Clinical efficacy of a stable prostacyclin analog, iloprost, in diabetic neuropathy

Iloprost, a stable prostacyclin analog, was evaluated clinically for its ability to ameliorate the symptoms of peripheral neuropathy associated with diabetes. In an open, nonrandomized trial, 13 diabetic patients with neuropathy but without proliferative retinopathy received an intravenous infusion of iloprost at a dose of 10 μg, at a rate of 0.1 μg/kg/h, twice daily for two weeks.The administration of Iloprost relieved the majority of such subjective symptoms as pain, numbness or sensation of cold and to a lesser extent, such autonomic symptoms as dizziness. In contrast, there was little evidence of objective improvement, e.g., in motor nerve conduction velocity. Iloprost treatment significantly inhibited the platelet aggregation rate stimulated by collagen . In the one patient tested, thermography revealed an increase in skin temperature by more than 2°C.Side effects associated with Iloprost included headache (3 patients) or aggravation of pain in the extremities (2 patients) and could be ameliorated by slowing the infusion rate or by discontinuing the drug (one patient). Iloprost appears to be safe and effective for relieving the symptoms of diabetic neuropathy. Our results provide the rationale for a double-blind, clinical trial in larger populations of diabetics with peripheral neuropathy.     

The experimental pathogenicity in Swiss mice of various strains of Nocardia isolated in soil of Tucumán (Argentina)

The isolation of Nocardia pathogenic for man in soil of Tucumán revealed the convenience of undertaking a study of their virulence, to evaluate any potential danger it may imply.Thirty three strains were studied: 28 N. brasiliensis, 3 N. asteroides and 2 N. caviae by inoculating albino Swiss mice into the footpads and intraperitoneally.The results obtained revealed the existence of Nocardia of variable aggresiveness in the soil of Tucumán.     

Intermolecular nuclear Overhauser effect and atomic pair potential approaches to wheat germ agglutinin-sugar binding

The detailed binding mechanism of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) was investigated using intermolecular 1H-1H nuclear Overhauser effect (NOE) and atomic pair potential (APP) calculations. Negative NOE was observed on the 1H spectrum of 1-O-methyl derivative of GlcNAc in a solution containing WGA, when the aromatic region of the WGA spectrum was irradiated. Analyses of the time dependence of NOE revealed that H2 and the N-acetyl methyl protons of the sugar are in close proximity to the aromatic protons of WGA in the bound state. This was confirmed and further elucidated by the APP calculations. According to the calculation, the major binding force comes from a hydrogen-bonding between C3-OH of sugar and an acidic residue present in each of the two binding sites of WGA: Glu115 in site 1 and Asp29 in site 2. The binding is further assisted by the N-acetyl group which interacts with a few more polar amino acid residues in the binding sites. The optimized binding mode suggested by the APP calculations supports the NMR results in that H2 and a part of the N-acetyl methyl protons are within 4.5 A distance from protons of both Tyr64 and Tyr73 in site 1 and of Tyr159 in site 2.     

Transport of physiologically active salmon gonadotropin into the circulation in goldfish,following oral administration of salmon pituitary extract

Summary Salmon pituitary gland extract was orally administered to goldfish. The resulting transport of gonadotropin (GtH) into the blood plasma and its biological activities were investigated.Plasma levels of salmon GtH assayed by radio-immunoassay increased acutely and reached maximum levels as early as 6 h after oral administration, when administered with phosphate buffer (PB) or 0.2% polyacrylic acid (PAA) gel bases. The sharp rise in salmon GtH was not observed when the pituitary extract was administered with 1% PAA. The changes occurred more slowly, and the plasma levels of GtH were lower than those of PB or 0.2% PAA groups. Plasma levels of goldfish GtH did not increase.The absorbed salmon GtH was found to retain its biological activities. Plasma levels of testosterone increased in proportion with GtH absorption, except in the 1% PAA group. Elevated plasma 17,20-dihydroxy-4-pregnen-3-one levels were also observed in the PB group. Milt production increased within the first 24 h after the administration of pituitary extract.When the pituitary extract was administered in 1% PAA, the period in time in which the absorbed GtH remained elevated was noticeably longer than when given with 0.2% PAA or PB.Abbreviations BSA bovine serum albumin - GtH gonadotropic hormone - PAA polyacrylic acid - 17,20-diOH P 17,20-dihydroxy-4-pregnen-3-one - PB phosphate buffer - RIA radioimmunoassay - ANOVA analysis of variance - SEM standard error of mean     

Expression of DNA-dependent protein kinase in human granulocytes

Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNAPK in PMN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration. In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.     

Effect of IL-1alpha on the expression of cartilage matrix proteins in human chondrosarcoma cell line OUMS-27

We examined the effect of the inflammatory mediator interleukin-1alpha (IL-1alpha) on cell proliferation, alkaline phosphatase (ALPase) activity, and the expressions of cartilage matrix proteins, bone morphogenetic protein-2 (BMP-2), and BMP-2 receptors in human chondrosarcoma cell line OUMS-27 (chondrocytes). The cells were cultured with Dulbecco's modified Eagle's medium containing 15% fetal bovine serum with 0, 1, 10, or 100 units/ml of IL-1alpha for up to 14 days. The expressions of cartilage matrix proteins, BMP-2, and BMP-2 receptors were estimated by determining mRNA levels using semiquantitative or real-time PCR and/or by determining protein levels using Enzyme-linked immunosorbent assay. Cell proliferation was decreased after 5 days in culture with IL-1alpha. The ALPase activity was decreased significantly in the presence of IL-1alpha until day 10 of culture. The expression of type II collagen was significantly decreased after 7 days in culture with IL-1alpha. The expressions of aggrecan and link protein were significantly decreased through day 14 of culture with IL-1alpha. The expression of BMP-2 was increased at days 3, 7, and 14 of culture with IL-1alpha, while the expression of type II receptor for BMP-2 was significantly decreased in the samples. These results suggest that IL-1alpha suppresses the expression of cartilage matrix proteins through a suppression of the autocrine action of BMP-2, brought about by the decrease in BMP-2 receptor expression in chondrocytes.     

Two new diterpenoids from Plectranthus species

Two new diterpenoids, ent-7α-acetoxy-15-beyeren-18-oic acid and (13S,15S)-6β,7α,12α,19-tetrahydroxy-13β,16-cyclo-8-abietene-11,14-dione, have been isolated from Plectranthus saccatus and Plectranthus porcatus, respectively, and their structures were established by 1D and 2D NMR spectroscopic studies. The new diterpenes showed no activity against Gram-negative bacteria and Candida albicans (yeast strain). Among Gram-positive bacteria, the lower MIC value was 62.50 μg/ml for the abietane derivative against Staphylococcus aureus ATCC 6538.     

Identification of a disulfide bridge essential for structure and function of the voltage-gated Ca(2+) channel α(2)δ-1 auxiliary subunit

Voltage-gated calcium (Ca(V)) channels are transmembrane proteins that form Ca(2+)-selective pores gated by depolarization and are essential regulators of the intracellular Ca(2+) concentration. By providing a pathway for rapid Ca(2+) influx, Ca(V) channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. Ca(V) channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α(1) subunit, which typically contains associated β and α(2)δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α(2)δ-1 is post-translationally cleaved into an extracellular α(2) polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α(2)δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α(2)) and Cys1047 (in the extracellular domain of δ) prevented the association of the α(2) and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α(2)δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (Ca(V)2.2α(1)/β(3)) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α(2)δ-1 function.     

Induction of methionine-sulfoxide reductases protects neurons from amyloid β-protein insults in vitro and in vivo

Self-assembly of amyloid β-protein (Aβ) into toxic oligomers and fibrillar polymers is believed to cause Alzheimer's disease (AD). In the AD brain, a high percentage of Aβ contains Met-sulfoxide at position 35, though the role this modification plays in AD is not clear. Oxidation of Met(35) to sulfoxide has been reported to decrease the extent of Aβ assembly and neurotoxicity, whereas surprisingly, oxidation of Met(35) to sulfone yields a toxicity similar to that of unoxidized Aβ. We hypothesized that the lower toxicity of Aβ-sulfoxide might result not only from structural alteration of the C-terminal region but also from activation of methionine-sulfoxide reductase (Msr), an important component of the cellular antioxidant system. Supporting this hypothesis, we found that the low toxicity of Aβ-sulfoxide correlated with induction of Msr activity. In agreement with these observations, in MsrA(-/-) mice the difference in toxicity between native Aβ and Aβ-sulfoxide was essentially eliminated. Subsequently, we found that treatment with N-acetyl-Met-sulfoxide could induce Msr activity and protect neuronal cells from Aβ toxicity. In addition, we measured Msr activity in a double-transgenic mouse model of AD and found that it was increased significantly relative to that of nontransgenic mice. Immunization with a novel Met-sulfoxide-rich antigen for 6 months led to antibody production, decreased Msr activity, and lowered hippocampal plaque burden. The data suggest an important neuroprotective role for the Msr system in the AD brain, which may lead to development of new therapeutic approaches for AD.