Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Proton motive force generation by citrolactic fermentation in Leuconostoc mesenteroides.

In Leuconostoc mesenteroides subsp. mesenteroides 19D, citrate is transported by a secondary citrate carrier (CitP). Previous studies of the kinetics and mechanism of CitP performed in membrane vesicles of L. mesenteroides showed that CitP catalyzes divalent citrate HCit2-/H+ symport, indicative of metabolic energy generation by citrate metabolism via a secondary mechanism (C. Marty-Teysset, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Biol. Chem. 270:25370-25376, 1995). This study also revealed an efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate cometabolism, suggesting that under physiological conditions, CitP may function as a precursor/product exchanger rather than a symporter. In this paper, the energetic consequences of citrate metabolism were investigated in resting cells of L. mesenteroides. The generation of metabolic energy in the form of a pH gradient (delta pH) and a membrane potential (delta psi) by citrate metabolism was found to be largely dependent on cometabolism with glucose. Furthermore, in the presence of glucose, the rates of citrate utilization and of pyruvate and lactate production were strongly increased, indicating an enhancement of citrate metabolism by glucose metabolism. The rate of citrate metabolism under these conditions was slowed down by the presence of a membrane potential across the cytoplasmic membrane. The production of D-lactate inside the cell during cometabolism was shown to be responsible for the enhancement of the electrogenic uptake of citrate. Cells loaded with D-lactate generated a delta psi upon dilution in buffer containing citrate, and cells incubated with citrate built up a pH gradient upon addition of D-lactate. The results are consistent with an electrogenic citrate/D-lactate exchange generating in vivo metabolic energy in the form of a proton electrochemical gradient across the membrane. The generation of metabolic energy from citrate metabolism in L. mesenteroides may contribute significantly to the growth advantage observed during cometabolism of citrate and glucose.     

The possible role of metallothioneins in copper tolerance of Silene cucubalus

Growth and copper-binding of a copper-tolerant and a copper-sensitive population of Silene cucubalus (L.) Wib. have been studied. The copper-tolerant plants showed a much lower uptake and a proportionally higher transport of copper from root to shoot. A copper-binding protein with an apparent M_r of 8500 resembling metallothionein has been isolated from the roots of copper-treated plants of the tolerant population. After 20 d, the protein was observed to be inducible upon copper supply in the copper-tolerant plants, but not yet in the sensitive ones. This could be an indication of a difference in metalregulated synthesis of the protein. Ion-exchange chromatography of the 8500 protein yielded a major copper-containing fraction eluting at high ionic strength. Other characteristics such as UV absorption and amino-acid composition resembled strongly those of metallothioneins. The involvement of metallothioneins in the detoxification of copper within Cu-tolerant plants is discussed in relation to other mechanisms.Abbreviation DEAE diethyloaminoethyl     

Detailed imaging and genetic analysis reveal a secondary BRAFL505H resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three‐dimensional imaging. In all patients, only a subset of lesions progressed. Next‐generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib‐resistant patient‐derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Sequence and hydropathy profile analysis of two classes of secondary transporters

A structural class in the MemGen classification of membrane proteins is a set of evolutionary related proteins sharing a similar global fold. A structural class contains both closely related pairs of proteins for which homology is clear from sequence comparison and very distantly related pairs, for which it is not possible to establish homology based on sequence similarity alone. In the latter case the evolutionary link is based on hydropathy profile analysis. Here, we use these evolutionary related sets of proteins to analyze the relationship between E-values in BLAST searches, sequence similarities in multiple sequence alignments and structural similarities in hydropathy profile analyses. Two structural classes of secondary transporters termed ST[3], which includes the Ion Transporter (IT) superfamily and ST[4], which includes the DAACS family (TC# 2.A.23) were extracted from the NCBI protein database. ST[3] contains 2051 unique sequences distributed over 32 families and 59 subfamilies. ST[4] is a smaller class containing 399 unique sequences distributed over 2 families and 7 subfamilies. One subfamily in ST[4] contains a new class of binding protein dependent secondary transporters. Comparison of the averaged hydropathy profiles of the subfamilies in ST[3] and ST[4] revealed that the two classes represent different folds. Divergence of the sequences in ST[4] is much smaller than observed in ST[3], suggesting different constraints on the proteins during evolution. Analysis of the correlation between the evolutionary relationship of pairs of proteins in a class and the BLAST E-value revealed that: (i) the BLAST algorithm is unable to pick up the majority of the links between proteins in structural class ST[3], (ii) ‘low complexity filtering’ and ‘composition based statistics’ improve the specificity, but strongly reduce the sensitivity of BLAST searches for distantly related proteins, indicating that these filters are too stringent for the proteins analyzed, and (iii) the E-value cut-off, which may be used to evaluate evolutionary significance of a hit in a BLAST search is very different for the two structural classes of membrane proteins.     

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d- galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 &mgr;m recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.