Lithium Enhances Accumulation of [3H]Inositol Radioactivity and Mass of Second Messenger Inositol 1,4,5-Trisphosphate in Monkey Cerebral Cortex Slices

We previously reported that lithium, in the presence of acetylcholine, increased accumulations of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in brain cortex slices from the guinea pig, rabbit, rat, and mouse. In the mouse and rat, the Li(+)-induced increases required supplementation of the medium with inositol. This probably relates to the following facts: (a) Brain cortices of the mouse and rat contain in vivo concentrations of inositol half of that of the guinea pig. (b) Incubated rat brain cortex slices are depleted of inositol by 80%. (c) The slices require 10 mM inositol supplementation to restore in vivo concentrations. We now show that in monkey brain cortex slices, therapeutic concentrations of Li+ increase accumulation of inositol 1,4,5-trisphosphate. The inositol 1,3,4,5-tetrakisphosphate level is not increased. Neither inositol nor an agonist is required. The same effects are seen whether inositol 1,4,5-trisphosphate is quantified by the [3H]inositol prelabeling technique or by mass assay, although mass includes a pool of inositol 1,4,5-trisphosphate that is metabolically inactive. Thus, in a therapeutically relevant model for humans, Li+ increases inositol 1,4,5-trisphosphate levels in brain cortex slices, as was previously seen in lower mammals at non-rate-limiting concentrations of inositol.     

Virions of primary human immunodeficiency virus type 1 isolates resistant to soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates.

Primary isolates of human immunodeficiency virus type 1 (HIV-1) are much less sensitive to neutralization by soluble CD4 (sCD4) and sCD4-immunoglobulin (Ig) chimeras (CD4-IgG) than are HIV-1 strains adapted to growth in cell culture. We demonstrated that there are significant reductions (10- to 30-fold) in the binding of sCD4 and CD4-IgG to intact virions of five primary isolates compared with sCD4-sensitive, cell culture-adapted isolates RF and IIIB. However, soluble envelope glycoproteins (gp120) derived from the primary isolate virions, directly by detergent solubilization or indirectly by recombinant DNA technology, differed in affinity from RF and IIIB gp120 by only one- to threefold. The reduced binding of sCD4 to these primary isolate virions must therefore be a consequence of the tertiary or quaternary structure of the envelope glycoproteins in their native, oligomeric form on the viral surface. In addition, the rate and extent of sCD4-induced gp120 shedding from these primary isolates was lower than that from RF. We suggest that reduced sCD4 binding and increased gp120 retention together account for the relative resistance of these primary isolates to neutralization by sCD4 and CD4-IgG and that virions of different HIV-1 isolates vary both in the mechanism of sCD4 binding and in subsequent conformational changes in their envelope glycoproteins.     

Hydration at the membrane protein-lipid interface.

Evidence has been found for the existence water at the protein-lipid hydrophobic interface of the membrane proteins, gramicidin and apocytochrome C, using two related fluorescence spectroscopic approaches. The first approach exploited the fact that the presence of water in the excited state solvent cage of a fluorophore increases the rate of decay. For 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-PC (DPH-PC), where the fluorophores are located in the hydrophobic core of the lipid bilayer, the introduction of gramicidin reduced the fluorescence lifetime, indicative of an increased presence of water in the bilayer. Since a high protein:lipid ratio was used, the fluorophores were forced to be adjacent to the protein hydrophobic surface, hence the presence of water in this region could be inferred. Cholesterol is known to reduce the water content of lipid bilayers and this effect was maintained at the protein-lipid interface with both gramicidin and apocytochrome C, again suggesting hydration in this region. The second approach was to use the fluorescence enhancement induced by exchanging deuterium oxide (D2O) for H2O. Both the fluorescence intensities of trimethylammonium-DPH, located in the lipid head group region, and of the gramicidin intrinsic tryptophans were greater in a D2O buffer compared with H2O, showing that the fluorophores were exposed to water in the bilayer at the protein-lipid interface. In the presence of cholesterol the fluorescence intensity ratio of D2O to H2O decreased, indicating a removal of water by the cholesterol, in keeping with the lifetime data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.

Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.     

Control of phytoseiids in a spider mite mass-rearing system (Acari: Phytoseiidae,Tetranychidae)

Phytoseiid mites which contaminated the spider mite colony and interfered with the mass-rearing of spider mites were controlled by dipping in hot water. Immersion for 60 s in water of 50°C killed all stages ofAmblyseius fallacis (Garman),A. womersleyi Schicha andPhytoseiulus persimilis (Athias-Henriot) (Acari: Phytoseiidae). Only approximately 0.3% of theA. womersleyi eggs hatched, and this seems negligible. The populations ofTetranychus kanzawai Kishida andT. urticae (Koch) (Acari: Tetranychidae) were reduced. However, they recovered well. Although this treatment resulted in the withering of some soybean seedlings, the next trifoliate leaf to be produced was normal. A very satisfactory result was obtained when this technique was applied to the mass-rearing system.     

Activation and inhibition of human alcohol dehydrogenase by monoclonal antibodies

The human class I alcohol dehydrogenase isozyme beta 2 beta 2 was used as the antigen to raise monoclonal antibodies. Altogether seven lines of hybridomas secreting monoclonal antibodies were obtained. None of the antibodies was isozyme specific and all of them exhibited a similar affinity against all isozymes of the human class I ADH. Five out of the seven monoclonal antibodies had no effect on beta 2 beta 2 activity. Antibody G3 acted as a non-competitive inhibitor with a KI of 3 micrograms/ml at pH 7.5. Increasing pH was effective in reducing the level of inhibition. On the other hand, antibody 1D4 exhibited a pH-dependent activation of ADH activity. In the presence of this antibody, the pH optimum of beta 2 beta 2 was shifted from 9 to 8.5 and total activity was increased by 70% at this optimal pH. Kinetic analysis indicates that 1D4 probably acts as a non-competitive activator and may exert its action by interacting with the coenzyme binding site.     

Low density lipoprotein receptor-related protein mediates endocytosis of monoclonal antibodies in cultured cells and rabbit liver

Monoclonal antibodies that bound to the external domain of the rabbit low density lipoprotein receptor-related protein (LRP) were taken into rabbit fibroblasts by receptor-mediated endocytosis. Uptake occurred in fibroblasts from Watanabe-heritable hyperlipidemic rabbits, which lack low density lipoprotein receptors, as well as in normal rabbit fibroblasts. The fate of the internalized antibodies differed, depending on the domain of LRP that was recognized. LRP is synthesized as a single polypeptide chain that is cleaved to form a heterodimer of two noncovalently bound proteins, 1) a 515-kDa subunit that contains the binding domain, and 2) an 85-kDa subunit that contains the membrane-spanning region and cytoplasmic tail. A monoclonal antibody directed against the 515-kDa subunit (anti-LRP 515) rapidly dissociated from LRP at pH 5.2. After uptake by cells this antibody dissociated from the receptor and was degraded in lysosomes. A second antibody directed against the external portion of the 85-kDa subunit (anti-LRP 85) failed to dissociate at acid pH. After uptake by cells this antibody was not degraded, but instead was released from the cells in an acid-precipitable form. When administered intravenously to rabbits, both 125I-labeled antibodies were rapidly cleared from the circulation, 75-95% of the uptake occurring in the liver. The anti-LRP 515 antibody was degraded and acid-soluble products appeared in the plasma. No significant acid-soluble products appeared when the anti-LRP-85 antibody was infused. We conclude that LRP can carry out receptor-mediated endocytosis and that its ligand-binding domain, like the similar domain of the low density lipoprotein receptor, undergoes an acid-dependent conformational change that ejects ligands within the endosome. We also conclude that in the body this endocytotic function is expressed primarily in the liver. Both of these conclusions lend support to the hypothesis that LRP may function in humans and animals as a receptor for apolipoprotein E-enriched lipoproteins, such as chylomicron remnants.     

Co-purification of proteases with basic fibroblast growth factor (FGF)

Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against anti-basic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparin-Sepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed.     

Stabilization of Z-DNA by demethylation of thymine bases: 1.3-A single-crystal structure of d(m5CGUAm5CG)

Methylation of cytosine bases at the C5 position has been known to stabilize Z-DNA. We had previously predicted from calculations of solvent-accessible surfaces that the methyl group at the same position of thymine has a destabilizing effect on Z-DNA. In the current studies, the sequence d(m5CGUAm5CG) has been crystallized and its structure solved as Z-DNA to 1.3-A resolution. A well-defined octahedral hexaaquomagnesium complex was observed to bridge the O4 oxygens of the adjacent uridine bases at the major groove surface, and four well-structured water molecules were found in the minor groove crevice at the d(UA) dinucleotide. These solvent interactions were not observed in the previously published Z-DNA structure of the analogous d(m5CGTAm5CG) sequence. A comparison of the thymine and uridine structures supports our prediction that demethylation of thymine bases helps to stabilize Z-DNA. A comparison of this d(UA)-containing Z-DNA structure with the analogous d(TA) structure shows that access of the O4 position is hindered by the C5 methyl of thymine due to steric and hydrophobic inhibition. In the absence of the methyl group, a magnesium-water complex binds to and slightly affects the structure of the Z-DNA major groove surface. This perturbation of the solvent structure at the major groove surface is translated into a much larger 1.41-A widening of the minor groove crevice, thereby allowing the specific binding of two water molecules at well-defined sites of each internal d(UA) base pair. Possible mechanisms by which modifications at the major groove surface of Z-DNA can affect the solvent properties of the minor groove crevice are discussed.     

Polarized electronic spectra of Z-DNA single crystals

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.