Histone neighbors in nuclei and extended chromatin

Histone neighbors in compact and extended chromatin have been investigated by cross-linking histones in nuclei and in nucleohistone extended with 6 M urea, using the bifunctional reversible reagent methyl-4-mercaptobutyrimidate (MMB). Similar histone dimers are found in both conformational states of chromatin. The dimers most frequently found are H2b-H2a, H2b-H3 and H3-H2a; dimers found less frequently are H3-H4, H3-H3 and H2b-H4. More H3-H3 is found in nuclei than in extended chromatin. H1 is found predominantly as poly-H1, although it can be cross-linked to H2b or H3. After reaction with MMB, native compact chromatin is no longer extendable in 6 M urea, which shows that the reagent is capable of linking together histones holding the chromatin in a compact conformation. Thus the histone propinquity in extended chromatin mimics and intimate histone associations in compact chromatin.     

Developmental arrest of germ cells in the pathogenesis of germ cell neoplasia

Clinical observations and epidemiological evidence suggest that important aetiopathological events that cause neoplastic transformation of the male germ cell may occur in fetal life or early infancy. The incidence of germ cell neoplasia is high in individuals with various disorders of gonadal development and sexual differentiation, such as gonadal dysgenesis or androgen insensitivity syndrome. Increased risk has also been noted in individuals with trisomy 21, idiopathic infertility and low birth weight. Infertility is sometimes associated with small aberrations of sex chromosomes (e.g. low frequency mosaicism XY/XO) which can also be found in patients with testicular cancer. The variety of conditions that predispose to testicular neoplasia and the rise in its incidence in many countries speaks for the influence of environmental factors which may affect genetically predisposed individuals. We hypothesise that if the development of the testis is disturbed or delayed, primordial germ cells or gonocytes undergo maturation delay or differentiation arrest which may render them susceptible to neoplastic transformation. Morphologically homogenous premalignant carcinoma in situ (CIS) cells have the potential to differentiate into a variety of histological forms of overt testicular tumours. Analysis of cell surface antigens expressed by CIS cells found in the vicinity of pure and mixed tumours demonstrates that CIS cells are phenotypically heterogeneous. Comparison of the phenotypes of CIS cells, primordial germ cells, human embryonal carcinoma cells and closely related primate embryonal stem cells reveals various similarities but also differences. We speculate that phenotypical heterogeneity of CIS cells may be associated with their potential to give rise to different tumour types, and may be related to the developmental stage of the early germ cell which has undergone malignant transformation.     

cDNA libraries from human tissues and cell lines

We report the construction of 34 cDNA libraries from human tissues and cell lines in lambda phage vectors (Charon BS, lambda gt11, lambda SHK, or lambda zapII). The cDNA was synthesized from poly A+ RNA, using oligo-dT as a primer, and size-selected before ligation to vector arms. High-complexity cDNA libraries from human tissues and cell lines should be a valuable resource for genome mapping studies and identification of disease genes.     

Medical and surgical management of pulmonary metastases from germ cell tumors

Campylobacter jejuni strains are able to produce at least two different cytotoxins called "cytolethal distending toxin" (CLDT) and "cytolethal rounding toxin" (CLRT). In this study, we investigated the corresponding changes in CHO-K1 cells using the cell counter and analyzer system CASY 1. Determination of the cell volume after toxin treatment of the cells is a useful criterion for differentiation between the cytotoxic activities produced by Campylobacter strains. Incubation of the cells with crude CLDT resulted in a decrease in the cell count combined with a dramatic increase of the mean cell volume in comparison to the control culture. A decrease in the cell count was also seen as a response to CLRT preparations, while this toxin had no effect on the mean cell volume determined. It was shown that only CLDT caused histone-associated DNA fragments in the cytoplasm of CHO-K1 cells indicating an apoptotic pathway of cell death. In addition, the polymerase chain reaction (PCR) was employed to screen Campylobacter strains for the presence of the cdtB gene sequence, which was detectable in all strains investigated.     

Induction of mosaic sex-linked recessive lethals in the different germ cell stages of Drosophila melanogaster by bleomycin

The dead-end filtration characteristics of the dimorphic yeast Kluyveromyces marxianus var. marxianus (formerly fragilis) NRRLy2415 were investigated for a range of mean cell morphologies, ranging from predominantly yeast-like to predominantly filamentous. Semiautomated image analysis was used to measure the mean cell specific surface area, Sv, and the mean ratio of cell length to equivalent cylindrical diameter, Ldm, in each broth. The method of Ju and Ho (Biotechnol. Bioeng. 1988, 32, 95-99) was used to show that for broths with Ldm values between 1.72 and 10.03, the voidage of cell pellets formed by centrifugation increased with increasing Ldm. In the pressure range 30-180 kPa, the specific filter cake resistance, alpha, was found to be related to pressure, DeltaP, through the equation alpha = alpha0(1 + kcDeltaP). The dependence of alpha0/Sv2 on Ldm was found to be qualitatively consistent with the pellet voidage data and the Carman-Kozeny equation. Considerably better agreement with the experimental data was obtained when the Kozeny constant, K, was treated as variable and related to Ldm through the equation K = 4.83 + 7.08 log10 Ldm. The cake compressibility constant, kc, was found to increase with increasing Ldm, a phenomenon consistent with the wide range of voidages that can be displayed by beds of long cylinders.     

Variations in the subunit content and catalytic activity of the cytochrome c oxidase complex from different tissues and different cardiac compartments

The objective of this study was to fully characterize normosmic perception of stimuli expected to cause widely varying degrees of olfactory and nasal trigeminal stimulation and to directly evaluate the possible role of olfactory nerve stimulation in nasal irritation sensitivity. During each of four identical test sessions, four anosmic and 31 normosmic participants were presented with a range of concentrations extending from peri-threshold for normosmics to supra-threshold for anosmics. For each session, odor (O) and nasal irritation (NI) sensitivities were summarized in terms of the concentrations required to produce four sensation levels ('iso-response' concentrations). Within-participant variation in these iso-response concentrations was < 10-fold for 95% of normosmics, for both O and NI. For O but not NI, these apparent fluctuations in sensitivity were largely accounted for by the uncertainty surrounding the iso-response concentrations calculated for each session. Anosmics exhibited minimal within- and between-participant variation in NI and required, for all but the highest perceptual level, a higher concentration than almost all normosmics. Between-participant variation, expressed in terms of 90% confidence interval widths, was approximately 0.5 log units for both O and NI for the highest perceptual level, but increased to approximately 0.8 and 1.8 log units, respectively, for the lowest (peri-threshold) level. Our findings suggest that: (i) most apparent variation over time in O sensitivity is actually a reflection of the uncertainty surrounding estimates of sensitivity obtained for each session; (ii) within- and between-participant variation in O sensitivity is far less than is commonly reported; and (iii) low to moderate levels of NI in normosmics are the result of relatively weak trigeminal stimulation combined with much greater olfactory activation.     

Spontaneous germ cell apoptosis in humans: evidence for ethnic differences in the susceptibility of germ cells to programmed cell death

Since the first clinical studies regarding sealing of arterial puncture sites with collagen with the use of the vascular hemostatic device (VHD) and the hemostatic puncture closing device (HPCD) in the early 1990s were performed, no analysis summarizing the published patients has been reported. Therefore we performed a Medline search of data as far back as 1990 and included abstracts presented at the major scientific meetings in the United States (American Heart Association, American College of Cardiology), Europe (European Society of Cardiology), and Germany (German Society of Cardiology). A total of 6007 patients were found to have been enrolled in studies with VHD (4448 patients) or with HPCD (1559 patients). Parameters analyzed in this review were hemostasis success rates and local complications. To assess the impact of the sealing devices on local complications, studies without control groups were excluded. The hemostasis success rates immediately after deployment seemed to be higher for HPCD, but at 2' to 5' after sheath removal, they were in the same range for VHD and HPCD. In controlled studies minor local complications occurred at a rate of 7.6% in the VHD group and in 6.7% of the HPCD group. Because the control group in the HPCD studies showed a considerably higher rate of minor complications than the VHD group (11.7% vs 5.7%), the reduction in minor complications was statistically significant for HPCD, whereas VHD did not reduce minor local complications. Major local complications were reported in 3.8% of the VHD group but in only 1.8% of the HPCD group. The increase of major local complications was statistically significant with VHD (control, 1.7%) but not with HPCD (control, 1.4%). Our analysis shows that some differences between collagen devices may exist, but neither device has been proven to reduce major local complications.     

Nitric oxide induces cell death in a catecholaminergic cell line derived from the central nervous system

The nitric oxide (NO) donors, sodium nitroprusside (SNP), 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium+ ++-1,2-diolate] (DETA NONOate), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) produce a dose-dependent increase in cell death in a catecholaminergic cell line (CATH.a) derived from the central nervous system. Cell death is associated with a decrease in mitochondrial membrane potential. Dopamine also induced cell death of CATH.a cells and this was potentiated by concentrations of SNP which alone did not produce cell death. Hemoglobin, a scavenger of NO radicals, blocked SNP- and SNAP-induced cell death. Catalase and superoxide dismutase, enzymes that metabolize H2O2 and superoxide, respectively, did not inhibit SNP- or SNAP-induced cell death. These data indicate that NO donors produce cell death in CATH.a cells through a mechanism related to the production of NO and the loss of the mitochondrial membrane potential but unrelated to the production of H2O2.     

Differential replication of ovine lentivirus in endothelial cells cultured from different tissues

Gastrin-like immunoreactive peptides were extracted from the gastric antrum of the American alligator (Alligator mississippiensis) and purified by fractionation using C18 Sep-Paks, Sephadex G-50, pH stable C8 reversed-phase HPLC, and C18 reversed-phase HPLC. Three major immunoreactive peaks were purified and found to correspond to 49, 45, and 34 residue peptides by microsequence analysis. The amino acid sequence of the largest peptide was DWLASLSQDQ KHLISKFLPH IYGELAN QEN YWQEDDALHD HDYPGWMDF-amide. The two smaller peptides corresponded to carboxyl-terminal 45 and 34 residue fragments of the 49 residue peptide. The putative proteolysis of the 49 residue peptide to the two shorter peptides occurs at cleavage sites that are unusual in biosynthetic processing. Mass spectral analysis confirmed the molecular weights that were predicted from the amino acid sequences, thus revealing the absence of any post-translational modifications, such as sulfation. Although the three alligator gastrins resemble mammalian cholecystokinin in having a tyrosine residue in the seventh position from the carboxyl terminus, this tyrosine is apparently nonsulfated as in turtle gastrin. When tested by radioreceptor assay, a synthetic replicate of alligator gastrin-49 exhibited a gastrin-like pattern of biological activity on mammalian CCK-A and CCK-B receptors. Comparison of the amino acid sequences of known peptides revealed that alligator gastrin is most similar to turtle gastrin (76% identical), followed by frog gastrin (51% identical), chicken gastrin (49% identical), and human gastrin (12% identical). These similarities closely reflect vertebrate phylogeny and support the hypothesis that functionally distinct gastrins evolved from CCK in early tetrapods. However, gastrin evolved via different mechanisms in the mammalian lineage (mechanism unknown) versus the amphibian and reptilian/avian lineages, in which two different single nucleotide base changes can account for the separate evolution of amphibian gastrin and reptilian/avian gastrin.     

Flow-microfluorometric analysis of nuclei isolated from various normal and malignant human epithelial tissues. A preliminary report

In order to obviate some of the technical problems associated with preparation of monocellular cell suspensions required for flow fluorometry, isolation of nuclei from several types of benign and malignant human tissues was undertaken. Satisfactory preparations of nuclei were obtained from epithelia of the uterine cervix and colon and from lung tissue using the citric acid method. The sucrose method was effective with colonic epithelium only. Distribution of deoxyribonucleic acid content in these nuclei was measured based on green fluorescence of acridine orange and red fluorescence of propidium iodide in a Bio-Physics Cytofluorograph. Essentially diploid patterns of deoxyribonucleic acid distribution were observed for all benign samples regardless of tissue origin whereas the malignant samples gave histograms suggestive of abnormal deoxyribonucleic acid distribution. Preliminary observations on distribution of single-stranded nucleic acids using acridine orange red fluorescence showed marked differences between populations of benign and malignant nuclei. Isolated nuclei appear to be suitable for flow-through microfluorometric analysis and offer some significant advantages over intact cells.     

The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis

Sertoli cells, the supportive cells in the seminiferous epithelium, orchestrate spermatogenesis by providing structural and nutritional support to germ cells. In the rat, physiological apoptosis occurs continuously to limit the size of the germ cell population to numbers that can be adequately supported. This form of germ cell death is exaggerated after testicular insults such as toxicant treatment, radiation, and heat exposure. The Fas system has been proposed as a key regulator of the activation of germ cell apoptosis. According to this model, Fas ligand (FasL) and Fas, expressed by Sertoli cells and germ cells, respectively, respond to environmental conditions and initiate germ cell death. To assess the role of the Fas system in various testicular injury models, a semiquantitative RT-PCR technique was used to evaluate the expression kinetics of both FasL and Fas after induction of massive germ cell death. Radiation exposure, which targets actively dividing germ cells, produced an up-regulation of Fas gene expression, but not FasL gene expression. However, administration of mono-(2-ethylhexyl)phthalate and 2,5-hexanedione, two widely studied Sertoli cell toxicants, resulted in up-regulated expression of both FasL and Fas. These data support the following hypotheses: 1) up-regulation of Fas is a common and critical step for initiating germ cell death in vivo; and 2) if Sertoli cells are injured, Sertoli cells up-regulate FasL to eliminate Fas-positive germ cells, which cannot be supported adequately.     

Flow cytometric and microscopic evaluation and effect on fertility of abnormal chromatin condensation in bovine sperm nuclei

The techniques of Feulgen staining, acridine orange staining, and a sperm chromatin structure assay using acridine orange and flow cytometry were compared for selective examination of bovine sperm nuclei. Twenty frozen semen samples were simultaneously analysed by all three methods. The prevalence of abnormally condensed DNA and its relationship to other semen traits were determined in ejaculates from 70 bulbs presented for routine examination for breeding soundness and in frozen semen from 348 bulls evaluated over five years. A breeding trial with 118 beef heifers using semen from six bulls with different degrees of nuclear abnormalities was performed to assess the importance of the defects with respect to fertility. The results indicate that few spermatozoa with abnormal DNA condensation are found in normal semen, but the incidence increases with disturbance of spermatogenesis. However, high numbers of abnormally condensed nuclei were found in the absence of an increase in other defects. This nuclear defect might be at least partially of epididymal origin; it can lower fertility and can be compensated for by increasing the numbers of normal spermatozoa in the insemination dose. The percentage of abnormally condensed sperm nuclei as detected by Feulgen staining was significantly correlated with that detected by microscopy after acridine orange staining and by the sperm chromatin structure assay. We therefore consider the Feulgen technique to be a valuable tool for assessing the nuclear integrity of bovine spermatozoa.     

Variations in mitochondrial size and ultrastructure during germ cell development

Size variations and ultrastructural changes in mitochondria of developing germ cells of the female hamster were analyzed. Mitochondria in oogonia of foetus and newborn were elongate with transverse cristae. During pre-dictyate meiotic prophase they became small, rounded, and electron-dense with pleomorphic cristae. These changes were largely reversed when dictyate was reached. Maximum mitochondrial size and complexity of cristae were reached just at the beginning of the phase of rapid oocyte growth, and thereafter declined. As mitochondrial size and number of cristae decreased in the rapidly enlarging oocyte, the ratio of length to width increased, as did electron density of the matrix, until the formation of an antrum within the follicle. After antrum formation, the mitochondria again became more rounded and cristae were seldom seen. An attempt is made to correlate changes of mitochondrial morphology with other events occurring during oogenesis.