Preparative electrophoresis on agarose submerged gels of two aggregating proteoglycan monomers from articular cartilage

Analytical electrophoresis on polyacrylamide-agarose gels of aggregating proteoglycan monomers from baboon articular cartilage produces two distinct bands, corresponding to two different aggregating monomer populations. A preparative electrophoresis procedure is described for isolating the two monomers. Proteoglycans were extracted from young baboon articular cartilage in 4 M guanidinium chloride containing proteolysis inhibitors and aggregated after hyaluronic acid addition. The aggregates were separated from non-aggregated proteoglycans by isopycnic centrifugation, followed by gel chromatography on Sepharose CL-2B. The monomers of the aggregates were obtained by isopycnic centrifugation under dissociative conditions. Two monomers were separated by preparative electrophoresis on 0.8 % agarose submerged gels. Approximately 60 % of the proteoglycans were recovered from the gel using a freeze-squeeze procedure. Aliquots of the separated monomers gave single bands when submitted to analytical polyacrylamide-agarose gel electrophoresis. Their migration and appearance were similar to that of the two bands present in the non separated preparation of monomers.     

Nucleolytic action of chondroitinase ABC on the lumbar disc of the rabbit

Nucleolysis using chondroitinase ABC was studied using the rabbit's intervertebral lumbar disc. The purpose was to find a possible alternative to chymopapain which is commonly used in the management of sciatica due to disc herniation. The injection of 1 U of the enzyme into the nucleus pulposus gave significant histological and biochemical changes in all twelve discs studied.     

Proteoglycan populations of baboon (Papio papio) articular cartilage.

Configurations were determined for previously identified amino components of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 8505. Glucosamine and galactosamine belong to the D-series, and alanine and aminogalacturonic acid to the L-series. An additional amino component was identified as 2,4-diamino-2,4,6-trideoxy-D-glucose. This compound may be a characteristic component of the O-specific chain in lipopolysaccharides of strains of Pseudomonas aeruginosa belonging to Habs sero-group 3.     

Release and specific binding of prostaglandins in bovine pineal gland

Incubated bovine pineal glands released prostaglandin E-and prostglandin F-like material (304 +/- 20 and 582 +/- 56 pg/mg dry tissue wt/h, respectively) and the release was increased 2.2 2.9-fold by adding 10(-4)-10(-6)M of norepinephrine to the medium. Binding assays revealed the existence of high affinity binding of 3H-prostaglandin E2 (3H-PGE2) and 3H-prostaglandin F1 alpha (3H-PGF2 alpha) in low speed supernatants of pineal homogenates. Binding was increased by increasing Ca++ concentration in medium up to 2 mM, was heat labile and was depressed following incubation with trypsin. In subcellular fractionation studies maximal 3H-PG binding was found in the 27000 x g pellet. Scatchard analysis of 3H-PGE2 binding revealed the presence of a single population of binding sites with a Kd= 1.2 nM and a binding site concentration of 1-2 pmoles/g protein. A single population of binding sites for 3H-PGF2 alpha was also detected with a Kd= 1.7 nM and a similar binding site concentration. Non-radioactive PGE1 and PGE2 were almost equally effective to compete for 3H-PGE1 binding sites (ED50= 5 and 2 nM, respectively). Unlabeled PGF1 was relatively ineffective to compete for 3H-PGE2 binding (ED50 greater than 1000 nM) but displaced effectively 3H-PGF2 alpha binding (ED20=1.2 nM).     

A new method for the quantification of superoxide dismutase mimics with an allopurinol–xanthine oxidase–lucigenin enhanced system

Allopurinol is a prodrug converted to oxypurinol by xanthine oxidase, a process followed by an efficient enzyme inhibition. Using a lucigenin-enhanced chemiluminescence method, we found that, under alkaline conditions, superoxide radicals are produced in large amounts in the first step of the interaction between the enzyme and the inhibitor. A comparison between lucigenin and cytochrome c as final detectors revealed that only the chemiluminescence technique is able to detect the superoxide anions from allopurinol oxidation. The allopurinol–xanthine oxidase–lucigenin system can be used for the quantification of various free-radical scavengers, in particular superoxide dismutase mimics. Three manganese compounds from different structural classes [manganese(II) chloride, manganese N,N′-bis(salicylidiene)ethylenediamine chloride, and manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin] were compared at five concentrations (0.01, 0.1, 1, 10, and 100 μM). The method is fast, 16 times more sensitive than the cytochrome c assay at pH 10.1 and could be used for in vivo investigations avoiding the lucigenin redox cycle. If the concentrations of the reagents are increased and Tween 20 is added, the method is also operative at pH 7.4.     

A DNA HIV-1 vaccine based on a fusion gene expressing non-structural and structural genes of consensus sequence of the A-C subtypes and the ancestor sequence of the F-H subtypes. Preclinical and clinical studies

A potent DNA vaccine against HIV, combining a vector that takes advantage of the segregation and compartmentalization effect of bovine papilloma virus E2 protein with MultiHIV insert, expressing a fusion gene coding for the non-structural and structural proteins was developed and tested for immunogenicity in mice and humans.     

On the presence of 3H-GABA uptake mechanism in bovine spermatozoa

In the present study, existence of (3)H-GABA uptake mechanism in bovine spermatozoa and the modulation of (3)H-GABA transport by GABA itself were evaluated. The hypothesis was tyrosine phosphorylation affects transporter (GAT) function. (3)H-GABA uptake assays were performed on bovine spermatozoa and it resulted to be temperature- and time-dependent and K(m) was 1.48muM. Uptake was inhibited by the metabolic inhibitor ouabain and different blockers of GAT-1 (beta-alanine, l-DABA, nipecotic acid, tiagabine). Extracellular GABA up-regulated GABA transport, while the addition of SKF89976A, a high affinity inhibitor of the rat brain GABA transporter, reduced GABA uptake. Tyrosine phosphorylation affects transporter function since genistein, a broad-spectrum tyrosine kinase inhibitor, decreased (3)H-GABA uptake. Reduction in uptake did not occur in the presence of daidzein, an inactive genistein analogue. Furthermore, the genistein-mediated reduction in transport could be prevented by the tyrosine phosphatase inhibitor pervanadate. The action of these drugs on GABA transport is likely mediated through the GABA transporter GAT-1 since SKF89976A blocked a majority of GABA uptake. Wash-out experiments indicated that the genistein effect was reversible. When the experiments were conducted using "in vitro" capacitated spermatozoa there was no detectable uptake. Present results demonstrate that the carrier-mediated GABA uptake system in bovine spermatozoa modulates its function in response to extracellular GABA, that changes in lipid distribution and membrane composition which occur during capacitation eliminates GABA uptake and suggest the involvement of tyrosine phosphorylation in GABA transport.     

Polymorphisms in the neural nicotinic acetylcholine receptor α4 subunit (CHRNA4) are associated with ADHD in a genetic isolate

The neural nicotinic acetylcholine receptor α4 subunit (CHRNA4), at 20q13.2-q13.3, is an important candidate gene for conferring susceptibility to attention deficit/hyperactivity disorder (ADHD). Several studies have already looked for association/linkage between ADHD and CHRNA4 in different populations. We used the Pedigree Disequilibrium Test to search for evidence of association between ADHD and six SNP marker loci in families from the isolated Paisa population. We found that the T allele of SNP rs6090384 exhibits a deficit of transmission in unaffected individuals (OR = 5.43, IC 1.54-19.13) (global P value = 0.014). We also found significant association and linkage to extended haplotypes rs2273502-rs6090384 (combination of variants C-T, respectively) (P = 0.02) and rs6090384-rs6090387 (P = 0.04) (combination of variants T-G, respectively). SNP rs6090384, variant T, has also been reported to be associated with inattention in a previous study. This makes ours the ninth study to examine the association of CHRNA4 with ADHD and the seventh one to find evidence for association in a population with a different ethnicity.