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)     

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.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

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.     

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.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.