Differences in polypeptide expression in thermoinhibited and germinating achenes of Tagetes minuta L.

When imbibed at their optimum germination temperature of25°C, achenes of Tagetes minutaL.germinate over a period of 48 h. At a temperature of36°C, the achenes become thermoinhibited and do notgerminate. This elevated temperature does not harm the achenes, however, and assoon as the temperature is reduced below 35°C,germination occurs, but is spread over a reduced period of only 24h. The polypeptide patterns of germinating and thermoinhibitedachenes were compared, to test whether thermoinhibition is the result ofdifferential gene expression or repression. Ten polypeptides which weredifferentially expressed in thermoinhibited achenes were observed. Thesepolypeptides were all extremely specific to thermoinhibited achenes anddeclinedrapidly when the incubation temperature was decreased, in a manner whichcorrelated with the increase in germinability of the achenes. The tightlyregulated nature of the expression of these polypeptides suggests thatthermoinhibition in T. minuta may be under positivecontrol. These proteins may therefore result in the prevention of radicleemergence at unfavourable temperatures.     

Evidence of Long-Lived Founder Virus in Mother-to-Child HIV Transmission

Exposure of the infant’s gut to cell-associated and cell-free HIV-1 trafficking in breast milk (BM) remains a primary cause of mother-to-child transmission (MTCT). The mammary gland represents a unique environment for HIV-1 replication and host-virus interplay. We aimed to explore the origin of the virus transmitted during breastfeeding, and the link with quasi-species found in acellular and cellular fractions of breast-milk (BM) and in maternal plasma. The C2–V5 region of the env gene was amplified, cloned and sequenced from the RNA and DNA of BM, the RNA from the mother’s plasma (PLA) and the DNA from infant’s dried blood spot (DBS) in 11 post-natal mother-infant pairs. Sequences were assembled in Geneious, aligned in ClustalX, manually edited in SeAL and phylogenetic reconstruction was undertaken in PhyML and MrBayes. We estimated the timing of transmission (ETT) and reconstructed the time for the most recent common ancestor (TMRCA) of the infant in BEAST. Transmission of single quasi-species was observed in 9 of 11 cases. Phylogenetic analysis illustrated a BM transmission event by cell-free virus in 4 cases, and by cell-associated virus in 2 cases but could not be identified in the remaining 5 cases. Molecular clock estimates, of the infant ETT and TMRCA, corresponded well with the timing of transmission estimated by sequential infant DNA PCR in 10 of 11 children. The TMRCA of BM variants were estimated to emerge during gestation in 8 cases. We hypothesize that in the remaining cases, the breast was seeded with a long-lived lineage latently infecting resting T-cells. Our analysis illustrated the role of DNA and RNA virus in MTCT. We postulate that DNA archived viruses stem from latently infected quiescent T-cells within breast tissue and MTCT can be expected to continue, albeit at low levels, should interventions not effectively target these cells.     

Inactivation of arginine esterase E-I of Bitis gabonica venom by irreversible inhibitors including a water-soluble carbodiimide, a chloromethyl ketone and isatoic anhydride.

1. Esterase E-I from Bitis gabonica was inactivated with irreversible inhibitors which included studies with a water-soluble carbodiimide, an affinity labelling peptide and a mechanism-based inactivator. 2. The reaction with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide was biphasic and the dominant part followed saturation kinetics. At pH 5.5 a rate constant of 0.4 min-1 for inactive enzyme formation was calculated and a dissociation constant (Ki) of 0.2 M for the enzyme-inhibitor complex. 3. Inactivation with D-Phe-Pro-Arg-chloromethyl ketone indicated a two-step mechanism, for which the reaction parameters at pH 8.0 were determined. The Ki value was 0.2 microM and the inactivation rate was 2.5 min-1. 4. With isatoic anhydride pseudo-first-order kinetics was observed. At pH 8.0 a rate constant of 0.9 min-1 and a Ki of 2.0 mM were obtained. The inactivation of the enzyme was found to be governed by a group in the enzyme showing a pK value of 7.3.     

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.     

Phylogenetic position of Cyperus clandestinus (Cypereae,Cyperaceae) clarified by morphological and molecular evidence

Extreme morphological reduction and convergent evolution can obscure taxonomic relationships. This phenomenon is frequently encountered in Cyperaceae, where characters traditionally used to diagnose genera have been shown to have evolved independently multiple times. The Ethiopian high‐altitude perennial first described as Cyperus clandestinus was subsequently moved to Ficinia because it has spiral glume arrangement, unlike typical Cyperus species, which have distichous glume arrangement. However, this position has remained uncertain as no nutlets have previously been studied to establish the presence or absence of the gynophore – the synapomorphy for Ficinia. We resolve this 140‐year‐old puzzle by describing the morphology of the nutlet, which lacks a gynophore, and use DNA sequence data to resolve the taxon within Cyperus. Cyperus clandestinus was found to be closely related to Remirea maritima and Cyperus cyperoides in the C₄Cyperus clade, whose members predominantly have distichously arranged glumes. This provides further evidence for the unreliability of glume arrangement as a character to distinguish between members of the Cyperus and Ficinia clades, whereas gynophore presence is more congruent with molecular data.     

Selenium-containing proteins of rat kidney

Rat kidney selenium (Se)-containing proteins were studied by isotopic labeling with [75Se]selenite or [75Se]selenomethionine via three routes: oral, intraperitoneal injection, and incubation of kidney slices with the isotope. The two major Se-containing proteins in kidney were fractionated and partially characterized. 75Se elution profiles from Sephadex G-150 chromatography were similar for each labeling protocol, except for the profile obtained following incubation of slices with [75Se]selenomethionine. Of the two major 75Se-containing proteins, the one eluting at the void volume during Sephadex G-150 fractionation had a subunit of 23,000 Mr. The 75Se-labeled tryptic peptide from this protein and a 75Se-containing tryptic peptide from glutathione peroxidase had the same elution time from an HPLC column. A 75,000 Mr 75Se-containing protein had a 65,000 Mr subunit, and the 75Se-labeled tryptic peptide from this protein eluted from the HPLC column before that of glutathione peroxidase. Glutathione peroxidase is the most abundant kidney selenoprotein. Injection of animals with 75Se is the method of choice for isotopic labeling of rat kidney Se-containing proteins. Appropriate methods were developed that can be used in future studies of kidney Se-containing proteins.