Formation of Virus Assembly Intermediate Complexes in the Cytoplasm by Wild-Type and Assembly-Defective Mutant Human Immunodeficiency Virus Type 1 and Their Association with Membranes

We have previously identified two distinct forms of putative viral assembly intermediate complexes, a detergent-resistant complex (DRC) and a detergent-sensitive complex (DSC), in human immunodeficiency virus type 1 (HIV-1)-infected CD4(+) T cells (Y. M. Lee and X. F. Yu, Virology 243:78-93, 1998). In the present study, the intracellular localization of these two viral assembly intermediate complexes was investigated by use of a newly developed method of subcellular fractionation. In wild-type HIV-1-infected H9 cells, the DRC fractionated with the soluble cytoplasmic fraction, whereas the DSC was associated with the membrane fraction. The DRC was also detected in the cytoplasmic fraction in H9 cells expressing HIV-1 Myr- mutant Gag. However, little of the unmyristylated Gag and Gag-Pol proteins was found in the membrane fraction. Furthermore, HIV-1 Gag proteins synthesized in vitro in a rabbit reticulocyte lysate system in the absence of exogenous lipid membrane were able to assemble into a viral Gag complex similar to that of the DRC identified in infected H9 cells. The density of the viral Gag complex was not altered by treatment with the nonionic detergent Triton X-100, suggesting a lack of association of this complex with endogenous lipid. Formation of the DRC was not significantly affected by mutations in assembly domains M and L of the Gag protein but was drastically inhibited by a mutation in the assembly I domain. Purified DRC could be disrupted by high-salt treatment, suggesting electrostatic interactions are important for stabilizing the DRC. The Gag precursor proteins in the DRC were more sensitive to trypsin digestion than those in the DSC. These findings suggest that HIV-1 Gag and Gag-Pol precursors assemble into DRC in the cytoplasm, a process which requires the protein-protein interaction domain (I) in NCp7; subsequently, the DRC is transported to the plasma membrane through a process mediated by the M domain of the matrix protein. It appears that during this process, a conformational change might occur in the DRC either before or after its association with the plasma membrane, and this change is followed by the detection of virus budding structure at the plasma membrane.     

Insulin-Like Peptides of the Cerebropleural Ganglion of the Mollusc Anodonta cygnea: Isolation, Purification, and Radioligand Analysis

Cerebropleural ganglia from 4000 individuals of the mollusc Anodonta cygnea were submitted to procedures developed for isolation of vertebrate pancreatic insulins: homogenization and extraction, stage-like isoelectrical sedimentation, and ion-exchange chromatography. As a result of purification of the obtained preparation, using high-effective liquid chromatography, there were identified 7 protein peaks differing by time of retention on the reverse-phase sorbent in acetonitryl gradient and designated as insulin-related peptides (IRP), IRP1-IRP7. The material was characterized by the peptide ability to inhibit specific binding of ¹²⁵I-insulin and of insulin-related factor-1 (¹²⁵I-IGF-1) by plasma membranes of the rat liver and brain. The IC₅₀ value of peptide concentration (nM) able to replace 50% of the labeled hormone bound with the receptor amounted in the insulin radioreceptor system for IRP1 to 330, for IRP3 to 130, for IRP4 to 17, for IRP5 to130, for IRP6 to 420 nM. Peptide IRP7 at a maximal concentration (10⁴ ng/ml) replaced less than 50% of labeled hormone, whereas in IRP2 no inhibitory ability was detected under these experimental conditions. The IC₅₀ value in the case of ¹²⁵I-IGF-1 amounted for IRP1, IRP4, and IRP5 to17, for IRP2 to 50, for IRP3 to 83, for IRP6 to 133 nM. IRP7 at a concentration of 10⁴ ng/ml replaced less than 50% of labeled hormone. The same high relative affinity of the peptide IRP4 (12% of activity of standard insulin and IGF-1) to both receptor types is revealed. The results of analysis in two types of hormonal test systems indicate the ability of the insulin-related peptides of the anodonta cerebropleural ganglion to interact with the vertebrate receptor of insulin and IGF-1. This gives grounds to suggest the presence of the metabolic and growth-stimulating properties in these peptides. For the first time, the IGF-1 activity is revealed in insulin-like molecules in invertebrates. Taking into account the chromatographically revealed differences of physicochemical characteristics of individual IRP as well as predominance of their IGF-1-binding properties, there is suggested another organization of the IRP receptor-binding domains in IPR of this mollusc species, as compared with mammalian insulins.     

The Role of the Vascular Factor in the Reorganization of Water Metabolism in Denervated Liver after Bacterial Endotoxin Poisoning

Intoxication with bacterial lipopolysaccharide (endotoxin) is accompanied by considerable rearrangements in the systems of blood microcirculation and water metabolism of the liver. These rearrangements are manifested as increased sinusoid area, changed total area of the cytoplasm and nuclei as well as the nucleocytoplasmic ratio in hepatocytes, increased content of total water in the organ, and changed magnetic relaxation properties (spin-lattice and spin-spin relaxation times). Preliminary parasympathetic denervation of the liver (vagotomy) changes the pattern of the organ response to bacterial endotoxin poisoning as indicated by the kinetics of studied morphological and biophysical parameters.     

Kinase Signaling in the Spindle Checkpoint

The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the “wait anaphase” signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects.     

Characteristics of interhemispheric relationships in the absence or presence of a skill

A comparative analysis of the time and amplitude characteristics of the negative N200 and positive P300 components of visual evoked potentials recorded at symmetric points of the frontal, parietal, temporal, and occipital areas of the right and left hemispheres of the cerebral cortex has been performed in subjects with or without the skill of operating a computer. Subjects inexperienced in an operator’s work exhibited an interhemispheric difference in the time and amplitude characteristics of the studied components. In subjects that had the skill of operating a computer, the interhemispheric difference was little, which suggests that the cortex plays only a small role in the cerebral control of this activity.     

Effects of two-vessel forebrain ischemia and of administration of indomethacin and quinacrine on Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity in various rat brain areas

The effect of a two-vessel forebrain ischemia (induced by occlusion of carotid arteries and hypotension), subsequent reperfusion, and administration of indomethacin and quinacrine on the Na⁺,K⁺-ATPase activity and diene conjugate content was studied in various rat forebrain fields. The most pronounced metabolic alterations were observed during ischemia and reperfusion. Under these effects, there was a statistically significant reduction of the Na⁺,K⁺-ATPase activity in the brain cortex and striatum and an increase of the diene conjugate content in the rat brain cortex in comparison with sham-operated animals. Injection of indomethacin, a cyclooxygenase inhibitor, to rats subjected to ischemia and reperfusion, resulted to a statistically significant increase of the Na⁺,K⁺-ATPase activity in the brain cortex, hippocampus, and striatum (p < 0.02) as compared with control animals. The diene conjugate content in the rat brain cortex during brain ischemia and reperfusion was statistically significantly lower in the rats injected with indomethacin. The effect of quinacrine (a blocker of phospholipase A₂) was similar to that of indomethacin in the rat cortex, whereas in the rat striatum and hippocampus, the quinacrine effect during ischemia and reperfusion was less marked than that of indomethacin. The obtained data indicate the ability of inhibitors of the arachidonic pathway of free radical formation to normalize the Na⁺, K⁺-ATPase activity during brain ischemia. There also revealed local peculiarities of metabolic disturbances in different regions of the rat forebrain during ischemia and reperfusion.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 33–38.Original Russian Text Copyright © 2005 by Molchanova, Moskvin, Zakharova, Yurlova, Nosova, Avrova.