Removal of swine zonae pellucidae with sequential exposures to pronase and acidified medium

Exposure to acidified PBS (pH 3) for 60 sec removed swine zonae pellucidae from 70.4% of 27 swine morulae, and 73.7% of these formed blastocysts in culture. Further investigations revealed that treating embryos with 0.5% pronase and acidified PBS (pH 3) for 30 sec each was more effective. Zonae were removed in 90.6% of 85 embryos (four-cell to morulae) treated. A total of 76.9% of 65 zona-free embryos and 81.6% of 38 untreated embryos formed blastocysts (P > 0.05). An additional 57 untreated and 49 zona-free embryos (morulae to blastocysts) were transferred to seven recipient sows. Four sows returned to estrus (18 to 27 days), but three others were pregnant when slaughtered at 38 to 42 days. One pregnant sow had received a combination of five zona-free and six untreated embryos and demonstrated the potential for further development of treated embryos in vivo.     

Gene mapping in marsupials and monotremes

Somatic cell genetic mapping of marsupial and monotreme species will greatly extend the power of comparative gene mapping to detect ancient mammalian gene arrangements. The use of eutherian-marsupial cell hybrids for such mapping is complicated by the frequent retention of deleted and rearranged marsupial chromosomes. We used staining techniques, involving the fluorochromes Hoechst 33258 and chromomycin A₃, to facilitate rapid and unequivocal identification of marsupial chromosomes and chromosome segments and to make chromosome assignment and regional localization of marsupial genes possible. Chromosome segregation in rodent-macropod hybrids was consistent with preferential loss of the marsupial complement. The extent of loss was very variable. Some hybrids retained 30% of the marsupial complement; some retained small centric fragments; and some, no cytologically identifiable marsupial material. We examined the chromosomes and gene products of a number of rodent-grey kangaroo Macropus giganteus hybrids, and have assigned the genes Pgk-A (phosphoglycerate kinase-A), Hpt (Hypoxanthine phosphoribosyl transferase), and Gpd (Glucose-6-phosphate dehydrogenase) to the long arm of the kangaroo X chromosome, and provisionally established the gene order Pgk-A -Hpt -Gpd.     

Target antigens in malaria transmission blocking immunity

Malaria transmission blocking immunity has been found to operate against two distinct phases of development of malaria parasites in the mosquito midgut: (i) against the extracellular gametes and newly fertilized zygotes shortly after ingestion by a mosquito of parasitized blood and (ii) against the zygotes during their subsequent development into ookinetes. Immunity is antibody-mediated and stage-specific. A set of three proteins, synthesized in the gametocytes, expressed on the surface of the gametes and newly fertilized zygotes and subsequently shed during later transformation of the zygotes, has been identified as the target antigens of anti-gamete fertilization blocking antibodies. A single protein, synthesized and expressed on the zygote surface during its development to ookinetes, has been identified as the target of antibodies which block the development of the fertilized parasites in the mosquito. Immunization of human populations against gamete or zygote antigens, while not directly protecting an immunized individual from inflection, would reduce the transfer of malaria within the population. Such immunity, in addition to reducing the overall rate of malaria transmission, would, if combined with a vaccine against the asexual (disease-causing) stages, reduce the chance of selection of parasites that are resistant to the asexual vaccine by preventing their entry into the mosquito population.     

In vitro stimulation of alkaline phosphatase activity in immature embryonic chick pelvic cartilage by adenosine

Cyclic AMP content in embryonic chick pelvic cartilage increases significantly as the embryo ages from 8 to 10 d. This in ovo elevation in cyclic AMP content precedes maximal cartilage alkaline phosphatase activity by some 24 h. We studied whether this temporal relationship may be causally related, using an in vitro organ culture. Incubation of pelvic cartilage from 9- and 10-d embryos in medium containing monobutyryl cyclic AMP (BtcAMP) resulted in significant increases in alkaline phosphatase activity (220 and 66 percent, respectively) as compared to that of cartilages incubated in medium alone. This stimulation was both concentration- and time-dependent with maximal response at 0.5 mM BtcAMP and 4-h incubation, respectively. Similar incubations of cartilage in medium containing 1-methyl-3-isobutyl xanthine (MIX), 0.25 mM, also resulted in increased alkaline phosphatase activity (114 percent). However, pelvic cartilage from 11-d embryos incubated in medium containing BtcAMP or MIX showed no increase in alkaline phosphatase activity. We postulated that developmental age was the factor responsible for this difference in response and that immature cartilage (that with little or no alkaline phosphatase activity) would respond to BtcAMP whereas mature cartilage (that with significant alkaline phosphatase activity) would not. This was tested by incubating end sections of 11-d cartilage, which have little alkaline phosphatase activity, and center sections, which have significantly alkaline phosphatase activity, with both BtcAMP and MIX. Alkaline phosphatase activity in end sections (immature cartilage) was stimulated by BtcAMP and MIX, whereas it was not stimulated in the center sections. Actinomycin D and cycloheximide inhibited BtcAMP and MIX stimulation of alkaline phosphatase activity. Thus, the in vitro data suggest that cyclic AMP is a mediator for the stimulation of alkaline phosphatase activity in embryonic cartilage.     

Rabbit-liver phosphorylase: improvement of the purification procedure and assay of the inactive b form

Continuous electrophoresis buffers are described for polyacrylamide gels at pH values ranging from 3.8 to 10.2. The buffers consist of an acidic and a basic component with pK values near the pH of the buffer. The pH is maintained to within 0.5 pH unit in the electrode compartments during prolonged electrophoresis. Some proteins produce clear bands on gels with each of the 10 buffers. The buffers provide an expansion of the pH range of gel electrophoresis and are likely to be useful in the detection of genetic variation in proteins and in other applications.     

CD2 antigen mediated activation of the guanine nucleotide binding proteins p21ras in human T lymphocytes.

T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism.     

Calcium and phosphate effects on growth and alkaloid production in Coffea arabica: experimental results and mathematical model

Plant, mammalian, and microbial cells are commonly immobilized in calcium alginate gels for the production of valuable secondary metabolites. However, calcium ions are known to inhibit growth in various types of cells, and calcium is an integral part of such gels. Therefore, an investigation was conducted to evaluate the effect of calcium on the growth and alkaloid production of a model cell-line, Coffea arabica, in suspension culture before, attempting to immobilize such cells in alginate. A kinetic model was then developed from the results to describe cell growth and alkaloid production and the mechanism by which calcium influences these variables. In addition, it was observed that there was a characteristic relationship between the concentration of calcium in the external medium and the concentration of extra cellular and intracellular phosphate. The intracellular phosphate level was, in turn, related to the production of alkaloids. Using these results, a dynamic mathematical model of cell growth and alkaloid production was developed based on the proposed roles of calcium and phosphate. The model showed satisfactory agreement with three sets of experiments at different calcium concentrations. A possible linkage between the calcium and phosphate results is postulated based on the limited solubility of calcium phosphate.