Maternal or fetal origin of rhesus monkey (Macaca mulatta) amniotic fluid leukocytes can be identified by polymerase chain reaction using the zinc finger Y gene.

Leukocytes can be found in substantial numbers within the intrauterine tissues and amniotic fluid of women, and play a central role in the pathophysiology of infection-related preterm labor by their production of proinflammatory mediators. It remains unclear whether these leukocytes represent a fetal immune response, a maternal response, or a combination of the two. The objective of this study was to develop a test in the rhesus monkey (Macaca mulatta) suitable for determining the percentage of male fetal cells present in a population of leukocytes recovered from blood or amniotic fluid. We found inadequate specificity for rhesus monkey cells using commercial human Y-chromosome paint kits (fluorescence in situ hybridization (FISH)). Human-specific primers for the repetitive Y chromosome DYZ-1 locus employed in the polymerase chain reaction (PCR) produced an unacceptable percentage of false positives. However, we successfully developed a PCR-based test using rhesus-specific primers for the zinc finger Y (ZFY) locus. Densitometry of PCR products from known ratios of male and female adult peripheral leukocytes generated a linear standard curve which provided quantitative results and required only 400 cells per sample. The rhesus beta globin (RBG) gene served as an internal control. The PCR test correctly discriminated the sex of peripheral leukocytes in 20 adult males, 20 adult females, two male fetuses, and one female fetus. Serial samples of amniotic fluid from four chronically catheterized rhesus monkeys bearing male fetuses were used to confirm the utility of this assay for quantifying fetal cells in amniotic fluid. In conclusion, we have developed a PCR test which is suitable for distinguishing male from female cells in adult and fetal blood and in amniotic fluid, which lends itself to a variety of diagnostic and biologic applications in the rhesus monkey and potentially in other nonhuman primates.     

Temperature sensitive mutant of Herpes simplex virus type 1. II. Neurovirulence and latency]

The course of acute infection of mice with ts mutant or the native strain DNA and the antigens of HSV in brain nerve cells were determined. Virus DNA was detected in brains of all mice in both animal groups while the virus antigens--only in cells of mice infected with the native strain. It can be suggested, therefore, that the ability of ts mutant to replicate in central nervous system of the infected mice is lacking or much lower. The detection of virus nucleic acid 3-5 months after virus infection might indicate a possibility of establishing latent infection. However, ts mutant showed a significantly lower possibility of latency induction, as compared with highly virulent strains. It was found that the mutant ability to induce latent infection was markedly increased when mice were treated with both ts mutant and Depo-Medrol as immunosuppressive agent. This finding shows both a possibility of increase of frequency of latent infections in the state of immunosuppression, and of activation of the latent infection (recurrence of acute form of infection).     

Variation in Quantitative Requirements for Na for Transport of Metabolizable Compounds by the Marine Bacteria Alteromonas haloplanktis 214 and Vibrio fischeri

The rates of uptake by Alteromonas haloplanktis of 19 metabolizable compounds and by V. fischeri of 16 of 17 metabolizable compounds were negligible in the absence of added alkali-metal cations but rapid in the presence of Na. Only d-glucose uptake by V. fischeri occurred at a reasonable rate in the absence of alkali-metal cations, although the rate was further increased by added Na, K, or Li. Quantitative requirements for Na for the uptake of 11 metabolites by A. haloplanktis and of 6 metabolites by V. fischeri and the characteristics of the Na response at constant osmotic pressure varied with each metabolite and were different from the Na effects on the energy sources used. Li stimulated transport of some metabolites in the presence of suboptimal Na concentrations and for a few replaced Na for transport but functioned less effectively. K had a small capacity to stimulate lysine transport. The rate of transport of most of the compounds increased to a maximum at 50 to 300 mM Na, depending on the metabolite, and then decreased as the Na concentration was further increased. For a few metabolites, the rate of transport continued to increase in a biphasic manner as the Na concentration was increased to 500 mM. Concentrations of choline chloride equimolar to inhibitory concentrations of NaCl were either not inhibitory or appreciably less inhibitory than those of NaCl. All metabolites examined accumulated inside the cells against a gradient of unchanged metabolite in the presence of Na, even though some were very rapidly metabolized. The transport of l-alanine, succinate, and d-galactose into A. haloplanktis and of l-alanine and succinate into V. fischeri was inhibited essentially completely by the uncoupler 3,5,3',4'-tetrachlorosalicylanilide. Glucose uptake by V. fischeri was inhibited partially by 3,5,3',4'-tetrachlorosalicylanilide and also by arsenate and iodoacetate.     

Gabaculine Inhibition of Chlorophyll Biosynthesis and Nodulation in Phaseolus lunatus L

Gabaculine (3-amino-2,3-dihydrobenzoic acid) was an inhibitor of in vivo chlorophyll biosynthesis in lima bean (Phaseolus lunatus L. cv Henderson). When applied to roots of 9-day-old plants, 10 micromolar gabaculine was sufficient to terminate biosynthesis of new chlorophyll. The trifoliolate leaves which emerged after gabaculine treatment were yellow. Gabaculine-treated plants had slightly lower dry weights; yet, overall plant size showed very little change. Chlorophyll fluorescence induction kinetics and CO₂ exchange measurements were used to monitor both immediate and long-term effects of gabaculine on photosynthesis. A lowered rate of the decline from the maximum level of fluorescence was observed after 10 hours for nitrate-supplemented plants, and all treated plants showed a slightly increased level of original fluorescence after 6 days. No change was observed in the rate of photosynthesis by unifoliolate leaves. The trifoliolate leaves, though not able to photosynthesize, were able to continue respiration. This suggested that heme biosynthesis for mitochondrial cytochromes was not abolished. In untreated lima bean, root nodules were induced by Rhizobium sp. 127E15. Following gabaculine treatment, root nodules formed, but were largely ineffective in nitrogen fixation. Nodule dry weight, nitrogen fixation activity, and leghemoglobin content were decreased by gabaculine.     

The relative biological effectiveness of 670 MeV/A neon as a function of depth in water for a tissue model

Linear energy transfer (LET infinity) spectra of identified charge fragments and primaries, produced by nuclear interactions of 670 MeV/A neon in water, were measured along the unmodulated Bragg curve of the neon beam. The relative biological effectiveness (RBE) values for spermatogonial cell killing, as reported on the basis of weight loss assay of mouse testes irradiated with beams of approximately constant single LET infinity, were summed over the particle LET infinity spectra to obtain an effective RBE for each charged-particle species, as a function of water absorber thickness. The resultant values of effective RBE were combined to obtain an effective RBE for the mixed radiation field. The RBE calculated in this way was compared with experimental RBEs obtained for spermatogonial cell killing in the mixed radiation field produced by neon ions traversing a thick water absorber. Discrepancies of 10-40% were observed between the calculated RBE and the RBE measured in the mixed radiation field. Part of this discrepancy can be attributed to undetected low-Z fragments, whose contribution is not included in the calculation, leading to an overestimated value for the calculated RBE. On the other hand, calculated values 10% greater than the measured RBE are explained as track structure effects due to the higher radial ionization density near neon tracks relative to the ionization density near the silicon tracks used to fit the RBE vs LET infinity data.     

The primary structure of the beta-subunit of the cell surface adhesion glycoproteins LFA-1, CR3 and p150,95 and its relationship to the fibronectin receptor.

The lymphocyte-function-associated antigen-1 (LFA-1), the complement receptor type 3 (CR3) and the antigen p150,95 are cell-surface glycoproteins. They are heterodimeric complexes, each containing a unique alpha-subunit noncovalently associated with a common beta-subunit. We have purified the beta-subunit from human spleen and obtained limited peptide sequences. What appears to be the complete primary structure for the fully processed beta-subunit was obtained by cDNA sequencing of clones from a phorbol ester (PMA) stimulated U937 cDNA library. There are five possible glycosylation sites and a transmembrane segment. The sequence contains a high level of cysteine (7.6%), with 24 of the 57 cysteine residues being found in three repeating units each with eight residues. The entire primary structure has 47% identity to a subunit of a fibronectin binding protein from chicken fibroblasts. It seems that LFA-1, CR3 and p150,95 antigens may belong to an extended family of cell surface molecules including the fibronectin binding protein.     

Adenosine and oxytocin reverse antagonism of cyclic AMP elevating agents to insulin activation of adipocyte pyruvate dehydrogenase

ACTH, isoprenaline, forskolin, and dibutyryl cyclic AMP prevented insulin from stimulating adipocyte pyruvate dehydrogenase in the presence of adenosine deaminase. Antagonism was reversed by N6-phenylisopropyladenosine as well as oxytocin. The stimulatory effects of insulin, adenosine and oxytocin on adipocyte pyruvate dehydrogenase appear to be through (a) mechanism(s) which is (are) similar or related.