A procollagen C-proteinase inhibitor diminishes collagen and lysyl oxidase processing but not collagen cross-linking in osteoblastic cultures

The deposition of insoluble functional collagen occurs following extracellular proteolytic processing of procollagens by procollagen N- and C-proteinases, fibril formation, and lysyl oxidase dependent cross-linking. Procollagen C-proteinases in addition process and activate lysyl oxidase. The present study evaluates a possible role for procollagen C-proteinases in controlling different aspects of collagen deposition in vitro. Studies determine whether inhibition of procollagen C-proteinase activity with a specific BMP-1 inhibitor results in perturbations in lysyl oxidase activation, and in collagen processing, deposition, and cross-linking in phenotypically normal cultured murine MC3T3-E1 cells. Data show that BMP-1 Inhibitor dose dependently inhibits lysyl oxidase activation by up to 50% in undifferentiated proliferating cells. In differentiating cultures, BMP-1 inhibitor decreased collagen processing but did not inhibit the accumulation of mature collagen cross-links. Finally, electron microscopy studies show that collagen fibril diameter increased. Thus, inhibition of procollagen C-proteinases results in perturbed collagen deposition primarily via decreased collagen processing.     

Genetic analysis reveals a role for the C terminus of the Saccharomyces cerevisiae GTPase Snu114 during spliceosome activation

Snu114 is the only GTPase required for mRNA splicing. As a homolog of elongation factor G, it contains three domains (III-V) predicted to undergo a large rearrangement following GTP hydrolysis. To assess the functional importance of the domains of Snu114, we used random mutagenesis to create conditionally lethal alleles. We identified three main classes: (1) mutations that are predicted to affect GTP binding and hydrolysis, (2) mutations that are clustered in 10- to 20-amino-acid stretches in each of domains III-V, and (3) mutations that result in deletion of up to 70 amino acids from the C terminus. Representative mutations from each of these classes blocked the first step of splicing in vivo and in vitro. The growth defects caused by most alleles were synthetically exacerbated by mutations in PRP8, a U5 snRNP protein that physically interacts with Snu114, as well as in genes involved in snRNP biogenesis, including SAD1 and BRR1. The allele snu114-60, which truncates the C terminus, was synthetically lethal with factors required for activation of the spliceosome, including the DExD/H-box ATPases BRR2 and PRP28. We propose that GTP hydrolysis results in a rearrangement between Prp8 and the C terminus of Snu114 that leads to release of U1 and U4, thus activating the spliceosome for catalysis.     

Low doses and high doses of heparin have different effects on osteoblast-like Saos-2 cells in vitro

Long-term treatment with heparin has been associated with an increased risk of osteoporosis. Given the importance of heparan sulfate proteoglycans for bone metabolism, it can be anticipated that heparin due to its structural similarity with heparan sulfate chains somehow interferes with the biological activities of these cell surface- and extracellular matrix-associated molecules. Initially in order to study the effect(s) of heparin on osteoblasts that possibly contribute to the development of heparin-induced osteoporosis, we treated osteoblast-like Saos-2 cells in monolayer culture for different periods of time with different concentrations of heparin. None of the heparin concentrations tested led to an inhibition of osteoblast proliferation during the early proliferative phase. After longer incubation times, however, cultures treated with higher concentrations of heparin (>/=5 microg/ml) exhibited a reduction in cell number as well as an inhibition of matrix deposition and mineralization. These effects could not be observed with lower heparin concentrations. On the contrary, low concentrations of heparin (5-500 ng/ml) even promoted matrix deposition and its subsequent mineralization. Apparently, heparin has a biphasic effect on osteoblast-like Saos-2 cells, being inhibitory at high concentrations but stimulatory at low concentrations. These results imply that heparin at concentrations well below those used for antithrombotic therapy might eventually turn out to be beneficial for bone formation.     

HIV drug resistance and optimization of antiviral treatment in resource-poor countries

HIV drug resistance has been associated with treatment failure in Western countries but the lessons learned can be useful in optimization of highly active antiretroviral treatment (HAART) in resource-poor settings. There is a need to improve access to HAART in such regions, but appropriate strategies must be rapidly implemented, such as adapted programs to facilitate adherence to therapy, rational use of genotypic drug resistance monitoring in specific situations, and use of alternative treatment regimens. The implications of HIV genetic diversity must also be considered in management of drug resistance.     

Identification of mutation of the phenylalanine hydroxylase gene using an automated DNA sequencer

Mutations were studied in phenylalanine hydroxylase gene of phenylketonuria patients from Kemerovo oblast and Altaiskii krai (15 and 2 families, respectively). The following mutations were identified in exons of this gene: R408W, R261Q, R243Q, Y414C, Y386C, P281L, Y168H, R68S (lead to amino acid substitutions), R243X (leads to stop codon formation), and three splice site mutations (IVS12nt 1g-->a, IVS2nt-13t-->g, IVS7nt 1g-->a).     

Toxicity and in vitro metabolism of t-permethrin in eastern subterranean termite (Isoptera: Rhinotermitidae)

Toxicity and metabolism of t-permethrin were evaluated in two colonies (UF and ARS) of the eastern subterranean termite, Reticulitermes flavipes (Kollar), collected in Gainesville, FL. The UF colony (LC50 = 1.86 micrograms per vial) was approximately twofold more tolerant of t-permethrin than the ARS colony (LC50 = 0.89 microgram per vial) at the LC50. The synergists piperonyl butoxide and S,S,S-tributylphosphorotrithioate increased t-permethrin toxicity four- and threefold (at the LC50) in the UF and ARS colonies, respectively. Despite these differences in t-permethrin susceptibility, microsomal oxidase activities toward surrogate substrate (aldrin epoxidase, and methoxyresorufin O-demethylase), cytochrome P450 content, and microsomal esterase activity toward alpha-naphthyl acetate did not differ significantly between the colonies. Moreover, no significant differences in qualitative and quantitative metabolism of [14C]t-permethrin were observed between the UF and ARS colonies for three enzyme sources (microsomal oxidase, microsomal esterase, and cytosolic esterase). Based on in vitro metabolism assays, the major detoxification route of t-permethrin in the UF and ARS termite colonies appears to be hydrolysis catalyzed by microsomal esterases.