Point mutation in precore region of hepatitis B virus: Sequential changes from 'wild' to 'mutant'

One point mutation to make a stop codon in the precore (pre-C) region of the hepatitis B virus DNA in anti-HBe-positive patients has been reported recently. This mutation disturbs the formation of the pre-C protein that is processed to make HBeAg. The relationship between the point mutation and HBe antigen antibody status was investigated in B-viral liver diseases. The pre-C region was amplified by a polymerase chain reaction (PCR) method and the nucleotide sequences were determined by a direct sequencing method. In seven cases who were persistently HBeAg-positive, the wild type (no mutation in pre-C region) was detected in all. In 20 cases who were anti-HBeAg-positive at diagnosis, the mutant type (point mutation at nucleotide 1896 in pre-C region, which makes a stop codon) was detected in 16 cases and the wild type in two cases. In HBe seroconversion (SC) cases, the types of virus were investigated in serial blood samples. No mutant type was detected in initial sera during the HBeAg-positive period. In two ‘natural’ SC cases, the mutant type appeared before anti-HBe formation. However, in three anti-viral ‘drug-induced’ SC cases, the mutant type appeared after the formation of anti-HBe. In two ‘reversed’ seroconversion cases only the wild type was detected throughout the follow-up period. These data suggest that the appearance of a pre-C mutant may help to predict seroconversion from HBeAg to anti-HBe and may help distinguish ‘natural’ and ‘drug-induced’ seroconversion of HBeAg.     

A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving

The epithelial Na+ channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial Na+ transport, mechanotransduction, and neurotransmission. The role of ENaC in Na+ homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC beta and gamma subunits causing hypertension. The function of ENaC in Na+ reabsorption depends critically on its ability to discriminate between Na+ and other ions like K+ or Ca2+. ENaC is virtually impermeant to K+ ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC alpha subunit (alphaS589), which when mutated allows larger ions such as K+, Rb+, Cs+, and divalent cations to pass through the channel. The relative ion permeability of each of the alphaS589 mutants is related inversely to the ionic radius of the permeant ion, indicating that alphaS589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate Na+ and Li+ ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration.     

Clinical features,radiological characteristics and offloading modalities in stage 0 Acute Charcot's neuroarthropathy - A single centre experience from South India

AimsStage 0 Acute Charcot's Neuroarthropathy (ACN)in Type 2 Diabetes patients is a challenging diagnosis with subtle clinical features and normal appearing plain radiographs of the affected foot. Delay in diagnosis can lead to progression of disease and irreversible deformities. There is a paucity of data on Stage 0 ACN from India. The aim of this study was to assess clinical and radiological characteristics and treatment outcomes in Indian Type 2 Diabetes patients with Stage 0 ACN.Materials and methodsA comparative, case-control study was carried out amongst patients attending the Integrated Diabetes Foot Clinic at a tertiary care South Indian hospital. During the 3-year study period, a total of 1811 patients with Type 2 Diabetes Mellitus were screened. Of these, n = 10 patients with stage 0 ACN Charcot's arthropathy were identified based on clinical features and MRI imaging of the foot for confirmation of diagnosis. These were compared with an age and duration of diabetes-matched group of n = 50 patients without ACN as controls.ResultsOur study identified 10 patients (0.5%) with Stage 0 Acute charcot neuroarthropathy (ACN) in the study population. Those with ACN had higher BMI, poorer glycaemic control and greater degree of peripheral neuropathy (p < 0.05). Clinically relative lack of pain and infrared thermometric temperature difference >2 °C in the affected foot were the most significant findings, while MRI foot was useful in early detection of active and severe stage 0 disease. Total contact cast was the preferred initial offloading modality, with delay in initiating complete immobilization leading to worse outcomes.ConclusionsThis is the first study to highlight the characteristic features of Stage 0 ACN in Indian Type 2 Diabetes patients. Thorough clinical evaluation, infrared thermometry and radiological findigs on MRI foot leads to early disease detection. Complete offloading, preferably with total contact casts can prevent disease progression and chronic deformities.     

Protein nitration is mediated by heme and free metals through Fenton-type chemistry: an alternative to the NO/O2- reaction

The chemical origins of nitrated tyrosine residues (NT) formed in proteins during a variety of pathophysiological conditions remain controversial. Although numerous studies have concluded that NT is a signature for peroxynitrite (ONOO(-)) formation, other works suggest the primary involvement of peroxidases. Because metal homeostasis is often disrupted in conditions bearing NT, the role of metals as catalysts for protein nitration was examined. Cogeneration of nitric oxide (NO) and superoxide (O(2)(-)), from spermine/NO (2.7 microM/min) and xanthine oxidase (1-28 microM O(2)(-)/min), respectively, resulted in protein nitration only when these species were produced at approximately equivalent rates. Addition of ferriprotoporphyrin IX (hemin) to this system increased nitration over a broad range of O(2)(-) concentrations with respect to NO. Nitration in the presence of superoxide dismutase but not catalase suggested that ONOO(-) might not be obligatory to this process. Hemin-mediated NT formation required only the presence of NO(2)(-) and H(2)O(2), which are stable end-products of NO and O(2)(-) degradation. Ferrous, ferric, and cupric ions were also effective catalysts, indicating that nitration is mediated by species capable of Fenton-type chemistry. Although ONOO(-) can nitrate proteins, there are severe spatial and temporal constraints on this reaction. In contrast, accumulation of metals and NO(2)(-) subsequent to NO synthase activity can result in far less discriminate nitration in the presence of an H(2)O(2) source. Metal catalyzed nitration may account for the observed specificity of protein nitration seen under pathological conditions, suggesting a major role for translocated metals and the labilization of heme in NT formation.     

Serum cholesterol changes from 1983-1984 to 1993-1994 in the People's Republic of China

BACKGROUND AND AIM: Increasing cardiovascular disease (CVD) mortality in the People's Republic of China (PRC) led to the 1981 establishment of the PRC-USA Study of Cardiovascular and Cardiopulmonary Epidemiology which, among other objectives, is concerned with the correlates of CVD morbidity and mortality in Chinese populations among other objectives. This report describes changes in total cholesterol (TC) levels in four PRC populations from 1983 to 1993 and identifies factors related to the changes. METHODS AND RESULTS: Population screenings carried out in 1983-1984, 1987-1988 and 1993-1994 involved the collection of demographic data, specimens (including blood), medical history and physical examination data. The data from cohort and independent samples were used to assess TC changes in urban and rural men and women over the decade, with and without adjustment for age and body mass index (BMI) changes. For Guangzhou men and women, the cohort analyses (aged 35-54 at baseline) showed increases in TC of 10-20 mg/dL after adjustment for age and changes in BMI; the independent sample analyses (aged 35-44) also showed higher average TC levels in 1993-1994 than in 1983-1984. For the Beijing cohorts, the results showed decreases in TC during the decade in men, an increase in TC in urban women and no change in rural women; the independent sample analyses indicated declines in TC for Beijing men and women. Possible reasons for the Guangzhou TC increases are economic growth, and dietary and BMI changes. The mean age-adjusted BMI significantly increased (5-10%) over the 10-year period in all of the studied groups. CONCLUSIONS: TC increased 10-20 mg/dL in Guangzhou men and women, probably as a result of socioeconomic development during the decade. The inconsistent patterns of TC changes in Beijing require further study.     

Double jeopardy from a single translocation: deletions of the derivative chromosome 9 in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is characterized by formation of a BCR-ABL fusion gene, usually as a consequence of the Philadelphia (Ph) translocation between chromosomes 9 and 22. Recently the development of new fluorescence in-situ hybridization (FISH) techniques has allowed identification of unexpected deletions of the reciprocal translocation product, the derivative chromosome 9, in 10% to 15% of patients with CML. These deletions are large, span the translocation breakpoint, and occur at the same time as the Ph translocation. Such deletions therefore give rise to previously unsuspected molecular heterogeneity from the very beginning of this disease, and there is mounting evidence for similar deletions associated with other translocations. Several studies have demonstrated that CML patients who carry derivative chromosome 9 deletions exhibit a more rapid progression to blast crisis and a shorter survival. Deletion status is independent of, and more powerful than, the Sokal and Hasford/European prognostic scoring systems. The poor prognosis associated with deletions is seen in patients treated with hydroxyurea or interferon, and preliminary evidence suggests that patients with deletions may also have a worse outcome than nondeleted patients following stem cell transplantation or treatment with imatinib. Poor outcome cannot be attributed to loss of the reciprocal ABL-BCR fusion gene expression alone, and is likely to reflect loss of one or more critical genes within the deleted region. The molecular heterogeneity associated with the Philadelphia translocation provides a new paradigm with potential relevance to all malignancies associated with reciprocal chromosomal translocations and/or fusion gene formation.     

A therapy-related point mutation changes the HLA restriction of an HIV-1 Pol epitope from A2 to B57 and enhances its recognition

In this study, we investigated CD8 T-cell recognition of wild-type HIV-1 Pol (RT 210-220) peptide LRWGFTTPDKK and of several variants incorporating common antiretroviral therapy-associated mutations (L210W, T215Y, Y215C). LRWGFTTPDKK was weakly and infrequently recognized in the context of HLA-A2. However, the 215C mutation created a strong, commonly recognized HLA-B57-restricted epitope. An antiretroviral therapy-associated mutation thus alters the HLA restriction of a weak Pol epitope, potentially enhancing CD8 T-cell recognition of HIV.     

The subcellular particulate NADPH-dependent O2.(-)-generating oxidase from human blood monocytes: comparison to the neutrophil system

Highly purified preparations of normal human monocytes obtained from peripheral blood were shown to contain a subcellular particulate O2.(-)- generating oxidase system. This O2.(-)-generating activity was present in particulate preparations from monocytes that had been previously stimulated with phorbol myristate acetate but was low or absent in control preparations from unstimulated monocytes or stimulated monocytes from a patient with chronic granulomatous disease. In the stimulated preparations from normal monocytes, O2.(-)-generation was linearly proportional to cell protein concentration, insensitive to inhibition by azide, and dependent on NADPH as substrate. These characteristics are similar to the O2.(-)-generating oxidase system from human neutrophils. A significant difference in the apparent Km for NADPH was shown between preparations from stimulated monocytes and neutrophils (monocyte 83 +/- 16 microM, neutrophil 31 +/- 5 microM, mean +/- SE). Additionally, affinity of the stimulated monocyte particulate preparation for NADH was unmeasurably low.     

From the Cover: A bacterial symbiont is converted from an inedible producer of beneficial molecules into food by a single mutation in the gacA gene

Stable multipartite mutualistic associations require that all partners benefit. We show that a single mutational step is sufficient to turn a symbiotic bacterium from an inedible but host-beneficial secondary metabolite producer into a host food source. The bacteria’s host is a “farmer” clone of the social amoeba Dictyostelium discoideum that carries and disperses bacteria during its spore stage. Associated with the farmer are two strains of Pseudomonas fluorescens, only one of which serves as a food source. The other strain produces diffusible small molecules: pyrrolnitrin, a known antifungal agent, and a chromene that potently enhances the farmer’s spore production and depresses a nonfarmer’s spore production. Genome sequence and phylogenetic analyses identify a derived point mutation in the food strain that generates a premature stop codon in a global activator (gacA), encoding the response regulator of a two-component regulatory system. Generation of a knockout mutant of this regulatory gene in the nonfood bacterial strain altered its secondary metabolite profile to match that of the food strain, and also, independently, converted it into a food source. These results suggest that a single mutation in an inedible ancestral strain that served a protective role converted it to a “domesticated” food source.     

Identifying the causes of the changes in the prevalence patterns of diabetes in older U.S. adults: A new trend partitioning approach

Aims

To identify how efforts to control the diabetes epidemic and the resulting changes in diabetes mellitus, type II (T2D) incidence and survival have affected the time-trend of T2D prevalence.

Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation.

Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as the terminal monooxygenase in the d-camphor monooxygenase system), in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction. The mutant enzymes were expressed in Escherichia coli and were purified by a conventional method. All the mutant enzymes in the presence of d-camphor exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms. In a reconstituted system with putidaredoxin and its reductase, the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to that of the wild-type enzyme (1330 per min per heme), whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme also exhibited an oxidase activity to yield H2O2 accompanied by a relative decrease in the monooxygenase activity. On the other hand, the serine enzyme exhibited essentially the same monooxygenase activity as that of the wild-type enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was produced by the substitution of an amino acid without a hydroxyl group. When binding of O2 to the ferrous forms was examined, the alanine and valine enzymes formed instantaneously an oxygenated form, which slowly decomposed to the ferric form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, respectively. Since these rates were too slow to account for the overall rates of O2 consumption, the formation of H2O2 was considered to proceed not by way of this route but through the decomposition of a peroxide complex formed by reduction of the oxygenated form by reduced putidaredoxin. Based on these findings, a possible mechanism for oxygen activation in this monooxygenase reaction has been discussed.     

Second-site revertants of an arginine-210 to lysine mutation in the a subunit of the F0F1-ATPase from Escherichia coli: implications for structure.

Arg-210 of the a subunit of the Escherichia coli F0F1-ATPase has been proposed previously as a component of the proton pore. A mutant in which lysine was substituted for Arg-210 was generated and was found to be unable to translocate protons. A plasmid carrying this mutation, along with wild-type genes encoding the c and b subunits, was unusual in that it failed to complement a chromosomal c-subunit mutation on succinate minimal medium. Three revertants on succinate minimal medium contained plasmids that showed complementation with chromosomal c-subunit but not with a-subunit mutations. One of these had a deletion in the a subunit. The other two were point mutations, resulting in the substitution of aspartic acid by Gly-53 and of arginine for Leu-211. The Gly-53 to aspartic acid change implied that Gly-53 and Arg-210 are normally in close proximity. To test this idea further, a series of mutants in which aspartic acid was placed in helix I at positions ranging from 42 to 57 was generated. Full complementation was regained only when the aspartic acid residue was present on the same side of a putative helix as Gly-53 over a span of three turns of the alpha-helix. These results and others suggest modifications of a previously proposed model for the transmembrane helices of the F0 portion of the F0F1-ATPase. The implications of these modifications for the mechanism of proton translocation are discussed.