Permeable collagen-glycosaminoglycan cross-linked copolymers for the study of biological responses of coculturede Sertoli and spermatogenic cells

A novel collagen-glycosaminoglycan (C-GAG) substrate was developed to overcome the optical opacity of a HATF nitrocellulose substrate and to provide a more physiological permeable substrate for cocultured Sertoli and spermatogenic cells. Cocultures were prepared on optically transparent C-GAG discs attached to a polyester mesh to facilitate handling. Sertoli cells displayed a cuboidal-to-columnar shape; a large number of spermatogonia and primary spermatocytes connected by intercellular bridges were associated with basolateral and apical surfaces of Sertoli cells up to 12 days after plating. Rat Sertoli-spermatogenic cell cocultures have been used for testing the effect of toxicants on rat spermatogenesis in vitro. In our initial studies, we tested the effects of the toxicant gossypol on spermatogenic cells cocultured with Sertoli cells on nonpermeable (plastic) and permeable substrates (HATF nitrocellulose) under both standard culture conditions and during perifusion after achieving a continuous electrical-resistant cell monolayer. A selective mitochondrial structural damage was observed in spermatogenic cells (spermatogonia and spermatocytes) but not in the coexisting Sertoli cells. This damage was time- (15–60 min) and dose-dependent (0.1–10µM) and developed more rapidly under perifusion conditions. Similar mitochondrial damage was reported in the intact animal but required higher concentrations (mg) and longer administration time (months) for detection. Studies are in progress to evaluate the effect of additional toxic chemical agents on functional properties of Sertoli and spermatogenic cells in cocultures prepared on various classes of C-GAG substrates.Abbreviations C-GAG, collagen type I-glycosaminoglycans - C-C6S, collagen type I-chondroitin-6-sulfate - C-H, collagen type I-heparin     

High intensity and blue light regulated expression of chimeric chalcone synthase genes in transgenic Arabidopsis thaliana plants

Summary To establish a genetic system for dissection of light-mediated signal transduction in plants, we analyzed the light wavelengths and promoter sequences responsible for the light-induced expression of the Arabidopsis thaliana chalcone synthase (CHS) promoter fused to the -glucuronidase (GUS) marker gene. Transgenic A. thaliana lines carrying 1975, 523, 186, and 17 by of the CHS promoter fused to the GUS gene were generated, and the expression of these chimeric genes was monitored in response to high intensity light in mature plants and to different wavelengths of light in seedlings. Fusion constructs containing 1975 and 523 by of CHS promoter sequence behaved identically to the endogenous CHS gene under all conditions. Expression of these constructs was induced specifically in response to high intensity white light and blue light. The response to blue light was seen in the presence of the P_fr form of phytochrome. Fusion constructs containing 186 by of promoter sequence showed reduced basal levels of expression and only weak stimulation by blue light but were induced significantly by high intensity white light. These analyses showed that the expression of the A. thaliana CHS gene is responsive to a specific blue light receptor and that sequences between — 523 and — 186 by are required for optimal basal and blue light-induced expression of this gene. The experiments lay the foundation for a simple genetic screen for light response mutants.     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

Phytoliths as indicators of prehistoric maize (Zea mays subsp.mays,Poaceae) cultivation

Maize (Zea mays L. subsp.mays) has been identified in archaeological contexts by a high proportion of large cross-shaped phytoliths. Given the numerous races of maize, this study was undertaken to determine if differences below the species level could be noted. It was also designed to see if phytoliths differed in various plant parts at various stages of growth. Several races were grown under experimental conditions. No significant differences were found. Furthermore, few phytoliths alleged to be diagnostic of maize were discovered. Systemic studies of maize and analyses of prehistoric cultivation by means of phytoliths seem not to be as promising as some researchers have argued.     

Phenotypic analysis of thymocytes that express homing receptors for peripheral lymph nodes

Thymocytes that express high levels of homing receptors for peripheral lymph nodes can be detected with the monoclonal antibody MEL-14. We have shown that in adult mice these rare MEL-14hi thymocytes a) are cortical in location and typically constitute 1 to 3% of the total thymocyte population, b) may be a major source of thymus emigrants, and c) contain a high frequency of precursors of alloreactive cytotoxic T lymphocytes. In this study we have analyzed the phenotype of the MEL-14hi thymocyte subset. Most normal adult MEL-14hi thymocytes are midsize and express the mature phenotype typical of thymus emigrants, medullary thymocytes, and peripheral T cells: they are predominantly PNAlo, H-2K+, Thy-1+, Ly-1hi, and either Lyt-2-/L3T4+ or Lyt-2+/L3T4-. These findings argue strongly for the presence of rare MEL-14hi immunocompetent cortical thymocytes that, aside from their homing receptor expression, are phenotypically indistinguishable from medullary thymocytes. However, a minority (20 to 30%) of MEL-14hi thymocytes are large and phenotypically nonmature: they express intermediate to high levels of PNA binding sites, and are H-2K- to H-2Klo, Thy-1hi, Ly-1+, and either Lyt-2+/L3T4+ or Lyt-2-/L3T4-. Through a technique that selectively labels outer cortical cells, phenotypically nonmature MEL-14hi thymocytes have been shown to be concentrated in the subcapsular blast region of the outer cortex. Although we have no direct evidence of a precursor-product relationship, we consider it likely that the phenotypically nonmature outer cortical MEL-14hi lymphoblasts give rise to phenotypically mature MEL-14hi cells located deeper in the cortex. These results are consistent with our previous proposal that MEL-14hi thymocytes are a major source of thymus emigrants, and indicate that expression of high levels of MEL-14-defined homing receptors may be closely linked to the intrathymic selection process.     

Molecular cloning of virulence genes from Erwinia stewartii.

A library of Erwinia stewartii DNA was constructed in cosmid pVK100 and used to complement spontaneous and Mu pf7701-induced (designated by the prefix MU) avirulent mutants. Plasmid pES4507 restored water-soaking ability and extracellular polysaccharide (EPS) synthesis to mutants MU14110 and MU2B70 (group I); pES1044 restored water-soaking ability to MU43, MU51, MU136, MU141, and RDF6011 (group II); and pES2144 complemented four spontaneous EPS- mutants (group III). Hybridization of labeled plasmid DNA to Southern blots of genomic DNA from the mutants revealed that a Mu pf7701 insertion was associated with the respective cloned region in all mutants except MU2B70 and MU223. In these strains, the plasmid may be suppressing the avirulent phenotype rather than complementing the mutation.     

Purification and characterization of endo-xylanases from Aspergillus niger. II. An enzyme of pl 4.5

A homogeneous endo-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) was obtained from a crude Aspergillus niger pentosanase by chromatography with Ultrogel AcA 54, SP-Sephadex C-25 at pH 4.5, DEAE-Sephadex A-25 at pH 5.4, Sephadex G-50, and SP-Sephadex C-25 with a gradient from pH 2.8 to pH 4.6. It was much more active on soluble than on insoluble xylan, yielding large amounts of unreacted xylan and a mixture of oligosaccharides with chain lengths from two to six. No xylose or L-arabinose was produced. There was high activity on a xylopentaose through xylononaose mixture, but not on xylobiose, xylotriose, or xylotetraose. The enzyme had slight activity on untreated cellulose, carboxymethylcellulose, and pectin. Molecular weight was ca. 1.4 x 10(4), with an isoelectric point of 4.5 and an amino acid profile high in acidic but low in sulfur-containing residues. In a 25-min assay at pH 4.7, this endo-xylanase was most active at 45 degrees C, with an activation energy from 5 to 35 degrees C of 33.3 kJ/mol. The optimum pH for activity was 4.9. Decay in buffer was first order, with an activation energy at pH 4.7 from 48 to 53 degrees C of 460 kJ/mol. Optimum pH for stability was about 5.6, where the half-life at 48 degrees C in buffer was ca. 40 h.     

Plasma membrane transglutaminase and cornified envelope competence in cultured human keratinocytes

When confluent cultures of the transformed human keratinocyte line SV-K14 are shifted to serum-free medium the cells achieve, within 4 days, the ability to synthesize a cornified envelope after challenge with the Ca2+ ionophore A23187. During these 4 days the enzyme transglutaminase (EC 2.3.2.13), which catalyses the cross-linking of different envelope precursor proteins, is partially transferred from the cytosolic pool into the plasma membrane. The association of the enzyme with the plasma membrane proves to be an essential step in the envelope formation since a direct correlation between plasma membrane-bound transglutaminase and envelope competence is observed. Retinoids block the insertion of the enzyme and therefore prevent envelope formation.     

Interaction of human spermatozoa with the human zona pellucida and zona-free hamster oocyte following capacitation by exposure to human cervical mucus

The functional capacity for sperm interaction with the human zona pellucida and zona-free hamster oocyte was tested after human spermatozoa were capacitated by passage through a column of human cervical mucus. The results were compared with those obtained when spermatozoa from an aliquot of the same semen sample were capacitated by the standard laboratory methods involving sequential washing by dilution and centrifugation of the semen. Washed-capacitated sperm suspensions were more successful than mucus-capacitated sperm in attaching to the zona-free hamster oocyte and in fusing with its oolemma. However, the ability of mucus-capacitated sperm to penetrate the human zona pellucida was equal to washed capacitated sperm. These experiments demonstrate an approach that may be useful in comparative studies of human sperm capacitation in vivo and in vitro.