HIV-1 Nef enhances both membrane expression and virion incorporation of Env products. A model for the Nef-dependent increase of HIV-1 infectivity

The expression of human immunodeficiency virus Nef increases the viral infectivity through mechanisms still not fully elucidated. Here we report that wild-type (wt) human immunodeficiency virus, type 1 (HIV-1), particles were neutralized by higher concentrations of either anti-Env glycoprotein (gp) 41 antibodies or recombinant soluble human CD4 compared with Deltanef HIV-1. This appeared to be the result of a Nef-induced increase of virion incorporation of both gp41 (transmembrane (TM)) and surface gp120 Env products likely originating from enhanced steady-state levels of cell membrane-associated Env products. This, in turn, seemed to be the consequence of a reduced retention of the Env precursor. Most interesting, we found that both the Nef-directed increase of Env membrane expression and the Nef-induced enhancement of HIV-1 infectivity relied on the presence of the intracytoplasmic domain of TM, supporting the hypothesis of a functional correlation between these effects. Mutagenesis studies allowed us to establish that the two leucine residues at the TM C terminus, which are part of a sorting motif involved in the control of Env membrane expression, and the 181-210-residue Nef C-terminal region were critically involved in the Nef/Env functional interaction. In conclusion, we propose that Nef increases the infectivity of HIV-1 at least in part by enhancing the amounts of Env products incorporated into virus particles.     

Kinetics and mechanism of the reaction between chromium(III) and 3,4-dihydroxyphenylpropionic (dihydrocaffeic) acid in weak acidic aqueous solutions

The reaction of 3,4-dihydroxyphenylpropionic acid (dihydrocaffeic acid, hydcafH3) with chromium(III) in weak acidic aqueous solutions has been shown to take place through various oxygen-bonded intermediates. The formation of the oxygen-bonded complexes upon substitution of water molecules of the chromium(III) coordination sphere takes place in at least three stages, the first of which has an observed rate constant k1(obs)=k1K0'[hydcafH3]/[H+] where K0' corresponds to the Cr(H2O)6(3+) complex dissociation equilibrium. The second and third stages are ligand concentration independent and are thus attributed to isomerisation and chelation processes. The corresponding activation parameters are DeltaH2(not equal)=78+/-3 kJmol(-1), DeltaS2(not equal)=-49+/-9 JK(-1)mol(-1), DeltaH3(not equal)=60+/-9 kJmol(-1) and DeltaS3(not equal)=-112+/-39 JK(-1)mol(-1). The kinetic results support associative mechanisms and the nature of the electronic spectra a catecholic-type of coordination at the pH and concentration range studied and reported in this paper. The associatively activated substitution processes are accompanied by proton release causing a pH decrease. At lower acid concentration oxidation of the ligand takes place with concomitant high increase in the UV and VIS absorbance.     

Simultaneous optimization of enzyme activity and quaternary structure by directed evolution

Natural evolution has produced efficient enzymes of enormous structural diversity. We imitated this natural process in the laboratory to augment the efficiency of an engineered chorismate mutase with low activity and an unusual hexameric topology. By applying two rounds of DNA shuffling and genetic selection, we obtained a 400-fold more efficient enzyme, containing three non-active-site mutations. Detailed biophysical characterization of the evolved variant suggests that it exists predominantly as a trimer in solution, but is otherwise similarly stable as the parent hexamer. The dramatic structural and functional effects achieved by a small number of seemingly innocuous substitutions highlights the utility of directed evolution for modifying protein-protein interactions to produce novel quaternary states with optimized activities.     

Identification of novel Asian Indian and Japanese mutations causing β-thalassaemia in the Egyptian population

β-thalassaemia is a major health problem in Egypt. It has been estimated that of the 1.5 million live births, 1000 children with β-thalassaemia major are born annually. Although the available treatment has increased the life expectancy of patients, it is still unsatisfactory and represents a significant drain on the country’s resources. National screening and prenatal diagnosis programmes can be provided in Egypt once the spectrum of β-thalassaemia mutations has been identified within the Egyptian population. We have examined 16 DNA samples with 21 β-thalassaemia mutations that remained unidentified in a study of 54 patients reported by Rady and colleagues in 1996. Using the polymerase chain reaction and single strand conformation analysis we identified the following changes: frameshift (FS) codon (CD) 8/9 (+G), 4 FS CD 29 (–G) and 2 novel mutations in exon I (15 CD 22 A-C and 1 FS CD 28 –C). In addition, a silent, probably polymorphic mutation, CD 17 G-A was present in all chromosomes. Received: 19 August 1996 / Revised: 21 September 1996     

Searching for tagged male-sterile mutants of arabidopsis

Although many male-sterile mutants have been identified inArbidopsis thaliana, few of the corresponding genes have been cloned. In order to facilitate cloning of a male sterility gene, 23 of Feldmann's T-DNA-generated, reduced-fertility lines were screened to identify a tagged male-sterile mutation. Malesterile mutants were identified, as well as mutants that were both male and female sterile. Segregation of the kanamycin marker gene in the progeny of 15 of these lines was studied. Forty percent had functional T-DNAs (encoding resistance to kanamycin) inserted at a single locus, the remainder segregating for two or more functional T-DNA inserts. Linkage between T-DNA inserts and mutant phenotype was tested for six lines. In three of these lines, mutations were not linked to a T-DNA insert. In three lines, the mutation segregated with a T-DNA insert.     

Inhibition of carbonic anhydrase isoforms I,II, IX and XII with secondary sulfonamides incorporating benzothiazole scaffolds

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO₂ hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of benzothiazole-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I and hCA II and the transmembrane hCA IX and hCA XII. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting hCA IX and hCA XII over the off-target ones hCA I and hCA II. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.     

pH sensitivity of chlorophyll fluorescence quenching is determined by the detergent/protein ratio and the state of LHCII aggregation

Here we show how the protein environment in terms of detergent concentration/protein aggregation state, affects the sensitivity to pH of isolated, native LHCII, in terms of chlorophyll fluorescence quenching. Three detergent concentrations (200, 20 and 6 μM n-dodecyl β-d-maltoside) have been tested. It was found that at the detergent concentration of 6 μM, low pH quenching of LHCII is close to the physiological response to lumen acidification possessing pK of 5.5. The analysis has been conducted both using arbitrary PAM fluorimetry measurements and chlorophyll fluorescence lifetime component analysis. The second led to the conclusion that the 3.5 ns component lifetime corresponds to an unnatural state of LHCII, induced by the detergent used for solubilising the protein, whilst the 2 ns component is rather the most representative lifetime component of the conformational state of LHCII in the natural thylakoid membrane environment when the non-photochemical quenching (NPQ) was absent. The 2 ns component is related to a pre-aggregated LHCII that makes it more sensitive to pH than the trimeric LHCII with the dominating 3.5 ns lifetime component. The pre-aggregated LHCII displayed both a faster response to protons and a shift in the pK for quenching to higher values, from 4.2 to 4.9. We concluded that environmental factors like lipids, zeaxanthin and PsbS protein that modulate NPQ in vivo could control the state of LHCII aggregation in the dark that makes it more or less sensitive to the lumen acidification. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Fine-scale sampling reveals distinct isolation by distance patterns in chum salmon (Oncorhynchus keta) populations occupying a glacially dynamic environment

Populations with spatially restricted gene flow are characterized by genetic differentiation that may be positively correlated with the geographic distance separating populations, a pattern known as isolation by distance (IBD). Here we examined the fine-scale genetic structure of 66 chum salmon (Oncorhynchus keta) populations spawning in Alaska waterways and explored patterns of IBD using 90 nuclear and 3 mitochondrial single nucleotide polymorphisms. Estimating population structure of chum salmon in Alaska is of increasing concern because of fluctuating census sizes and the uncertain effects of harvest on specific populations. We hypothesized that IBD would be present because chum salmon spawn in coastal rivers that are distributed along a linear array and gene flow is spatially restricted due to homing. Evidence of very weak IBD was found throughout the region (R² = 0.06, p < 0.0001) but the strength of the IBD relationship varied greatly over different spatial scales and geographic regions. Decomposed pairwise regression analyses identified nine outlier populations to regional IBD patterns, suggesting that geographic distance is not the only factor influencing genetic differentiation in the region. Instead, population structure appears to be heavily influenced by glacial history of the region and the presence of a glacial refugium on Kodiak Island.     

GIST suture-line recurrence at a gastrojejunal anastomosis 8 years after gastrectomy: can GIST ever be described as truly benign? A case report

Background

A synovial sarcoma (SS) is an aggressive soft tissue tumor that classically occurs in the extremities near, but rarely within large joints, in young adults. Variable symptoms and clinical manifestations may be encountered and a definite diagnosis should depend on pathological results. This poses certain difficulties in arriving at a prompt diagnosis and appropriate treatment.

Case presentation

We report the case of a 68-year-old woman patient who presented an inguinal mass with swelling and pain in the right lower limb. She underwent surgery, and later received systematic intravenous chemotherapy. The pathological studies, especially the specific chromosomal translocation of a t(X;18) (p11.2;q11.2), confirmed the diagnosis as a synovial sarcoma. To the best of our knowledge, this is the first report of a monophasic synovial sarcoma in the inguinal region.

Conclusion

Besides making the readership aware of the rarity of location and age of this present case, this report distinctly highlights the great value of a molecular analysis of an SYT associated genetic alteration in the diagnosis of synovial sarcoma occurring at rare sites especially when immunochemical results are equivocal.