indicating DNA synthesis were observed in nuclei. As compared with those of unlabelled cells in the basal layer, the nuclei and nucleoli of labelled cells were larger, with fewer cell organelles. The present study provides, for the first time, the basic quantitative data regarding cell proliferation of mouse esophageal epithelium from neonatal to senescent stage.

Human papillomavirus type 18 DNA in so-called HEP-2, KB and FL cells--further evidence that these cells are HeLa cell derivatives.

Abstract: The contamination of HeLa cells into many culture cells has been suspected. We detected HPV type 18 DNA in two HeLa cell sublines, Hep-2 laryngeal cancer and KB oral cancer cell lines as well as FL cell line derived from normal amniotic membrane which have been extensively used so far in experimental virology. On the other hand, we detected HPV type 18 DNA neither in other epithelial cell lines nor in fibroblastic human embryonic cells. The digestion analysis with several restriction enzymes of the HPV type 18 DNA in HeLa, Hep-2, KB and FL cell lines were almost the same. The molecular size of episomal DNA could not be demonstrated by digestion of these DNAs with XhoI, a non-cut enzyme for HPV type 18 DNA, suggesting all HPV types 18 DNA were integrated. Interestingly enough, the HPV type 18 specific DNA in these cells was cleaved into two fragments with another non-cut enzymes, B g/II and HindIII, suggesting the presence of a single cutting site for each enzymes presumably generated by rearrangements or mutations. These results provide further evidence that the so-called Hep-2, KB and FL cells are all HeLa cell derivatives.

Light and electron microscopic radioautographic studies on the RNA synthesis of peri-implanting pregnant mouse uterus during activation of receptivity for blastocyst implantation.

Abstract: The uterine receptivity for blastocyst implantation in the rodents is activated by nidatory estradiol and is maintained for a restricted period. To establish the variation of the RNA synthesis related to the activation of the uterine receptivity for blastocyst implantation, 1 hr. pulse 3H-uridine incorporation in the mouse uterus at time 0, 3, 6, 12 and 18 hrs. after nidatory estradiol treatment was analyzed by light (LM-RAG) and electron microscopic (EM-RAG) radioautography. The qualitative and quantitative differences of 3H-uridine incorporation in the luminal epithelium and endometrial stroma of three regions of the uterus were evaluated. In the LM-RAM, both the luminal epithelium and stromal cells around the conceptus showed a high incorporation 6 hrs. after estrogen treatment. After the peak of incorporation, the RNA synthesis both of the epithelial and stromal cells of antimesometrial side of the implantation site (AI) decreased abruptly and the grain count values become lower than seen in the other two sites. Both the epithelial and stromal cells of the IN sites showed a gradual but low increase of 3H-uridine incorporation through the time analyzed. The cells of the MI sites also showed a peak of incorporation 6 hrs. after estradiol treatment, but less intense than the AI sites. The EM-RAM showed the silver grains distributed over the cell structures related to the mRNA and rRNA metabolism both on the epithelial and stromal cells. The labeling were also seen on the trophoblastic cells, endothelial cells, smooth muscle cells of the myometrium and mesothelial cells. These results proved the strong influences of the estrogen on the pregnant uterus and showed the topological differences on the responses of the endometrium according to their relation to the implanting blastocyst, and suggest a time restricted responses of the endometrium for activation of uterine receptivity for blastocyst implantation.

Proliferative activity in the kidneys of aging mice evaluated by PCNA/cyclin immunohistochemistry.

Abstract: The change of proliferative activity in mouse kidney cortex cells due to aging was studied by PCNA/cyclin immunostaining. Mouse kidney tissues of various ages: late fetal, newborn, suckling, weaning, adult and senescent were used for this experiment. Small pieces of kidney tissues were fixed in methacarn solution and embedded in paraffin. Sections were stained with PCNA/cyclin monoclonal antibody. The reaction product for PCNA/cyclin was observed mainly on nuclei. The ratio of the PCNA/cyclin positive nuclei to the total number of nuclei were calculated. PCNA/cyclin positive ratios in glomeruli and uriniferous tubules in the superficial layer were higher than those in the deep layer from late fetal period to suckling period. They decreased due to aging after birth and became to nearly zero after weaning period.

Biochemical and morphological studies on the effects of anthocyans and vitamin E on carbon tetrachloride induced liver injury.

Abstract: The effects of the natural antioxidants - anthocyans and vitamin E (in a solubilized pharmaceutical form) on carbon tetrachloride - induced liver injury in rats are studied. The changes in the activity of serum transaminases (ALAT and ASAT), the content of the reduced glutathione and cytochrome P-450 as well as the intensity of the processes of lipid peroxidation are assessed. The anthocyans exert a protective effect comparable to that of vitamin E on liver cells. The favorable effects of the combination of the antioxidants on the content of the reduced glutathione and on the processes of lipid peroxidation are more intensely expressed. The morphological changes occurring in hepatocytes correlate with the results of the biochemical studies. It is evident that both substances have a marked hepatoprotective activity

Embryonic skin fibroblasts release TGF alpha and TGF beta able to influence synthesis and secretion of GAG.

Abstract: Conditioned medium (CM), collected from 7 and 14 days-old chick embryo skin fibroblasts and added to the same cells, increases glycosaminoglycans (GAG) intra- and extracellular accumulation. The factors responsible for GAG enhancement are TGF alpha and TGF beta because they are trypsin and dithiothreitol sensitive, stable or enhanced by heat and transient acidification. Moreover, Sephadex G-75 fractions of CM active on GAG synthesis contain, when analysed on SDS-polyacrylamide gel electrophoresis, two bands that comigrate with TGF alpha and TGF beta and induce NRK cells clone 49F to form large colonies of mean size > 8.000 microns 2 in soft agar. Since both the factors must be present to induce the formation of large colonies we come to the conclusion that CM contains TGF alpha and TGF beta. The two growth factors have different effects on the accumulation of individual classes of GAG in the ECM. In particular, TGF beta stimulates a marked increase of CS and DS, TGF alpha of HA and DS in the medium. The contemporaneous addition of TGF alpha and TGF beta to 7 days-old fibroblasts produces a pattern of GAG response similar to CM. These embryonic fibroblasts may control their own GAG synthesis and secretion through autocrine TGF alpha and beta activity.

Electron microscopic radioautographic study on DNA synthesis in perinatal mouse retina.

Abstract: For the purpose of comparing the quantitative changes in both labelled and unlabelled retinal cells with 3H-thymidine radioautography, normal ddY mice from early embryonic stage (E 9.5) to postnatal 2 weeks were utilized as materials. About 200 electron microscopic radioautograms were taken from the middle portions of retina in the early embryonic stages and from the inner half of outer neuroblastic layers of retina of 9 groups of litter mice at the late embryonic and postnatal ages, and the enlarged photographs were quantitatively analyzed by image analysis. The results showed that the area of nuclei, cytoplasm, mitochondria and the number of mitochondria, per retinal cell, decreased from early embryonic stages to postnatal ages in both labelled and unlabelled cells (p < 0.05). However, no significant changes occurred in the area of ER per retinal cell in both labelled and unlabeled cells. Significant differences of the ultrastructural changes between labelled and unlabelled cells were not statistically detected except the area of nuclei at P 1 and the number of mitochondria at P 3.

Synaptic and non-synaptic immunolocalization of GABA and glutamate acid decarboxylase (GAD) in cerebellar cortex of rat

Abstract: The existence of a large number of GABA receptors in the cerebellar molecular layer, and the observation of numerous punctate immunoreactive deposits of GABA synthesizing enzyme (GAD) throughout this layer, could indicate the existence of numerous axon terminals that may be involved in neurotransmission modulated by GABA. These axon terminals may be different from those considered classically as cerebellar GABAergic axon terminals. Therefore, we have reinvestigated the localization of GABA- and GAD-immunoreactivities in the cerebellar cortex of the rat with the PAP method, using different antisera obtained from rabbits immunized with GABA, baclofen and GAD. The results observed in our investigation have demonstrated GABA- and GAD-immunoreactivities in the axon terminals considered classically as GABAergic, as well as in others which, until now, have not been considered GABAergic. This fact leads us to think that the distribution of GABA or molecules structurally similar to GABA is far more extended than previously thought in the cerebellum. We have also observed both GABA- and GAD-immunoreactivities within dendrites and glial cells. These facts suggest us a possible extrasynaptic release of GABA.

Light microscopic radioautographic study on DNA synthesis in aging mice corneas.

Abstract: The morphological change and DNA synthesis of the aging mice corneal cells were investigated by light microscopic radioautography after injection of tritiated thymidine The result showed that the incorporation of tritiated thymidine in corneal cells changed with aging The sites of tritiated thymidine incorporation were located in the epithelium from postnatal day 19 to 1 year after birth, in the stroma and endothelium from prenatal day 19 to postnatal day 8 only The labeling index in the epithelial cells reached its highest value at 1 month after birth Labelled stromal and endothelial cells reached their peaks simultaneously on the third day after birth and disappeared completely from postnatal 1 month onwards The thickness of the cornea increased obviously at one month and there were no notable morphological change thereafter Our investigation provides for the first time a systematic study on the age-related changes of DNA synthesis and construction in the aging mice corneas

The effects of endotoxin administration on blood amino acid concentrations: similarities with sepsis.

Abstract: Following an acute endotoxin (LPS) administration (1 mg/kg body weight) to rats, there was a decrease in the blood concentration of most gluconeogenic amino acids, alanine, glycine, serine, threonine and proline. While the administration of the endotoxin induced no changes in the concentrations of aromatic amino acids, it decreased the concentration of both branched-chain amino acids (leucine, isoleucine and valine) and basic amino acids (lysine, arginine, histidine and ornithine). On a global basis, the endotoxin significantly decreased the total blood amino acid concentration 2 hrs. after the administration, the effect lasting as long as 8 hrs. after endotoxin treatment. This decrease was mainly associated with a lower blood concentration of essential amino acids. Rats induced septic, by cecal ligature and puncture, showed a blood amino acid pattern most similar to those acutely treated with LPS.

Actin, tropomyosin and alpha-actinin as markers of differentiation in human rhabdomyosarcoma cell lines induced with dimethyl sulfoxide.

Abstract: Most rhabdomyosarcomas are poorly differentiated malignant tumors. Dimethyl sulfoxide has been shown to modulate cell differentiation in cultured human cells. We induced differentiation in human rhabdomyosarcoma cell lines A-673, RD and A-204 with 1.25% dimethyl sulfoxide, and used desmin, the protein most frequently used as a marker of muscle cell differentiation, to trace this process. As alternative markers of the degree of differentiation, we quantified the expression of the proteins actin, tropomyosin and alpha-actinin in these cell lines, and followed the changes in expression of these proteins after induction for 8, 12, 24, 48 and 72 hrs. In the process of differentiation, protein expression in both the cytoplasm and cytoskeleton was significantly increased by treatments lasting 12 hrs. (alpha-actinin) and 24 hrs. (actin). On the basis of our results, alpha-actinin can be considered as an earlier marker of differentiation than actin in human rhabdomyosarcoma cell lines. However, the earliest indication of differentiation was a modification in desmin expression (8 hrs.). Because changes in tropomyosin expression were less marked, we consider this protein as a poor marker of rhabdomyosarcoma cell differentiation.

Endocytosis of lipopolysaccharide in mouse macrophages.

Abstract: Lipopolysaccharide binding sites of mouse peritoneal macrophages were demonstrated by means of immunogold technique. Resident peritoneal macrophages identified by peroxidatic activity in the nuclear envelope and in the rough endoplasmic reticulum show moderate and constant specific binding of bacterial lipopolysaccharide from E. coli to cell surface structures. Labeling of peritoneal macrophages with LPS-gold particles (LPS-Au) at 4 degrees C followed by incubation of the cells at 37 degrees C permits the investigation of LPS endocytosis. After various incubation times LPS-Au was detected in different endocytic compartments. LPS was internalized via coated pits and coated and uncoated vesicles (5 min.). After 60 min, incubation time LPS-Au occurred in electron lucent endosomes, multivesicular bodies, tubulo-reticular structures and in lysosomes. Gold particles appeared mainly in lysosomes after a longer incubation time (240 min.). The results of LPS binding and internalization are in accordance with a postulated LPS-receptor binding.

Light microscopic radioautographic study of DNA synthesis in the kidneys of aging mice.

Abstract: The change of proliferative activity in mouse kidney cortex cells during development and aging was studied by detecting S-phase cells by light microscopic radioautography using 3H-thymidine (3H-TDR). Mouse kidney tissues of various ages: prenatal 13.5, 15.5 and 19.0 days of gestation, postnatal 1 and 8 days, 1 and 2 months, and 1 year were used for this experiment. The kidney cortex tissues were labelled with 3H-TDR either in vitro or in vivo and they were processed for light microscopic radioautography. The labeling indices of the respective cell types were calculated. The labeling indices in glomeruli and uriniferous tubules in the superficial layer were higher than those in the deep layer of metanephric cortex through the developmental stages. However, they decreased rapidly after birth and reached low levels (below 4%) from 1 day to 1 year.

Timely and topologically defined protein synthesis in the peri-implanting mouse endometrium revealed by light and electron microscopic radioautography

Abstract: Many efforts have been made to correlate the morphological and biochemical evidences with changes on physiological state of the uterus during activation of the implantation window. However, the exact mechanism involved in such a phenomenon remains to be determined. The present experiment used the radioautographic approach to determine whether, chronological and/or topological variations of proteins synthesis occur in the peri-implanting endometrium. Pregnant mice submitted to ovariectomy, received exogenous supply of nidatory estradiol. After 0 to 18 hrs. of time-lapsed estradiol effects, each animal received intraperitoneally, 1 hr. pulse 3H-leucine. The uterine fragments embedded in epoxy resin, were processed for light and electron microscope radioautography. The pattern of 3H-leucine incorporation in the luminal epithelium varied according to their relation with the blastocyst present in the uterine lumen. The highest incorporation ratio was seen in the epithelium just around the conceptus 6 hrs. after estradiol treatment, while in the cells localized at interimplantation site no peak of incorporation was seen. At ultrastructural level, cell organelles involved in protein synthesis were found to be labelled. Accumulation of silver grains occurred at apical portion of the epithelial cells showed accumulation of silver grains after 6 hrs. of estradiol treatment, but not on cell surface. The endometrial stromal cells localized around the blastocyst also showed a peak of 3H-leucine incorporation 6 hrs. after estradiol, but not in the cells localized at interimplantation sites. No increased labeling was seen on the components of extra-cellular matrix at ultrastructural level. The present radioautographic experiment showed that epithelial and stromal cells localized in the endometrium of implantation chamber, changed their pattern of protein synthesis under nidatory estradiol effects. This evidence suggests that the phenomenon of implantation window induced by estradiol, is not a systemic response of the whole pregnant endometrium. The activation involves only a specific population of the endometrial cells localized just around the conceptus.

Gene-specific modulation of RNA synthesis and degradation by extremely low frequency electromagnetic fields

Abstract: Pulse-labeling studies from our laboratory and others have shown that extremely low frequency (ELF) electromagnetic fields can produce a transient increase in gene transcription. In this study, the synthesis, degradation and processing, and steady state levels of specific RNA species during exposure to ELF radiation were determined in human leukemia HL-60 cells. The overall steady state RNA levels, assessed by continuous and equilibrium labeling with 3H-uridine, were not affected by ELF exposure. Northern blot analysis using probes specific for c-myc, beta-actin, and 45S ribosomal RNA gene products revealed that ELF did not alter the steady state levels of these RNAs. Examination of gene-specific transcription by a novel nuclease protection assay revealed that while ELF did not substantially alter the transcription rates for c-myc and beta-actin, transcription of the 45S ribosomal RNA gene was increased by 40-50%. To explain the observed increase in the synthesis of 45S ribosomal RNA without an associated increase in its steady state level, the degradation and processing of the ribosomal gene transcript in the presence and absence of an ELF field were followed by pulse-chase 3H-uridine labeling. This revealed that ELF radiation accelerated both the processing and degradation of the ribosomal RNA transcript. During ELF exposure, the half-life of the 45S ribosomal RNA was decreased from 115 min. to 85 min. These results show that ELF can selectively affect RNA levels by modulating either the transcription rate and/or RNA post-transcriptional processing and turnover.

Biochemical characterization of a reverse transcriptase activity associated with retroviral-like particles isolated from human placental villous tissue.

Abstract: The presence of budding type-C retroviral-like particles in normal placental trophoblast, particularly at the basal surface of the placental syncytiotrophoblast, is well documented. Retroviral-like particles were isolated from human placental villous tissues using isopycnic sucrose gradient centrifugation. Reverse transcriptase activity (RTase) associated with isolated retroviral-like particles was characterised using a combination of synthetic template-primers. These studies showed that RTase activity was more specific with poly(rC).oligo(dG)12-18 than poly(dC).oligo(dG)12-18. Furthermore, activity was detected with poly(rCm).oligo(dG)12-18, a template-primer which has previously been shown to be specific for retroviral RTase. Maximum activity appeared at a sucrose density between 1.15-1.17 g/ml, characteristic of enveloped retroviral particles. Electron microscopy examination of the gradient purified particles revealed morphology and size similar to other retroviruses. Endogenous retroviral particles were isolated from 26 out of 32 (81%) first-trimester placental villous tissue extracts. These particles are likely to be product of endogenous proviral sequences present in the germline of humans. Although these studies showed presence of intact retroviral particles in placental tissues, it was not possible to propagate the isolated particles in vitro. All attempts to propagate placental retroviral particles by co-cultivation with human cells (U937 and JAr choriocarcinoma cells) and long term placental villous tissue explant cultures were unsuccessful. Subsequently, there was no evidence of retroviral-like particles or RTase activity in these cell cultures, including after stimulation with 5'-azacytidine or dexamethasone, chemical agents known to stimulate particle production in virus-infected lines.

Observation on incorporation of 3H-taurine in mouse skeletal muscle cells by light and electron microscopic radioautography.

Abstract: Taurine, one of the sulfur-containing amino acids, exist abundantly in the skeletal muscle tissues. The physiological function and ultrastructural cellular localization of taurine in the skeletal muscle cells are not clear. In this report, two experiments by radioautography were made to elucidate intracellular localization of this amino acid in the skeletal muscle cells. First, muscle tissue pieces obtained from normal and muscular dystrophy mice were cultured in a medium containing 3H-taurine and radioautographed. The number of silver grains appeared on muscle cells increased depending on the duration of the incubation time. Secondly, 3H-taurine was injected intraperitonealy in normal and muscular dystrophy mice. Then muscle specimens were fixed by two fixative procedures, one by a chemical fixation with glutaraldehyde and osmium tetroxide and another by cryo-fixation. Silver grains appeared over muscle cells prepared with both procedures. Silver grains were localized on myofilaments, sarcoplasmic membranes, sarcoplasmic reticulum, mitochondria, endothelial cells of blood capillaries and the cells of perineural sheath, but did not appear on Golgi apparatus, nuclei of muscle cells or adipose cells by any procedures. No difference in localization of silver grains was observed between normal and muscular dystrophy mice.

Glutathione reductase in human and murine lung tumors: high levels of mRNA and enzymatic activity.

Abstract: The enzyme glutathione reductase (GR) (GSSG+NADPH+H+-->2 GSH+NADP+) plays a key role in the cellular defense against oxidative stress. High levels of GR activity are often associated with tumor growth and/or resistance mechanisms against drug and radiation therapy. In order to investigate the molecular basis of elevated glutathione reductase activities we studied the enzyme at the DNA, mRNA and protein levels in murine experimental tumor cell lines and in human lung tumors. A modified ultracentrifugation procedure was developed which allowed the simultaneous isolation of DNA and total cellular RNA. Out of 11 human bronchial carcinomas obtained from patients without prior chemotherapy, five tumors showed a GR activity which was 2.4 to 3.8 times higher than in the respective control tissues. In each case the elevated enzyme activity was accompanied by an elevated GRmRNA levels. For none of the tumors, GR gene rearrangement or amplification was observed by Southern blot analyses. The mouse tumor cell lines ASB XIV, Lewis lung carcinoma and EAT cells, also showed high levels of GRmRNA whereas this mRNA was hardly detectable in normal mouse lung tissue.

Effect of denervation on lamellar cells of Meissner-like sensory corpuscles of the rat. An immunohistochemical study.

Abstract: The denervation-induced changes on S-100 protein, glial fibrillary acidic protein (GFAP) and vimentin immunoreactivity (IR) of the lamellar cells from cutaneous Meissner-like sensory nerve formations (SNF), or corpuscles, of the adult rat hind limb foot-pads were studied, using combined immunohistochemical and image analysis (optic microdensitometry) techniques. Animals were allowed to survive for 1, 3 and 7 days following sciatic and saphenous nerves transection. Lamellar cells of Meissner-like corpuscles displayed S-100 protein- and vimentin-IR, but not GFAP-IR. Denervation caused a marked time-dependent decrease of S-100 protein IR whereas vimentin-IR did not change or weakly increased. No positive GFAP-IR was observed in denervated SNF. These findings suggest that continuity of SNF with nerve fibers supplying them is necessary to maintain some of the immunohistochemical characteristics of the non-neuronal cells of SNF.

Lectin histochemistry of cell-surface glycoconjugates in the primary olfactory projections of the newt.

Abstract: The binding of 14 lectins were performed on paraffin-embedded sections of the olfactory bulb of Triturus to identify specific glycoconjugates on the cell surface of primary olfactory projections. The histochemical lectin staining patterns indicate that the membrane of olfactory neurons terminating in the main olfactory bulb contained prevalently oligosaccharides with alpha-acetyl-D-galactosamine as terminal residues. In the accessory olfactory bulb, instead, the primary olfactory projections possess a high density of alpha-D-galactose as sugar residues. The selective lectin binding on the surface of primary olfactory axons suggests that specific cell surface glycoproteins may have a role in the axonal growth due to the continual cycle of proliferation and death of olfactory receptors.

Study on the macromolecular synthesis in aging mouse seminiferous tubules by light and electron microscopic radioautography.

Abstract: Morphologic alternation and the changes of DNA, RNA and protein syntheses of the seminiferous tubules of aging mice were studied by light and electron microscopic radioautography. The morphology varied characteristically during the developmental periods. At embryonic and neonatal stages, the labelled myoid cells and Sertoli cells with 3H-thymidine were well observed, while at adult period, the labeling indices of both cells decreased to low levels. In contrast to this, the radioautograms showed that the gonocytes did not incorporate the labelled thymidine at embryonic day 19. As development proceeded to the 4th day after birth, the activity of DNA synthesis of gonocytes started. The labeling indices of spermatogonia reached the first peak at 3 weeks and were nearly constant thereafter. On the other hand, the activity of RNA synthesis of the cells in seminiferous tubules was not prosperous at embryonic day 19 and the neonatal stage. It was vigorous at adult and maintained a relatively high level at old period. The aging change of protein synthesis was detected in all kinds of the cells one hour after the 3H-leucine labeling. These results indicated that the old mice still kept adequate spermatogenesis. The Sertoli and myoid cells may play an important role in spermatogenesis in the whole life.

Radioautographic study on the intracellular localization of a hypolipidemic agent, bezafibrate, a peroxisome proliferator, in cultured rat hepatocytes.

Abstract: In order to demonstrate the intracellular localization of the peroxisome proliferator in the hepatocytes, we have examined the localization of silver grains due to 14C-labelled bezafibrate in cultured rat hepatocytes by means of light and electron microscopic radioautography. As it results from the difference between chemical fixation and freeze-substitution by light microscopic radioautography, more numerous silver grains, about twice, were observed in freeze-substitution specimens in comparison with chemical fixation. On light microscopic radioautograms of the epoxy resin sections during each experimental condition, about 90% of all the silver grains were localized over the cytoplasm. Then, statistical significance was evaluated on grain density in the cytoplasm. On electron microscopic radioautograms of whole mount cultured cells, silver grains were localized not on the peroxisome but on the cytoplasmic matrix specially over the endoplasmic reticulum. From these results, it is concluded that bezafibrate was localized over endoplasmic reticulum. This fact suggests that the receptor of the peroxisome proliferator should be associated with the endoplasmic reticulum or that the receptor exists on the endoplasmic reticulum. Thus, it is demonstrated that the peroxisome proliferator acts on the endoplasmic reticulum of hepatocytes to proliferate peroxisomes.

Radioautographic study on the aging change of 3H-glucosamine uptake in mouse ileum.

Abstract: The changes of the localization of incorporated glucosamine in mice ilea due to aging were studied by light microscopic radioautography in 10 groups of mice from fetus (19th day of embryo) to senescent (postnatal 2 years). After intraperitoneal injection of 6-3H-D-glucosamine the ilea of respective age groups were taken out and processed for light microscopic radioautography. The numbers of silver grains were counted at each part of ileal mucosal cells. The results show that the silver grains of columnar epithelial cells were localized mainly on brush border and Golgi region, and the grains of goblet cells were localized on Golgi region and mucous granules. Comparing the intestinal villi and crypts, the uptake of columnar epithelial cells and villi goblet cells was higher than these cells in crypt. The number of grains in respective age groups increased from perinatal to mature adults due to aging.