Liver cytosolic aldehyde dehydrogenases from "alcohol-drinking" and "alcohol-avoiding" mouse strains: purification and molecular properties

Liver cytosolic aldehyde dehydrogenases (AHD-2) have been isolated in a highly purified state from "alcohol-drinking" (C57BL/6J) and "alcohol-avoiding" (DBA/2J) strains of mice. The purified enzymes were resolved into three major and one minor form of activity by isoelectric focusing (IEF) techniques and showed similar zymogram patterns. The enzymes had identical subunit sizes on SDS-polyacrylamide gels: 53,000. Gel exclusion chromatography, using Ultrogel AcA34, indicated that the enzymes were dimers. The enzymes exhibited biphasic kinetic characteristics and were readily distinguished from each other. The purified forms of AHD-2 from C57BL/6J and DBA/2J mice exhibited two apparent Km values in each case: 10 microM/100 microM and 30 microM/330 microM respectively. AHD-2 exhibited a broad pH optimum in the range 7.0-9.0 and was very sensitive towards disulphuram inhibition, with 50% inhibition occurring at 0.17 microM. The kinetic results support proposals that AHD-2 may be the primary enzyme for oxidizing acetaldehyde during ethanol oxidation in vivo.     

Sensitivity to X-irradiation of peripheral blood lymphocytes from ageing donors

The proliferation of human blood lymphocytes from ageing donors, responding to concanavalin A, showed greater sensitivity to inhibition by X-rays than similar cells from younger donors. This increased sensitivity was associated with deficiency in repair of X-ray-induced damage to nuclear material, as measured by density in sucrose gradients, and with increased incidence of chromosomal damage following exposure of freshly isolated lymphocytes. There was also an increased frequency of spontaneous chromosomal aberrations in ageing subjects whose lymphocytes were deficient in repair of DNA damage.     

Sexual maturation in male Belding's ground squirrels: influence of body weight

The relation between body weight and sexual maturation was examined in a hibernator, Belding's ground squirrel, by manipulating the availability of food to weaned juvenile males. Following body weight manipulation in the summer, testicular growth, serum testosterone, and spermatogenesis were monitored during the subsequent year, which included 7 mo when males were in the coldroom (ca. 8 degrees C), followed by 5 mo in the laboratory (ca. 20 degrees C). Juveniles (less than 1 yr old) maintained on a restricted diet entered the coldroom at normal body weights for their age class in nature and had immature gonads throughout the year, which is characteristic of this group in the field. In contrast, juveniles given abundant food during the summer entered the coldroom at body weights typical for free-living yearlings and exhibited mature gonads shortly after males were removed from the cold (high relative testis weights, high serum testosterone levels, and all stages of spermatogenesis). The high level of gonadal activity in overfed males was confined to a period of a few weeks in the spring, which coincided with the time when mating occurs in nature. The ability of male Belding's ground squirrels to accumulate body weight prior to hibernation seems important to sexual maturation in this seasonally breeding rodent.     

Bone mineral content of limb bones of male weanling rats subjected to 30 and 60 days of simulated increases in body weight

The purpose of this study was to subject groups of male rats each to a specific 10% simulated increase in body weight, ranging from 1.1 to 2.0 g. Constant centrifugation was employed. After 30 and 60 days, rats were sacrificed and perfused with 10% BNF. The humerus, radius, ulna, femur, and tibia were removed, cleared of all soft tissues, weighed, decalcified with EDTA, and reweighed. Bone mineral content (BMC) was determined using the formula: [(Wu - Wd) divided by Wu] X 100. Tukey's multiple range test was used. The data suggest that male weanling rats subjected to simulated increases in body weight, within the range used in this study, undergo enhanced BMC, a bimodal curve describing the relationship between BMC and simulated increases in body weight.     

Melittin lysis of red cells

Summary This paper describes experiments designed to explore interactions between human red blood cell membranes and melittin, the main component of bee venom. We found that melittin binds to human red cell membranes suspended in isotonic NaCl at room temperature, with an apparent dissociation constant of 3×10^–8 m and maximum binding capacity of 1.8×10⁷ molecules/cell. When about 1% of the melittin binding sites are occupied, cell lysis can be observed, and progressive, further increases in the fraction of the total sites occupied lead to progressively greater lysis in a graded manner. 50% lysis occurs when there are about 2×10⁶ molecules bound to the cell membrane. For any particular extent of melittin binding, lysis proceeds rapidly during the first few minutes but then slows and stops so that no further lysis occurs after one hour of exposure of cells to melittin. The graded lysis of erythrocytes by melittin is due to complete lysis of some of the cells, since both the density and the hemoglobin content of surviving, intact cells in a suspension that has undergone graded melittin lysis are similar to the values observed in the same cells prior to the addition of melittin. The cells surviving graded melittin lysis have an increased Na and reduced K, proportional to the extent of occupation of the melittin binding sites. Like lysis, Na accumulation and K loss proceed rapidly during the first few minutes of exposure to melittin but then stops so that Na, K and hemoglobin content of the cells remain constant after the first hour. These kinetic characteristics of both lysis and cation movements suggest that melittin modifies the permeability of the red cell membrane only for the first few minutes after the start of the interaction. Direct observation of cells by Nomarsky optics revealed that they crenate, become swollen and lyse within 10 to 30 sec after these changes in morphology are first seen. Taken together, these results are consistent with the idea that melittin produces lysis of human red cells at room temperature by a colloid osmotic mechanism.     

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments.

In the murine coronavirus mouse hepatitis virus, a single glycoprotein, E2, is required both for attachment to cells and for cell fusion. Cell fusion induced by infection with mouse hepatitis virus strain A59 was inhibited by the addition of monospecific anti-E2 antibody after virus adsorption and penetration. Adsorption of concentrated coronavirions to uninfected cells did not cause cell fusion in the presence of cycloheximide. Thus, cell fusion was induced by E2 on the plasma membrane of infected 17 Cl 1 cells but not by E2 on virions grown in these cells. Trypsin treatment of virions purified from 17 Cl 1 cells quantitatively cleaved 180K E2 to 90K E2 and activated cell-fusing activity of the virions. This proteolytic cleavage yielded two different 90K species which were separable by sodium dodecyl sulfate-hydroxyapatite chromatography. One of the trypsin cleavage products, 90A, was acylated and may be associated with the lipid bilayer. The other, 90B, was not acylated and yielded different peptides than did 90A upon limited digestion with thermolysin or staphylococcal V8 protease. Thus, the cell-fusing activity of a coronavirus required proteolytic cleavage of the E2 glycoprotein, either by the addition of a protease to virions or by cellular proteases acting on E2, which was transported to the plasma membrane during virus maturation. There is a striking functional similarity between the E2 glycoprotein of coronavirus, which is a positive-strand RNA virus, and the hemagglutinin glycoprotein of negative-strand orthomyxoviruses, in that a single glycoprotein has both attachment and protease-activated cell-fusing activities.     

Studies of the GTPase domain of archaebacterial ribosomes

Ribosomes from the methanogens Methanococcus vannielii and Methanobacterium formicicum catalyse uncoupled hydrolysis of GTP in the presence of factor EF-2 from rat liver (but not factor EF-G from Escherichia coli). In this assay, and in poly(U)-dependent protein synthesis, they were sensitive to thiostrepton. In contrast, ribosomes from Sulfolobus solfataricus did not respond to factor EF-2 (or factor EF-G) but possessed endogenous GTPase activity, which was also sensitive to thiostrepton. Ribosomes from the methanogens did not support (p)ppGpp production, but did appear to possess the equivalent of protein L11, which in E. coli is normally required for guanosine polyphosphate synthesis. Protein L11 from E. coli bound well to 23S rRNA from all three archaebacteria (as did thiostrepton) and oligonucleotides protected by the protein were sequenced and compared with rRNA sequences from other sources.     

A correlative study of the binding of dexamethasone in hypothalamic blocks in vitro with its ability to inhibit the release of bioactive corticotrophin-releasing factor

Rat hypothalamic blocks incubated in vitro were used to study the characteristics of binding of [3H]dexamethasone and other steroids to cytosolic binding sites. Cytosols prepared following incubation of the tissue with [3H]dexamethasone for 2 h contained specifically bound steroid in amounts that depended upon the concentration of potassium (but not sodium) ions in the extracting buffer. There was an increase in bound [3H]dexamethasone extracted as the potassium ion concentration increased up to 0.1 M, but not beyond. Dexamethasone, when added to hypothalami in vitro caused a biphasic inhibition of bioactive corticotrophin-releasing factor (CRF) release, and the extent of the second phase of inhibition was dose-related. 11-Epicortisol, when added in a 100-fold molar excess over dexamethasone was able to prevent the second phase of inhibition caused by the latter steroid, as in the binding studies it was able to cause a 50% reduction in the binding of [3H]dexamethasone. In the functional studies it was shown that 11-epicortisol was able to "rescue" the tissue from dexamethasone-mediated delayed inhibition of CRF secretion if added to the blocks 30 min (but not later) after the agonistic steroid.     

Electrolysis-mediated irreversible inactivation of lipoxygenase directed toward electroaffinity labelling

Irreversible inhibition of soybean lipoxygenase-1 (SL-1) was accomplished via a controlled potential oxidative electrolysis of 1,5-dihydroxynaphthalene (1,5-DHN) at +0.8 V vs SCE. The inactivation of SL-1 with this known inhibitor was greatly enhanced under these electrolytic conditions to which the enzyme itself was stable. Electrolyses were run at 0 degree C in a 0.05 M phosphate buffer, pH 7.0, using graphite cloth electrodes. The rate of inactivation was observed to be limited by and dependent on the anodic oxidation of 1,5-DHN. The non-oxidizable (at this potential) inhibitor indomethacin was shown to protect the enzyme from irreversible inactivation, however, an external nucleophile (2-mercaptoethanol) had little effect. These initial studies support the capability of such electrochemical methods for the site-specific covalent modification (affinity labelling) of lipoxygenase, and perhaps other enzymes.     

Fluid transport and mechanical properties of articular cartilage: a review

This review is aimed at unifying our understanding of cartilage viscoelastic properties in compression, in particular the role of compression-dependent permeability in controlling interstitial fluid flow and its contribution to the observed viscoelastic effects. During the previous decade, it was shown that compression causes the permeability of cartilage to drop in a functional manner described by k = ko exp (epsilon M) where ko and M were defined as intrinsic permeability parameters and epsilon is the dilatation of the solid matrix (epsilon = tr delta u). Since permeability is inversely related to the diffusive drag coefficient of relative fluid motion with respect to the porous solid matrix, the measured load-deformation response of the tissue must therefore also depend on the non-linearly permeable nature of the tissue. We have summarized in this review our understanding of this non-linear phenomenon. This understanding of these flow-dependent viscoelastic effects are put into the historical perspective of a comprehensive literature review of earlier attempts to model the compressive viscoelastic properties of articular cartilage.