Quantitative aspects of the feeder cell phenomenon: mechanistic implications

It has been proposed that feeder cells function by supplying lymphocytes with the amino acid cysteine (a thiol compound). The results presented here indicate that thiols are the critical element of the feeder cell phenomenon. Specifically, we noted that the rank of thiol production by four different feeder cell lines corresponds to their relative abilities to support a lymphocyte cell line, CTLL-2. In addition, increasing thiol production by the feeder cells with lipopolysaccharide increased their support of CTLL-2 cells and decreasing it with homocysteate decreased support of CTLL-2 cells. However, it was also noted that substantial (up to 79% maximal) support of CTLL-2 growth was provided by feeder cell concentrations which could not produce detectable levels of free thiols. This prompted us to propose an alternative mechanism for the feeder effect which would explain these apparently paradoxical findings.     

The physiology of sperm recovered from the human cervix: acrosomal status and response to inducers of the acrosome reaction

Cervical mucus was collected from 35 women after artificial insemination. Mucus collections were performed at 1 h, 1 day, 2 days, or 3 days following insemination. Sperm viability was greater than 80% at all recovery times as assessed by exclusion of the supravital dye Hoechst 33258. Virtually 100% of the viable sperm were acrosome-intact at all times as assessed with a fluorescein isothiocyanate-conjugated pea lectin. Sperm were recovered from the mucus after migration into the Biggers, Whittin, and Whittingham medium in vitro. Sperm did not undergo the acrosome reaction in response to human follicular fluid immediately after migration from the mucus but did respond to this agonist after 6 h of incubation in vitro. Sperm recovered at all times after insemination had the same pattern of response to follicular fluid. Sperm that penetrated a column of cervical mucus in vitro also responded to follicular fluid with an increase in acrosome reactions after migration from the mucus and incubation for 6 h in vitro. Unlike the sperm that migrated from cervical mucus, sperm that were separated from semen by Percoll density centrifugation did not undergo the acrosome reaction when challenged with follicular fluid after 6 h but did respond after 24 h incubation. Sperm that migrated from cervical mucus had a similar increase in acrosome reactions after 6 h incubation, regardless of whether the acrosome reaction agonist was follicular fluid or disaggregated human zona pellucida.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermochemiluminescent assay of porcine, rat, and human erythrocytes for antioxidative deficiencies

The thermal induction of chemiluminescence of luminol-horseradish peroxidase-labeled erythrocytes from pigs, rats, and man was studied. The luminescent responses of rat, porcine, and human erythrocytes to heating were linear in respect to logs of counts per minute versus temperature. Landrace-Duroc crossbred pigs with a history of malignant hyperthermia (porcine stress syndrome) and Poland-China-miniature pigs inbred for malignant hyperthermia (MH) yielded erythrocytes with high-level thermochemiluminescence (TCL). Sprague-Dawley rat erythrocytes were intermediate in their production of TCL. Normal human and MH-resistant miniature swine erythrocytes produced low-level TCL. However, pretreatment of human erythrocytes with 1-chloro-2,4-dinitrobenzene (CDNB) resulted in high-level TCL. Furthermore, halothane enhanced the TCL of CDNB-treated human erythrocytes and Landrace-Duroc porcine erythrocytes that were not treated with CDNB. Red blood cells from pigs susceptible to the porcine stress syndrome demonstrated a TCL response very similar to CDNB-treated erythrocytes.     

Neonatal rat hepatocytes in primary tissue culture free from density-dependent regulation of growth

Summary Studies employing [³H]thymidine and radioautography as well as colchicine and Feulgen staining of DNA showed that up to 19-fold increases in the degree of cell crowding in vitro, i.e. from 1.45 to 27.55×10⁴ cells per specimen, did not change the rates of entry into DNA synthesis and mitosis of cultivated primary neonatal rat hepatocytes.     

Citric Acid Fermentation by Aspergillus niger on Low Sugar Concentrations and Cotton Waste

The possible use of cotton waste as a carbohydrate source of citric acid production by Aspergillus niger was examined. No citric acid was produced when A. niger was grown on cotton waste as a sole carbon source. In two-stage fermentations, however, mycelium obtained from surface cultures in cotton waste medium yielded more citric acid when transferred to sucrose-containing media than when directly inoculated to sucrose-containing media. It is concluded that cotton waste can be used for saving sucrose and for increasing yields of citric acid fermentation by A. niger.     

T cell responses to cleaved rabies virus glycoprotein and to synthetic peptides

The antigenic structure of the rabies virus glycoprotein has been studied. A limited number of fragments were obtained by cyanogen bromide (CNBr) cleavage of viral glycoprotein, and eight large peptides were isolated by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These were tested for their capacity to stimulate the proliferation of nylon wool-purified T cells obtained from spleens of rabies-immune A/J mice. Three peptides (Cr1, Cr2 plus Cr2A, and Cr3) stimulated antigen-specific proliferation, indicating that at least three T cell determinants of the native molecule are sequential or continuous in nature. Stimulation was also obtained with 27-residue and 13-residue synthetic peptides (designated R21 and R20, respectively) that included sequences towards the carboxy terminal end of Cr1, but not with synthetic peptides that included sequences of Cr2 and Cr3 (which are both glycosylated in virus-derived material). The intact viral glycoprotein and synthetic peptide R21 stimulated T lymphocytes with surface characteristics of helper cells, and induced the production of interleukin 2 by these lymphocytes. Synthetic peptides R20 and R21 also stimulated a minor population of Lyt-2-positive cells, which were not yet identified as either suppressor or cytotoxic T lymphocytes.     

Release of ribosome-bound ribosome recycling factor by elongation factor G

Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.     

Phylogeography of the Petunia integrifolia complex in southern Brazil

In this study, we evaluated the genetic diversity of the Petunia integrifolia species group using a phylogeographical approach, and attempted to understand better its diversification and taxonomy. Plants from five morphological groups were collected, covering a large part of the geographical distribution of most of the species. Two major clades were found in the phylogenetic tree, and an additional lineage, corresponding to P. inflata, was found in the haplotype network obtained for plastid markers. All three lineages are clearly delimited geographically, but, with the exception of P. inflata, the morphological groups were not genetically distinct. Our results suggest that a population expansion after a size reduction resulted in the establishment of two distinct and allopatric groups c. 0.5 Mya, one group occurring in a geologically ancient area, and the other occurring in areas that were under the influence of a series of marine transgressions/regressions at the end of the Pleistocene. These two clades are evolutionarily significant units with significantly different allele frequencies in their nuclear genome and reciprocal monophyly in maternal, uniparentally inherited markers. All our results suggest that the morphology‐based taxonomy in this group does not reflect its evolutionary history, and revision of its species limits should incorporate the distribution of the genetic diversity. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 199–213.