The highly modified membrane of cornified cells in stratified squamous epithelia: A comparison of heterogenous deposits in keratinized and nonkeratinized epithelia

Summary The chemical nature of the thickened plasma membrane of cornified cells in stratified squamous epithelium was investigated in comparison with that in noncornified epithelium. Localizations of transglutaminase, molecular weight 92000 daltons, and detection of epidermal cysteine proteinase inhibitor were effected with a monoclonal antibody and a monospecific rabbit anti-inhibitor immunoglobulin, respectively, directed to the antigens. N-(7-dimethylamino-4-methylcoumarinyl) maleimide was used to demonstrate S-S cross-linking. In all keratinizing epithelia, the enzyme and inhibitor were deposited on membranes of granular cells. S-S bonds were formed in cornification with the appearance of electron-dense material by the inner leaflet. Both enzyme and inhibitors occurred on the corneal epithelium, but S-S linkage and the thickened plasma membrane did not form even at the last stage of maturation. On the other hand, the internal vaginal epithelium in the proestrous stage without keratinization contained the enzyme, but neither inhibitor nor S-S linkage. Both antigens and S-S bonds were detected when keratinization proceeded during estrus. The staining patterns in the epithelium near the vaginal introitus were identical to those in the skin. Cuboidal and simple epithelia exhibited none of those constituents. The findings indicated that heterogenous components contribute to modification of the plasma membrane of cornified cells, but S-S cross-linkages are associated exclusively with formation of the ultrastructurally unique membrane structure. In addition, findings suggested hormonal regulation in the chemical modification of the membrane in estrogen-sensitive internal vaginal epithelium.     

Measurement of in vivo HGPRT-deficient mutant cell frequency using a modified method for cloning human peripheral blood T-lymphocytes

Approximately 80% of human peripheral blood T-lymphocytes could be cloned in the presence of crude interleukin-2, phytohemagglutinin, and X-irradiated autologous lymphocytes and Raji B-cells. This modified cloning method was used to measure the in vivo frequency of HGPRT-deficient mutant T-lymphocytes. Repeated experiments using blood from the same individuals revealed that the frequency of mutant cells was almost constant for each individual even though the cloning efficiency of lymphocytes varied somewhat from experiment to experiment. Approximately 80% of both wild-type unselected and 6-thioguanine-resistant colonies had helper/inducer and about 20% had suppressor/cytotoxic T-lymphocyte markers. No difference was observed in the distribution of lymphocyte subsets in relation to colony type.     

Induction of endoreduplication in Chinese hamsters V79 cells by cytosine arabinoside

Endoreduplication (ER) could be induced very effectively in Chinese hamster V79 cells exposed to cytosine arabinoside (1-β-D-arabinofuranosylcytosine; Ara-C). Cells were cultured for 48 hours in Ara-C containing medium. ER frequency increases rapidly after Ara-C release. About 60% of metaphase cells were endoreduplicated at 8–10 hours after release from Ara-C (5 μg/ml). Induction of ER also depends on Ara-C concentrations.     

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Cellular localization of thiol-proteinase inhibitor in the epidermis of the newborn rat

Summary In cichlid, poecilid and centrarchid fishes luteinizing hormone releasing hormone (LHRH)-immunoreactive neurons are found in a cell group (nucleus olfactoretinalis) located at the transition between the ventral telencephalon and olfactory bulb. Processes of these neurons project to the contralateral retina, traveling along the border between the internal plexiform and internal nuclear layer, and probably terminating on amacrine or bipolar cells. Horseradish peroxidase (HRP) injected into the eye or optic nerve is transported retrogradely in the optic nerve to the contralateral nucleus olfactoretinalis where neuronal perikarya are labeled. Labeled processes leave this nucleus in a rostral direction and terminate in the olfactory bulb. The nucleus olfactoretinalis is present only in fishes, such as cichlids, poecilids and centrarchids, in which the olfactory bulbs border directly the telencephalic hemispheres. In cyprinid, silurid and notopterid fishes, in which the olfactory bulbs lie beneath the olfactory epithelium and are connected to the telencephalon via olfactory stalks, the nucleus olfactoretinalis or a comparable arrangement of LHRH-immunoreactive neurons is lacking. After retrograde transport of HRP in the optic nerve of these fishes no labeling of neurons in the telencephalon occurred. It is proposed that the nucleus olfactoretinalis anatomically and functionally interconnects and integrates parts of the olfactory and optic systems.     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

A new type of inversion of a human Y chromosome.

Using chromosome banding techniques, a phenotypically normal male was found to have an abnormal banding pattern of the Y chromosome. By the constitutive heterochromatin staining method, a darkly stained band was located on the short arm and the proximal region of the long arm. The quinacrine staining method also showed a similar abnormal banding pattern: a brightly fluorescing band was seen on the short arm and the proximal region of the long arm. By the conventional Giemsa staining method, however, no specific morphological abnormality was detected in the aberrant Y. On detailed karyotype analyses no recognizable abnormality of banding patterns of any other chromosome was found aside from the abnormal Y. The abnormality was determined to be a complex inversion of the Y chromosome, which is described as 46,X,inv(Y)(pter leads to p11::q11 leads to q12::cen::q12 leads to qter).     

Genetic Diversity and Population Structure Analyses of Wild Relatives and Cultivated Cowpea (Vigna unguiculata (L.) Walp.) from Senegal Using Simple Sequence Repeat Markers

Cowpea (Vigna unguiculata (L.)) is an important crop for food security in Senegal; therefore, understanding the genetic diversity of local germplasm is relevant for crop improvement and genetic maintenance in the era of climate change. For this purpose, 15 microsatellite markers were used to estimate the genetic diversity of Senegalese cowpea germplasm, including 671 accessions grown in eight regions and 66 wild relatives and intermediate forms (weedy). For the cultivated, the main expected heterozygosity (mHe) ranged between 0.317 (Fatick) and 0.439 (South). A narrow genetic variation between accessions from the different regions was observed with genetic similarity ranging from 0.861 to 0.965 and genetic differentiation indices (Fst) between 0.018 and 0.100. The accessions from southern Senegal (Kédougou, Sédhiou, and Kolda regions) are more diverse than the others. However, the accessions from the North (Saint-Louis) are genetically different from other regions. The diversity analysis in wild relatives from Senegal, which had never been performed before, revealed that the wild/weedy forms remain more diverse than the cultivated with genetic diversity values (He) of 0.389 and 0.480, respectively. STRUCTURE software divided the Senegalese germplasm into five subpopulations. Three of them (i, ii, and iii) included only cultivated accessions from several regions, one (v) mainly from Saint-Louis, and one (iv) the wild/weedy with some cultivated accessions. Our results support the hypothesis that Vigna unguiculata var. spontanea is the wild progenitor of cowpea. The accessions from the South, the northern recession accessions, and the wild/weedy could serve as sources of new genes for the genetic improvement of cowpea in Senegal.

     

α-Amylase expressed in human small intestinal epithelial cells is essential for cell proliferation and differentiation

α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other tissues, but it is unclear whether the α-amylase is endogenously expressed in each tissue or mixed exogenously with one expressed by the pancreas or salivary glands. Furthermore, the biological significance of these α-amylases detected in tissues other than the pancreas and salivary glands has not been elucidated. We discovered that human α-amylase is expressed in intestinal epithelial cells and analyzed the effects of suppressing α-amylase expression. α-Amylase was found to be expressed at the second-highest messenger RNA level in the duodenum in human normal tissues after the pancreas. α-Amylase was detected in the cell extract of Caco-2 intestinal epithelial cells but not secreted into the culture medium. The amount of α-amylase expressed increased depending on the length of the culture of Caco-2 cells, suggesting that α-amylase is expressed in small intestine epithelial cells rather than the colon because the cells differentiate spontaneously upon reaching confluence in culture to exhibit the characteristics of small intestinal epithelial cells rather than colon cells. The α-amylase expressed in Caco-2 cells had enzymatic activity and was identified as AMY2B, one of the two isoforms of pancreatic α-amylase. The suppression of α-amylase expression by small interfering RNA inhibited cell differentiation and proliferation. These results demonstrate for the first time that α-amylase is expressed in human intestinal epithelial cells and affects cell proliferation and differentiation. This α-amylase may induce the proliferation and differentiation of small intestine epithelial cells, supporting a rapid turnover of cells to maintain a healthy intestinal lumen.