Electrophoretic pseudopolymorphism: the role of modifications illustrated by proteolysis

The term "pseudopolymorphism" refers to a situation, where there is no simple correspondence between genotype and phenotype: a single genotype may be moulded into several phenotypes. It is known that broad substrate specificity of enzymes may be one of the causes for pseudopolymorphism. This article deals with the other cause for this phenomenon--a consequence of post-translation modifications, such as limited proteolysis. Variability of some enzymes of grass carp Ctenopharyngodon idella Val. (Pisces, Cyprinidae) was studied by gel electrophoresis. It was found that variability of isozyme patterns of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-PDH), malic enzyme (ME) and esterase (EST) is connected with the differences in protease activity of grass carp liver homogenates. The fish isozyme patterns of high (and, partially, intermediate) proteinase activity had some anomalies: displacement of fractions, one or several additional fractions, decreased activity of single fractions or the whole spectrum. In some cases, this variability looked like a classical polymorphic system specified by two alleles of one locus. The effect of enzymes' and proteins' modifications on electrophoretical pseudopolymorphism is discussed.     

Kinetics of ligand binding to a cluster of membrane-associated receptors

The process of ligand binding to a cluster of membrane-associated receptors is examined theoretically. The theoretical model proposed involves the diffusion of ligands from the solution to the disc-like cluster of receptors on the surface of the spherical cell. When the ligand hits the internal part of the disc-like cluster, it begins to move laterally until it leaves the disc through its outer surface or is bound by one of the receptors inside the disc. If the ligand leaves the cluster, it returns to the solution and hits the disc again after a certain period, etc. According to our model the transition from a diffusion-limited to a reaction-limited process of binding is determined by the dimensionless parameter Dt _c/a ², where D is the lateral diffusion coefficient,t _c is the characteristic time of reaction, anda is the radius of the disc-like cluster. The forward rate constantk _f turns out to be a function of . Comparing the results of our calculations ofk _f with some experimental data we found that agreement is achieved at high , i.e. the process of ligand binding by clustered receptors is predominantly reaction-limited.     

Asymmetry of syringomycin E channel studied by polymer partitioning

To probe the size of the ion channel formed by Pseudomonas syringae lipodepsipeptide syringomycin E, we use the partial blockage of ion current by penetrating poly(ethylene glycol)s. Earlier experiments with symmetric application of these polymers yielded a radius estimate of approximately 1 nm. Now, motivated by the asymmetric non-ohmic current-voltage curves reported for this channel, we explore its structural asymmetry. We gauge this asymmetry by studying the channel conductance after one-sided addition of differently sized poly(ethylene glycol)s. We find that small polymers added to the cis-side of the membrane (the side of lipodepsipeptide addition) reduce channel conductance much less than do the same polymers added to the trans-side. We interpret our results to suggest that the water-filled pore of the channel is conical with cis- and trans-radii differing by a factor of 2-3 and that the smaller cis-radius is in the 0.25-0.35 nm range. In symmetric, two-sided addition, polymers entering the pore from the larger opening dominate blockage.     

Kinetics of opening and closure of syringomycin E channels formed in lipid bilayers

A cyclic lipodepsipeptide, syringomycin E (SME), incorporated into planar lipid membranes forms two types of channels ("small" and "large") different in their conductance by approximately a factor of six (Biophys. J. 74:2918-2925 (1998)). We analysed the dynamics of the SME-induced transmembrane current under voltage-clamp conditions to clarify the mechanisms of formation of these channels. The voltage-dependent opening/closure of SME channels in lipid bilayers are interpreted in terms of transitions between three types of clusters including 6-7 SME molecules and some lipid molecules. The initial cluster, the precursor of the other two, was in equilibrium with SME monomer molecules at the membrane surface. The other two types of clusters (State 1 and State 2) were formed from the precursor and also during their interconversions (the consecutive-parallel mechanism of transitions). State 1 was a non-conducting state in equilibrium with small channels, which partially determined the ionic conductance of lipid bilayers modified by SME. State 2 corresponded to large SME channels, major contributors to the conductance of a bilayer. The results of the theoretical analysis based on the chemical kinetics concepts were consistent with experimental observations. Such properties of the SME-induced channels as cluster organisation, voltage dependence and the existence of a non-conducting state are all features shared by many ion channels in biological membranes. This makes it possible to use SME channels as a model to study naturally occurring ion channels.     

Survival of civilian and prisoner drug-sensitive, multi- and extensive drug- resistant tuberculosis cohorts prospectively followed in Russia

Objective and Methods

A long-term observational study was conducted in Samara, Russia to assess the survival and risk factors for death of a cohort of non-multidrug resistant tuberculosis (non-MDRTB) and multidrug resistant tuberculosis (MDRTB) civilian and prison patients and a civilian extensive drug-resistant tuberculosis (XDRTB) cohort.

Results

MDRTB and XDRTB rates of 54.8% and 11.1% were identified in the region. Half (50%) of MDRTB patients and the majority of non-MDRTB patients (71%) were still alive at 5 years. Over half (58%) of the patients died within two years of establishing a diagnosis of XDRTB. In the multivariate analysis, retreatment (HR = 1.61, 95%CI 1.04, 2.49) and MDRTB (HR = 1.67, 95%CI 1.17, 2.39) were significantly associated with death within the non-MDR/MDRTB cohort. The effect of age on survival was relatively small (HR = 1.01, 95%CI 1.00, 1.02). No specific factor affected survival of XDRTB patients although median survival time for HIV-infected versus HIV-negative patients from this group was shorter (185 versus 496 days). The majority of MDRTB and XDRTB strains (84% and 92% respectively) strains belonged to the Beijing family. Mutations in the rpoB (codon 531 in 81/92; 88.8%), katG (mutation S315T in 91/92, 98.9%) and inhA genes accounted for most rifampin and isoniazid resistance respectively, mutations in the QRDR region of gyrA for most fluroquinolone resistance (68/92; 73.5%).

Conclusions

Alarmingly high rates of XDRTB exist. Previous TB treatment cycles and MDR were significant risk factors for mortality. XDRTB patients'' survival is short especially for HIV-infected patients. Beijing family strains comprise the majority of drug-resistant strains.