Pinocytosis in the rat visceral yolk sac effects of temperature, metabolic inhibitors and some other modifiers

Low temperature, 2,4-dinitrophenol and moniodoacetate could each completely abolish the pinocytic uptake of ¹²⁵I-labelled polyvinylpyrrolidone, ¹²⁵I-labelled bovine serum albumin or colloidal ¹⁹⁸Au by 17.5-day rat visceral yolk sac cultured in vitro. Cytochalasin B and colchicine caused a partial and dose-dependent inhibition. It is concluded that the mechanism of pinocytic uptake of these substrates is not micropinocytosis as conventionally defined. Removal of extracellular calcium or the oresence of theophylline inhibited liquid-phase pinocytosis by the rat yolk sac, whereas addition of ouabain caused a biphasic response: a slight stimulation of pinosome formation at a low concentration, and an inhibitory effect at a higher concentration.     

Spore coat protein and enterotoxin synthesis in Clostridium perfringens.

Polyacrylamide gel profiles of Clostridium perfringens spore coat protein revealed four and occasionally five components. Pulse-chase experiments indicated that synthesis of coat protein polypeptide and enterotoxin was an early sporulation event. However, maximum synthesis occurred coincident with the onset of heat resistance.     

Analysis of the major histocompatibility complex in Syrian hamsters. III. Cellular and humoral immunity to alloantigens.

Among the genetic loci incorporated into the major histocompatibility complex in every species studied to date have been prominent genes encoding for strong histocompatibility determinants that elicit detectable alloantibody responses and which are the chief antigenic targets of cell-mediated cytotoxicity reactions. The K and D regions of the H-2 complex in the mouse and the A, B, and C regions of the HLA complex in man are representative examples. Syrian hamsters, as described in this report, do not make alloantibodies to antigens of this type and only very poorly do they carry out in vitro cell-mediated cytotoxicity to target cells putatively bearing these antigens. Since hamsters are quite capable of discriminating analogous antigenic differences in xenogeneic species, and xenogeneic sources cannot distinguish immunologically between the antigens encoded by the two hamster major histocompatibility alleles. Hm-1a and Hm-1b, we conclude that the hamster strains we work with are serologically indistinguishable by the methods used here. However, they obviously differ for determinants which elicit T cell-mediated responses, as evidenced by their ability to express acute skin graft rejection, mixed lymphocyte reactivity, graft-vs-host reactions, and cell-mediated cytotoxicity reactions. Such alloreactivity may reflect a mutation at an SD locus, affecting antigenic sites recognized only by T cells, or that the available hamster strains are SD identical, but differ at loci similar to the I region loci in mice. Alternatively, we cannot exclude the possibility that Syrian hamsters somehow fail to express properly the genes coding for SD determinants.     

A fluorometric method for monitoring the enzymic hydrolysis of terminal galactose from GM1-ganglioside.

A fluorometric method for monitoring the enzymic hydrolysis of the terminal galactose from GM₁-ganglioside has been developed. The released galactose is oxidized with galactose dehydrogenase and NAD and the fluorescence of the product NADH measured. This method can detect as little as 0.1 nmol of galactose. β-Galactosidase from the gastropod Turbo cornutus was employed for the hydrolysis reaction. The rate of GM₁-ganglioside hydrolysis is linearly proportional to incubation time for 30 min under the assay conditions employed. In addition to galactose, the other product of hydrolysis, GM₂-ganglioside, is identified by thin-layer chromatography. This procedure provides a convenient and specific method for measuring the release of galactose from GM₁-ganglioside.     

Role of calcium ions in the control of embryogenesis of Xenopus. Changes in the subcellular distribution of calcium in early cleavage embryos after treatment with the ionophore A23187.

Treatment of stage 5 Xenopus embryos with the ionophore A23187 for only 10 min, in the absence of extracellular Mg2+ and Ca2+, causes cortical contractions and a high incidence of abnormal embryos during subsequent development. Cation analysis shows that divalent ions are not lost from the embryos, but that Ca2+ is redistributed within the subcellular fractions. Ca2+ is probably released from yolk platelets and/or pigment granules by the action of A23187, [Ca2+] rises in the cytosol, and the mitochondria attempt to take up this free Ca2+. The mitochondria concomitantly undergo characteristic ultrastructural transformations, changing towards energized-twisted and energized-zigzag conformations. A23187 allows these changes to be demonstrated in situ. Extracellular divalent cations (10(-4) M) interfere with this intracellular action of A23187. Intracellular accumulation of Na+ (by treatment with ouabain) or Li+ also causes abnormal development, probably by promoting a release of Ca2+ from the mitochondria. It is suggested (a) that all these treatments cause a rise in [Ca2+]i which interferes with normal, integrated cell division, so causing, in turn, abnormal embryogenesis, (b) that levels of [Ca2+]i are of importance in regulating cleavage, (c) that the mitochondria could well have a function in regulating [Ca2+]i during embryogenesis in Xenopus, and (d) that vegetalizing agents may well act by promoting a rise in [Ca2+]i in specific cells in the amphibian embryo.     

DNA intermediates at the Escherichia coli replication fork. II. Studies using dut and ung mutants in vitro.

The incorporation of uracil into and excision from DNA were studied in vitro using lysates on cellophane discs made from Escherichia coli strains with defects in the enzymes dUTPase (dut) and uracil-DNA glycosylase (ung).Results with dut ung lysates indicate that dUTP is competitively incorporated with dTTP at the replication fork. Such incorporation is not due to DNA polymerase I. There is a mild discrimination (2.5-fold) against incorporation of dUTP versus dTTP. These data, together with in vivo uracil incorporation data (Tye et al., 1978) permit a rough estimate of the pool of dUTP in vivo (~0.5% of the dTTP pool).These in vitro data indicate that uracil-DNA glycosylase is the initial step in at least 90% of uracil excision events. However, in a strain defective in uracil-DNA glycosylase (ung-1), uracil-containing DNA is still more subject to single-strand scission than non-uracil-containing DNA, albeit at a rate at least tenfold less than in an ung⁺ strain.A number of qualitative statements may also be made about different steps in uracil incorporation and subsequent excision and repair events. When high levels of dUTP are added in vitro, a dut ung⁺ strain has a higher steady-state level of uracil in newly synthesized DNA than does an isogenic dut⁺ ung strain. Thus the dUTPase in these lysates has a higher capacity to be overloaded than does the excision system (i.e. uracil DNA glycosylase). However, the DNA sealing system (presumably DNA polymerase I and DNA ligase) apparently can handle all single-strand interruptions being introduced by uracil excision at the maximal rate, at least so that DNA synthesis can continue.     

Hemotypes and genetic structure of Camargue horses

The nature and distribution of hemotypes constituted by electrophoretic phenotypes in six loci, in a group of 183 Camargue horses, have been compared with those of five breeds of horses and ponies. The genetic similarities have been observed mainly with New Forest and Haflinger ponies, less with Barbs and Thoroughbreds, and the least with Arab horses.     

Effects of guanine nucleotides on cns neuropeptide receptors

The effect of nucleotides on central nervous system neuropeptide receptor binding was investigated. The guanine nucleotides, guanosine-5′-triphosphate and guanylyl-5′-imidodiphosphate, significantly inhibited the binding of radiolabeled vasoactive intestinal polypeptide but not that of [Tyr⁴]bombesin to rat brain membranes. Vasoactive intestinal polypeptide binding was inhibited by guanine nucleotides in a dose-dependent manner. Using a 20 μM dose, 60% of the specific vasoactive intestinal polypeptide binding was inhibited by guanylyl-5′-imidodiphosphate, which was more potent than guanosine-5′-triphosphate, whereas other nucleotides were not effective. This reduction in binding was a consequence of lower affinity of the receptor for vasoactive intestinal polypeptide, which in turn resulted from an increased rate of dissociation.     

Vitamin D-dependent calcium binding protein in the chick brain

A radioimmunoassay for chick CaBP has been used to measure the distribution of the protein in the chick central nervous system. The cerebellum contained 7.4 μg/mg protein, six times more than the medulla, and eleven times more than the cerebral cortex. Most of the CaBP was found in the supernate, after ultracentrifugation, with only trace amounts in the secretosome, microsome and other fractions. Slices of cerebellum from 19 and 21 days $\text{in}$$\text{ovo}$ chicks were maintained in organ culture. Treatment with kainic acid resulted in a loss of CaBP from the slices while metabolites of vitamin D were without effect.