Specific asparagine-linked oligosaccharides are not required for certain neuron-neuron and neuron-Schwann cell interactions

To determine whether specific asparagine-linked (N-linked) oligosaccharides present in cell surface glycoproteins are required for cell-cell interactions within the peripheral nervous system, we have used castanospermine to inhibit maturation of N-linked sugars in cell cultures of neurons or neurons plus Schwann cells. Maximally 10-15% of the N-linked oligosaccharides on neuronal proteins have normal structure when cells are cultured in the presence of 250 micrograms/ml castanospermine; the remaining oligosaccharides are present as immature carbohydrate chains not normally found in these glycoproteins. Although cultures were treated for 2 wk with castanospermine, cells always remained viable and appeared healthy. We have analyzed several biological responses of embryonic dorsal root ganglion neurons, with or without added purified populations of Schwann cells, in the presence of castanospermine. We have observed that a normal complement of mature, N-linked sugars are not required for neurite outgrowth, neuron-Schwann cell adhesion, neuron-induced Schwann cell proliferation, or ensheathment of neurites by Schwann cells. Treatment of neuronal cultures with castanospermine increases the propensity of neurites to fasciculate. Extracellular matrix deposition by Schwann cells and myelination of neurons by Schwann cells are greatly diminished in the presence of castanospermine as assayed by electron microscopy and immunocytochemistry, suggesting that specific N-linked oligosaccharides are required for the expression of these cellular functions.     

Characteristics of free water distribution in the cytoplasm of cryotolerant cells of Cryptococcus laurentii

The resistance to freezing-thawing was studied with Cryptococcus laurentii cultivated at a near-zero plus temperatures in a minimal or a rich medium. At the transition into the stationary phase, the resistance of the cells to freezing increased 20 times in the culture grown in the minimal medium and 8 times in the culture grown in the rich medium. Free water localization in the cell cytoplasm was determined by electron microscopy. In yeast cells with the maximal cryotolerance, free water was found mainly between glycogen granules. The authors discuss the role of glycogen as of a possible factor making the cells resistant to low temperatures.     

Differences in the stress response of prolactin in young and aged female rats

The drawing of blood by orbital sinus puncture (OSP) under ether anesthesia is known to produce a marked increase in serum prolactin (PRL levels in young cycling female rats. The effect of this stressful procedure on PRL release was compared in young and aged female rats. Nonstressed PRL levels were obtained from blood drawn by decapitation. Whereas OSP with a one-minute ether exposure induced a marked increase in PRL levels in young rats on all days of the estrus cycle, older cycling female rats on the day of diestrus -1 and aged rats exhibiting prolonged diestrus (PD) showed virtually no increase above nonstressed levels. However, increasing the ether exposure time to five minutes did produce a rise in PRL levels. Old cycling female rats on the day of estrus and aged rats exhibiting constant estrus (CE) did show a PRL increase comparable to that seen in young animals. Ovariectomy (OVX) completely abolished the stress response seen in aged CE rats. The response, though markedly decreased, was still present in young ovariectomized rats. These experiments show that the stress-induced rise in PRL promoted by OSP under either anesthesia is markedly diminished in aged rats exhibiting a diestrus state. The attenuated response seen in these rats is believed due to factors characteristic of the diestrous state of aging.     

A new radiochemical assay for argininosuccinase with purified [14C]argininosuccinate

A sensitive and simple radiochemical assay is described to measure argininosuccinase activity in crude tissue homogenates and cultured cells. The method depends on the use of argininosuccinate labeled uniformly with ¹⁴C in the six carbons of the arginine moiety. On incubation in the presence of excess arginase, the [U-¹⁴C]arginine formed is measured as the sum of radioactivity in [U-¹⁴C]ornithine and [¹⁴C]urea. Separation from the substrate is accomplished on a small Domex 1-acetate column eluted with 25 mm acetic acid; ornithine and urea emerge in the first few milliliters while unutilized substrate remains on the column. [¹⁴C]Argininosuccinate was synthesized enzymatically from l-[U-¹⁴C]arginine and fumarate and isolated and purified as the barium salt. Development of a new purification step has brought the amino acid to a purity of 97% as judged by chromatographic and barium analysis. With the present specific radioactivity, as little as 5 to 10 nmol of product can be accurately measured under kinetically optimum conditions.     

Enlarged model of lambda phage ontogenesis

An enlarged threshold model of the Regulatory System of Development of λ-Phage (RSDP λ-2) is built. It includes 15 synthetic blocks of proteins and mRNAs and four blocks corresponding to the other ontogenetic processes: two-stage replication, integration and excision of phage genome, formation of oligomers of regulatory proteins, regulation of bacteriallysis. By way of computer simulation of the RSDP λ-2 model the dynamics of concentrations of all main proteins, respective fractions of mRNAs and DNA are described in the lytic and lysogenic regimes of phage ontogenesis. Results obtained are in good agreement with available experimental data. The dependence of a portion (%) of lysogenic responses on the multiplicity $\text{k}$ of phage infection of bacterial culture, is built. This curve has a maximum point in accordance with the experimental data of P. Kourilsky (1973).     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

Simian immunodeficiency virus negative factor suppresses the level of viral mRNA in COS cells.

The nef gene is conserved among all human and simian lentiviruses. However, the amino acid similarity between simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 NEF is only 38%. To assess the role of SIV NEF on virus replication and compare its activity with that of its human immunodeficiency virus type 1 counterpart, we examined the activity of an intact nef gene from proviral clone pSIV 102, an isolate from SIV-MAC-251-infected cells. Proviral clone pSIV BA was constructed by introducing a premature termination codon at codon 40 of the nef gene without altering the predicted amino acid sequence of the overlapping env gene. These two clones were transfected into CD4- COS cells, and virus replication was monitored by p27 enzyme-linked immunosorbent assay kits. In seven independent experiments, clone pSIV BA afforded two- to sixfold greater levels of viral antigen compared with those in clone pSIV 102 and two- to sixfold-increased levels of viral mRNAs as indicated with Northern (RNA) blot and S1 nuclease protection analyses. Nuclear run-on assays demonstrated a two- to threefold increased rate of RNA synthesis with nuclei isolated from cells transfected with pSIV BA compared with that from cells transfected with pSIV 102. In contrast, there was no apparent destabilization of SIV mRNAs by NEF, as measured in dactinomycin-treated cells. This study demonstrates that SIV NEF is a negative regulator of virus replication and acts by suppressing the level of mRNA synthesis and accumulation in COS cells.     

A phylogenetic analysis of genes of the influenza virus. Phylogenetic trees and fixation rates

The phylogenetic trees of influenza virus genes of hemagglutinins, neuraminidases, and of NS genes were composed. Considering properties of synonimic replacements to be neutral and their rates constant at each tree, the dates of ancestor branch points were calculated, and the rates of fixation of synonimic (Ks) and non-synonimic (Kns) replacements estimated. The epidemic branches were mostly shown to be "deadlocks", non-epidemic ones being internal or "roots." The ratios of the numbers of synonimic to non-synonimic replacements (vs/vns) were correspondingly 1.32+/-0.42 and 4.78+/-1.28 for all trees, the difference being significant. It was shown that the dated branch points for hemagglutinins are non-randomly clustered around the initial points of the main genetic shifts of the A-type virus, corresponding to the influenza pandemics. It seems that these ancestor forms of virus behave similar to the "train" of these shifts, reproducing together with the pandemic forms under conditions of decreased immune resistance of host population. The rates of fixation of non-synonimic replacements in the epidemic branches of this tree are 4 times increased, as compared to non-epidemic ones.     

PC12 cells as a source of neurite-derived cell surface mitogen, which stimulates Schwann cell division

Primary cultures of rat dorsal root ganglion Schwann cells were used to assay the efficacy of PC12 cells in stimulating Schwann cell proliferation. Co-cultures of PC12 cells and Schwann cells assayed by [3H]thymidine labeling followed by autoradiography showed proliferation of Schwann cells only where contact occurred between PC12 neurites and Schwann cells. Membranes derived from PC12 cells were shown to have many characteristics similar to membranes derived from sensory neurons; both could mimic whole cells in stimulating Schwann cell division; both were inactivated by mild heat treatment and by trypsinization, and both elevated intracellular cyclic AMP concentrations in Schwann cells 16 h after addition of membranes. We conclude that PC12 cells will be a valuable source for the isolation of the neuronal cell surface component which controls proliferation of Schwann cells during development of the peripheral nervous system.