A novel isotope labeling protocol for bacterially expressed proteins

Summary A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).     

Placental alkaline phosphatase expression at the apical and basal plasma membrane in term villous trophoblasts.

Human placental alkaline phosphatase (PLAP) was localized at the apical and basal plasma membrane of syncytiotrophoblasts and at the surface of cytotrophoblasts in term chorionic villi using immunoelectron microscopy. Similarly, apical and basolateral PLAP expression was found in polarized trophoblast-derived BeWo cells. Trophoblasts isolated from term placentas exhibited mainly vesicular PLAP immunofluorescence staining immediately after isolation. After in vitro differentiation into syncytia, PLAP plasma membrane expression was upregulated and exceeded that observed in mononuclear trophoblasts. These data call for caution in using PLAP as a morphological marker to differentiate syncytiotrophoblasts from cytotrophoblasts or as a marker enzyme for placental brush-border membranes. (J Histochem Cytochem 49:1155-1164, 2001)     

Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS

This study aims at substituting the essential functions of photoreceptors in patients who are blind owing to untreatable forms of hereditary retinal degenerations. A microelectronic neuroprosthetic device, powered via transdermal inductive transmission, carrying 1500 independent microphotodiode-amplifier-electrode elements on a 9 mm² chip, was subretinally implanted in nine blind patients. Light perception (8/9), light localization (7/9), motion detection (5/9, angular speed up to 35 deg s⁻¹), grating acuity measurement (6/9, up to 3.3 cycles per degree) and visual acuity measurement with Landolt C-rings (2/9) up to Snellen visual acuity of 20/546 (corresponding to decimal 0.037 or corresponding to 1.43 logMAR (minimum angle of resolution)) were restored via the subretinal implant. Additionally, the identification, localization and discrimination of objects improved significantly (n = 8; p < 0.05 for each subtest) in repeated tests over a nine-month period. Three subjects were able to read letters spontaneously and one subject was able to read letters after training in an alternative-force choice test. Five subjects reported implant-mediated visual perceptions in daily life within a field of 15° of visual angle. Control tests were performed each time with the implant''s power source switched off. These data show that subretinal implants can restore visual functions that are useful for daily life.     

Dimethyl Disulfide Produced by the Naturally Associated Bacterium Bacillus sp B55 Promotes Nicotiana attenuata Growth by Enhancing Sulfur Nutrition

Bacillus sp B55, a bacterium naturally associated with Nicotiana attenuata roots, promotes growth and survival of wild-type and, particularly, ethylene (ET)–insensitive 35S-ethylene response1 (etr1) N. attenuata plants, which heterologously express the mutant Arabidopsis thaliana receptor ETR1-1. We found that the volatile organic compound (VOC) blend emitted by B55 promotes seedling growth, which is dominated by the S-containing compound dimethyl disulfide (DMDS). DMDS was depleted from the headspace during cocultivation with seedlings in bipartite Petri dishes, and ³⁵S was assimilated from the bacterial VOC bouquet and incorporated into plant proteins. In wild-type and 35S-etr1 seedlings grown under different sulfate (SO₄⁻²) supply conditions, exposure to synthetic DMDS led to genotype-dependent plant growth promotion effects. For the wild type, only S-starved seedlings benefited from DMDS exposure. By contrast, growth of 35S-etr1 seedlings, which we demonstrate to have an unregulated S metabolism, increased at all SO₄⁻² supply rates. Exposure to B55 VOCs and DMDS rescued many of the growth phenotypes exhibited by ET-insensitive plants, including the lack of root hairs, poor lateral root growth, and low chlorophyll content. DMDS supplementation significantly reduced the expression of S assimilation genes, as well as Met biosynthesis and recycling. We conclude that DMDS by B55 production is a plant growth promotion mechanism that likely enhances the availability of reduced S, which is particularly beneficial for wild-type plants growing in S-deficient soils and for 35S-etr1 plants due to their impaired S uptake/assimilation/metabolism.     

Complications of Antiretroviral Therapy Initiation in Hospitalised Patients with HIV-Associated Tuberculosis

Background

HIV-associated tuberculosis is a common coinfection in Sub-Saharan Africa, which causes high morbidity and mortality. A sub-set of HIV-associated tuberculosis patients require prolonged hospital admission, during which antiretroviral therapy initiation may be required. The aim of this study was to document the causes of clinical deterioration of hospitalised patients with HIV-associated tuberculosis starting antiretroviral therapy in order to inform healthcare practice in low- to middle-income countries.

Methods

Prospective, observational cohort study of adult inpatients with HIV-associated tuberculosis starting antiretroviral therapy in a dedicated tuberculosis hospital in Cape Town, South Africa. Causes of clinical deterioration and outcome were recorded in the first 12 weeks of antiretroviral therapy. Patients with rifampicin-resistant tuberculosis were excluded.

Results

Between May 2009 and November 2010, 112 patients (60% female), with a median age of 32 years were enrolled. At baseline the median CD4 count was 55 cells/mm³ (IQR 31–106) and HIV viral load 5.6 log copies/mL. All patients had significant comorbidity: 82% were bed-bound, 65% had disseminated tuberculosis and 27% had central nervous system tuberculosis. Seventy six patients (68%) developed 144 clinical events after starting antiretroviral therapy. TB-IRIS, hospital-acquired infections and significant drug toxicities occurred in 42%, 20.5% and 15% of patients respectively. A new opportunistic disease occurred in 15% of patients and a thromboembolic event in 8%. Mortality during the 12 week period was 10.6%.

Conclusions

High rates of TB-IRIS, hospital-acquired infections and drug toxicities complicate the course of patients with HIV-associated tuberculosis starting antiretroviral therapy in hospital. Despite the high morbidity, mortality was relatively low. Careful clinical management and adequate resources are needed in hospitalised HIV-TB patients in the 1^st three months following ART initiation.     

Alcohol Affects the Brain's Resting-State Network in Social Drinkers

Acute alcohol intake is known to enhance inhibition through facilitation of GABA_A receptors, which are present in 40% of the synapses all over the brain. Evidence suggests that enhanced GABAergic transmission leads to increased large-scale brain connectivity. Our hypothesis is that acute alcohol intake would increase the functional connectivity of the human brain resting-state network (RSN). To test our hypothesis, electroencephalographic (EEG) measurements were recorded from healthy social drinkers at rest, during eyes-open and eyes-closed sessions, after administering to them an alcoholic beverage or placebo respectively. Salivary alcohol and cortisol served to measure the inebriation and stress levels. By calculating Magnitude Square Coherence (MSC) on standardized Low Resolution Electromagnetic Tomography (sLORETA) solutions, we formed cortical networks over several frequency bands, which were then analyzed in the context of functional connectivity and graph theory. MSC was increased (p<0.05, corrected with False Discovery Rate, FDR corrected) in alpha, beta (eyes-open) and theta bands (eyes-closed) following acute alcohol intake. Graph parameters were accordingly altered in these bands quantifying the effect of alcohol on the structure of brain networks; global efficiency and density were higher and path length was lower during alcohol (vs. placebo, p<0.05). Salivary alcohol concentration was positively correlated with the density of the network in beta band. The degree of specific nodes was elevated following alcohol (vs. placebo). Our findings support the hypothesis that short-term inebriation considerably increases large-scale connectivity in the RSN. The increased baseline functional connectivity can -at least partially- be attributed to the alcohol-induced disruption of the delicate balance between inhibitory and excitatory neurotransmission in favor of inhibitory influences. Thus, it is suggested that short-term inebriation is associated, as expected, to increased GABA transmission and functional connectivity, while long-term alcohol consumption may be linked to exactly the opposite effect.     

Relationship of F9 antigen and genes of theT/t complex

Serological studies relating F9 antigen of embryonal carcinoma cells to at the murineT/t complex have been extended and confirm that only the lethal haplotype t¹²- and none of the other five lethal haplotypes-affects the quantitative expression of F9 antigen on sperm. Cytotoxicity tests on preimplantation embryos show that t¹² homozygotes are less susceptible to antiF9 serum than t^w5 homozygotes, and that using specific antimutant haplotype antisera prepared against sperm, t¹² antigen is detectable on morulae, whereas t^w5 antigen is not.     

Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion

Two highly conserved NPA motifs are a hallmark of the aquaporin (AQP) family. The NPA triplets form N-terminal helix capping structures with the Asn side chains located in the centre of the water or solute-conducting channel, and are considered to play an important role in AQP selectivity. Although another AQP selectivity filter site, the aromatic/Arg (ar/R) constriction, has been well characterized by mutational analysis, experimental data concerning the NPA region--in particular, the Asn position--is missing. Here, we report on the cloning and mutational analysis of a novel aquaglyceroporin carrying one SPA motif instead of the NPA motif from Burkholderia cenocepacia, an epidemic pathogen of cystic fibrosis patients. Of 1357 AQP sequences deposited in RefSeq, we identified only 15 with an Asn exchange. Using direct and phenotypic permeability assays, we found that Asn and Ser are freely interchangeable at both NPA sites without affecting protein expression or water, glycerol and methylamine permeability. However, other mutations in the NPA region led to reduced permeability (S186C and S186D), to nonfunctional channels (N64D), or even to lack of protein expression (S186A and S186T). Using electrophysiology, we found that an analogous mammalian AQP1 N76S mutant excluded protons and potassium ions, but leaked sodium ions, providing an argument for the overwhelming prevalence of Asn over other amino acids. We conclude that, at the first position in the NPA motifs, only Asn provides efficient helix cap stabilization and cation exclusion, whereas other small residues compromise structural stability or cation exclusion but not necessarily water and solute permeability.