Some factors influencing the proportion of periplasmic hepatitisB virus pre-S2 antigen in the recombinant yeastHansenula polymorpha

Summary A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitisB pre-S2 antigen in the recombinant yeastHansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that is should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast.     

Expression of a novel nuclear protein is correlated with neuronal differentiation in vivo

We report the production of a monoclonal antibody (MAb 526) that recognizes a novel, developmentally regulated nuclear protein expressed in neurons throughout the rat nervous system. Analysis of whole brain and cell nuclear extracts by SDS-PAGE and immunoblotting determined that MAb 526 recognizes a single nuclear protein (np) of apparent molecular weight 42 kD, designated np526, as well as a slightly larger (ca. 44 kD) cytoplasmic protein. Light microscopic immunocytochemistry showed np526 to be present in neurons of all types throughout the central and peripheral nervous systems. Nuclei of both fibrous and protoplasmic astrocytes were also immunoreactive, but oligodendrocyte nuclei were negative. Positive, but highly variable immunocytochemical staining of nonneural cell nuclei in a variety of other tissues was also observed. Electron microscopic (EM) immunocytochemistry using pre-embedding peroxidase methods revealed that np526 is associated with euchromatin or with the edges of condensed chromatin bundles in neurons, indicating that it is likely to be a chromosomal protein. Most interestingly, the expression of np526 was found to be developmentally regulated in brain. Immunocytochemical analysis of the developing cerebral cortex from embryonic day (E) 16 to postnatal day (P) 4 and cerebellum from P4 to P18 revealed that np526 first appears in central neurons following the cessation of mitosis and that the intensity of nuclear staining increases during subsequent neuronal maturation. To our knowledge, np526 is the first presumptive chromosomal protein whose expression has been precisely correlated with the early postmitotic differentiation of mammalian neurons.     

An analog of ACTH/MSH (4–9), ORG-2766, reduces cerebral uptake of morphine

The uptake of morphine was significantly reduced in most regions of the brains of conscious, unrestrained rats within 10 minutes after treatment with an analog of ACTH/MSH (4–9), ORG-2766. The effect was most obvious in regions with significant densities of enkephalin receptors, namely basal ganglia, hippocampus and cortex. The results explain, in part, how some fragments and analogs of ACTH/MSH may antagonize behavioral actions of morphine, even though some of these peptides lack significant opiate receptor binding properties. We believe that this effect of ORG-2766 is related to an action on the permeability characteristics of the brain microvasculature. The underlying mechanism is unknown.     

Relationship Between Plasma and Brain Tryptophan in Pigs During Experimental Hepatic Coma Before and After Hernodialysis with Selective Membranes

Experimental acute liver ischemia in pigs induces an increment in plasma free tryptophan with decreased total tryptophan. Brain tryptophan is elevated in all brain areas. A slight, but significant increase of brain serotonin is demonstrated in the striatum only, while 5-HIAA (5-hydroxyindoleacetic acid) is significantly lower in the hypothalamus. Other brain areas do not show significant changes in serotonin and 5-HIAA levels. Neither the high plasma free tryptophan levels, nor the decreased sum of neutral competitive amino acids are consistent with such an elevation of brain tryptophan. Hemodialysis was carried out with two different kinds of membranes: cuprophan (with an efficient removal of molecules up to molecular weight 1300) and AN 69 polyacrylonitrile (efficient removal up to 15,000). Ammonia and aminoacid clearance are similar for both membranes. After AN 69, plasmatic free tryptophan and brain tryptophan are lower than after liver devascularization, but still higher than normal. Serotonin significantly increases in the cortex, midbrain and hypothalamus without concomitant rise of 5-HIAA levels. After cuprophan hemodialysis, plasma total tryptophan is lower than in normal and even comatose animals, whereas free tryptophan is normal. Intracerebral tryptophan is similar to AN 69 dialysed animals, but in the hypothalamus it is similar to nondialysed animals. Brain serotonin levels are not modified. 5-HIAA decreases in the hypothalamus. This finding suggests that middle molecules (which are not cleared out with cuprophan hemodialysis) are involved in the intracerebral transfer of tryptophan and the metabolism of serotonin, mainly in the hypothalamus.     

The influence of the social environment on sexual maturation of female deermice (Peromyscus maniculatus bairdi)

Population Ecology - Female deermice housed from weaning with groups of five females, five males or five males plus five females had significantly smaller uteri at 35–38 days of age compared...     

A method for preventing sorbitol interference with the determination of inorganic phosphate

A technique is described for preventing interference of sorbitol with the assay of P₁ by modifying the procedure of B. N. Ames (1966, in Methods in Enzymology, E. F. Neufeld and V. Ginsburg, eds., Vol. 8, pp. 115–118, Academic Press, New York). The new method relies on the ability of precipitated protein to bind phosphomolybdate and so allow separation of the P₁ from the soluble sorbitol. The conditions for the formation and precipitation of phosphomolybdate-protein complex and for the subsequent assay of P₁ are described. No unique set of conditions could be found which prevented interference at all sorbitol concentrations tested. Instead, conditions for the elimination of interference by particular sorbitol concentration ranges were established. The application of the procedure to samples containing 0–150 nmol of P₁ and 10–100 μmol of sorbitol is described. Complete recovery of P₁ was achieved after precipitation. Standard plots were linear. Coefficients of variation ranged from 9% with low amounts of P₁ (≤25 nmol) to 2.5% at higher levels (150 nmol). One hundred nanomoles of P₁ gave an absorbance at 700 nm of 0.87. Modifications are described to extend the technique to different sorbitol concentration ranges and other applications of the method are mentioned.     

The cultivation of the exoerythrocytic stages ofPlasmodium berghei from sporozoites

Summary Cultures of embryonic rat brain and liver, and embryonic turkey brain were inoculated with sporozoites ofPlasmodium berghei. Sporozoites succeeded in establishing exoerythrocytic infections in approximately 10% of the cultures. The exoerythrocytic parasites developed to a late schizont stage with some showing early segmentation although free merozoites were not observed. The morphology and rate of development of the exoerythrocytic parasites in culture appear similar to that seen in vivo. This work was supported by ONR Contract No. N00014-76-C-1132 and Naval Medical Research and Development Command, Research Work Unit No. M0095PN.002.5058. the opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in theGuide for the Care and Use of Laboratory Animals, Institute of Laboratory Resources, National Research Council, DHEW, Pub. No. (NIH) 74-23.     

Cellular events in protein-tolerant inbred rats. III. Antigen-reactive B cells in rats tolerant to human serum albumin.

Rats tolerant to human serum albumin (HSA) were injected with selected lymphocyte populations and challenged with HSA plus adjuvant to test for loss of tolerance. Thoracic duct lymphocytes (TDL) from normal or immune rats, either untreated or depleted of Ig-bearing cells or HSA-binding cells by affinity chromatography were all equally effective in restoring the HSA antibody response in previously tolerant recipients. In contrast, recirculating B cells (TDL from B rats) were not effective. The results indicated that unresponsiveness to HSA was a lesion of the T- but not the B-cell compartment. However, antibody affinity failed to mature to a high level in tolerant rats that were restored with T cells, and the response of transferred primed B cells into unresponsive recipients was inhibited, suggesting that the tolerant state was not merely due to a T-cell deletion.     

The ultrastructure of topical cutaneous calcinosis

Summary Mineralized plaques, which develop at the site of repeated subcutaneous injections of 100 g KMnO₄/0.2 H₂O in rats, were investigated by electron microscopy. The newly formed, delineated, white plaque tissue at the injection site consisted of numerous, mostly unaltered fibroblasts and collagen fibers, without participation of inflammatory cells. Some signs of cell injury were found in the center of the lesions. Numerous, irregularly distributed, small, mineralized foci were seen near the fibroblasts. These were formed by aggregation of small needle-like units (50 Å in diameter and 0.05–2.0 m long). These needle-shaped units were found either in vesicular, cell derived structures, considered to be shed cell processes or cell fragments, or on collagen fibers. Intramitochondrial deposits of such needle-like units were seen frequently. Fusion of smaller mineralized foci to larger plaques occured and then needle-shaped units were seen at the periphery of the electron-dense lesions. Hypotheses concerning the mechanism of experimental cutaneous calcinosis (soft tissue mineralization) are discussed and related to the findings of this study. Probable intracellular crystal deposition and mineralization in cell-derived structures were shown for the first time in topical cutaneous calcinosis.     

Optimization of integrated alkaline–extrusion pretreatment of barley straw for sugar production by enzymatic hydrolysis

In this work, an integrated one-step alkaline–extrusion process was tested as pretreatment for sugar production from barley straw (BS) biomass. The influence of extrusion temperature (T) and the ratio NaOH/BS dry matter (w/w) (R) into the extruder on pretreatment effectiveness was investigated in a twin-screw extruder at bench scale. A 2³ factorial design of experiments was used to analyze the effect of process conditions [T: 50–100 °C; R: 2.5–7.5% (w/w)] on composition and enzymatic digestibility of pretreated substrate (extrudate). The optimum conditions for a maximum glucan to glucose conversion were determined to be R = 6% and T = 68 °C. At these conditions, glucan yield reached close to 90% of theoretical, while xylan conversion was 71% of theoretical. These values are 5 and 9 times higher than that of the untreated material, which supports the great potential of this one-step combined pre-treatment technology for sugar production from lignocellulosic substrates. The absence of sugar degradation products is a relevant advantage over other traditional methods for a biomass to ethanol production process since inhibitory effect of such product on sugar fermentation would be prevented.