Dynamics of the immune system response in cerebrospinal fluid and blood of SIVmac-infected rhesus monkeys

Paired sera and CSF samples were collected from SIV_mac-infected macaques. Animals infected with SIV_mac251 maintained low gag and high env-specific antibody levels in plasma. Increasing env-specific antibody titers in CSF were associated in one animal with strong intrathecal synthesis. SIV_mac239-infected monkeys revealed high antibody titers of gag and env-specificity, in one animal accompanied by weak intrathecal synthesis of virus-specific antibodies. In all animals, the CD4/CD8 ratio in CSF decreased faster compared to blood.     

The influence of inorganic silicon (Si) on pituitary-thyroid axis

The influence of silicon-treatment on the levels of TSH and thyroid hormones was studied in rats. Concentrations of thyrotropin (TSH), triiodothyronine (T₃), and thyroxine (T₄) were estimated in sera of rats receiving per os a soluble silicon compound—sodium metasilicate nonahydrate (Na₂SiO₃·9H₂O), dissolved in the animals' drinking water. An increase in the TSH level in the tested group was observed, without statistically significant differences in T₃ and T₄ concentrations between the two groups of animals. The results provide evidence for the influence of silicon on the endocrine balance. They could also prove that this chemical element is capable of modifying the rate of some hormones' synthesis.     

Enhancing PCR amplification and sequencing using DNA-binding proteins

The polymerase chain reaction (PCR) is a powerful core molecular biology technique, which when coupled to chain termination sequencing allows gene and DNA sequence information to be derived rapidly. A number of modifications to the basic PCR format have been developed in an attempt to increase amplification efficiency and the specificity of the reaction. We have applied the use of DNA-binding protein, gene 32 protein from bacteriophage T4 (T4gp32) to increase amplification efficiency with a number of diverse templates. In addition, we have found that using single-stranded DNA-binding protein (SSB) or recA protein in DNA sequencing reactions dramatically increases the resolution of sequencing runs. The use of DNA-binding proteins in amplification and sequencing may prove to be generally applicable in improving the yield and quality of a number of templates from various sources.     

4-Hydroxy-2-Nonenal Levels Increase in the Plasma of Patients with Adult Respiratory Distress Syndrome as Linoleic Acid Appears to Fall

Gas chromatography-mass spectrometry has been applied to the analysis of plasma linoleic acid and one of its oxidation products, 4-hydroxy-2-nonenal (HNE), in adult patients with the acute respiratory distress syndrome (ARDS). Peak areas of total ion chromatograms showed there to be negative correlations between loss of linoleic acid and formation of HNE (measured by selective ion monitoring) in 7 out 10 patients studied. When HNE was quantitated by selective ion monitoring, with reference to a pure standard of HNE and an internal standard of nonanoic acid, ARDS patients showed significantly increased levels of HNE (0.412 ± 0.023 nmol/ml) compared with normal healthy controls (0.205 ± 0.018 nmol/ml).     

Volume regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the activation of the Cl− efflux

Trypsinized human skin fibroblasts in suspension perform regulatory volume decrease (RVD) after cell swelling in hypotonic medium. During RVD, ³⁶Cl^– efflux is dramatically increased and the cell membrane is depolarized, indicating the activation of Cl^– channels. This activation of Cl^– channels depends on extracellular as well as on intracellular Ca²⁺. The swelling-induced Cl^– efflux and the RVD response are inhibited by the 5-lipoxygenase inhibitor ETH 615-139. Finally, following hypotonic treatment, cellular pH decreases. The pH decrease does not involve the Cl^–/HCO ₃ ^– exchange because it is independent of the external Cl^– concentration.T. Mastrocola was recipient of a scientific fellowship from the Italian Consiglio Nazionale delle Ricerche (C.N.R.). This work was supported by Progetto Finalizzato Ingegneria Genetica, C.N.R., Roma, and by the Danish Natural Research Council.     

Cationic and Anionic Requirements for the Binding of 2β-Carbomethoxy-3β-(4-Fluorophenyl)[3H]tropane to the Dopamine Uptake Carrier

Abstract: The present study reports the ion dependency of 2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane ([³H]- CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [³H]CFT binding to synaptosomal P₂ membranes requires low concentrations of Na⁺ (peak binding between 20 and 50 m M Na⁺), is stimulated by phosphate anion or l^-, but is unaffected or only slightly affected by F^-, Cl^-, Br^-, NO₃^-, or SO₄^2-, Concentrations of Na⁺ of >50 m M become inhibitory except in the presence of l^-, which shifts peak binding levels toward higher Na⁺ concentrations and also elevates the peak binding level. K⁺ strongly decreased [³H]CFT binding with a shallow inhibition curve, and Na⁺ could not overcome this effect. Saturation analysis of [³H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 m M ; 1 mM plus 49 mM NaCIversus 10 m M plus 40 m M NaCI; or 1 mM plus 129 m M Nal versus 10 m M plus 120 m M Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.     

Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides

A -galactosidase (EC 3.2.1.23) capable of degrading a number of fruit cell-wall polysaccharides in vitro, was isolated from ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson cv. Hayward). The enzyme has a molecular weight of approximately 60 kDa by gel permeation and consists of several basic isoforms. Several polypeptides were enriched during purification, with 33-, 46- and 67-kDa bands being predominant after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme against p-nitrophenyl--d-galactopyranoside was at pH 3.2, but against a galactan purified from kiwifruit cell walls, it was at pH 4.9. The enzyme was specific for galactosyl residues in the -configuration, releasing galactose from a variety of kiwifruit cell-wall polysaccharide fractions including cell wall material, Na₂CO₃-soluble pectin, high-molecular-weight galactan, xyloglucan, and galactoglucomannan. A galactosylated glucuronomannan found throughout the kiwifruit plant was also a substrate for the enzyme. The results indicate that the enzyme attacks the non-reducing end of galactose side chains, cleaving single galactose residues which may be attached to the 2, 3, 4, or 6 position of the aglycone. Activity of the enzyme in-vitro was too low to account for the total loss of galactose from the cell walls during ripening. If the -galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening then its in-vivo activity must be much greater than that observed in-vitro.Abbreviations CWM cell wall material - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Bronwyn Culling and Teresa Wegrzyn for assistance and acknowledge a contribution towards the cost of the research from the New Zealand Kiwifruit Marketing Board.     

Factor in urinary extracts from pregnant women that inhibit mouse oocyte maturation in vitro

Mouse oocyte maturation inhibitory factors, on the basis of inhibitory activity of spontaneous germinal vesicle breakdown (GVBD) of denuded mouse oocytes in culture, were extracted and partially purified by reversed-phase resin adsorption and Sephadex G-100 and G-50 column chromatographies from the urine of pregnant women. Denuded oocytes obtained from ovaries of ICR mice underwent spontaneous GVBD by cultivation for 3 h in modified Krebs–Ringer's buffered solution, while this spontaneous GVBD was found to be inhibited by adding the final preparation (U-D-4) of urine. The inhibition was dose dependent, ranging from 0.6 to 10 μg protein/ml medium. Oocytes treated with U-D-4 and resuspended in control medium resumed GVBD. The molecular mass of U-D-4 was estimated to be less than 2,000 Da with gel filtration. Ether treatment failed to extract inhibitory factor(s) from U-D-4 and pepsin treatment inactivated U-D-4, indicating that inhibitory factor(s) in U-D-4 are peptide-like substances. The inhibitory effect of U-D-4 on spontaneous GVBD was partially reversed in the presence of naloxone, a potent opioid antagonist. U-D-4s obtained from urine samples of pregnant women, nonpregnant women, and men showed the inhibitory effect on spontaneous GVBD; however, the activity of U-D-4 obtained from pregnancy urine was significantly more potent than those of the other urine samples. © 1993 Wiley-Liss, Inc.