Affinity isolation of albumin-binding proteins using nitrocellulose-bound albumin

Albumin immobilized on a nitrocellulose membrane was used as an affinity matrix to purify albumin-binding proteins (ABP) from extracts of lung, heart, thymus, and isolated microvascular endothelial cells. Albumin was immobilized onto nitrocellulose either (i) directly (physically adsorbed), (ii) cross-linked by treatment with 0.25% glutaraldehyde, or (iii) covalently coupled to the matrix using NaIO4 and Na-borohydride. The affinity support was incubated with a membrane-enriched fraction (obtained from tissue homogenates) in the presence of protease inhibitors; specific binding of ABP occurred within 30 min of incubation. The adsorbed proteins were eluted with 0.5% sodium dodecyl sulfate (SDS) and analyzed by SDS-polyacrylamide gel electrophoresis and ligand blotting. Analysis of electrophoretic mobility of eluted proteins showed that they consisted exclusively of the two sets of polypeptides of 31 000 Da and 18 000 Da previously identified as ABP (N. Ghinea et al., J. Cell Biol. 107, 231-239 (1988]. As demonstrated by ligand blotting, the ABP purified on nitrocellulose-bound albumin maintain the ability to interact specifically with albumin. Preliminary experiments showed that the method employed may be of a broader use for the isolation of receptor proteins from tissue extracts by incubating the latter with the cognate ligand immobilized on nitrocellulose membranes.     

Expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in human uterine leiomyomas and myometrium during the menstrual cycle and after menopause

To investigate the expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak proteins in human uterine leiomyomas and homologous myometrium during the menstrual cycle and after menopause.The expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in leiomyomas (n=24) and myometrial samples (n=22) from women with leiomyomas was measured by immunohistochemistry and Western blot. Measured by immunohistochemistry, a significant difference between leiomyomas and myometrium was observed only for the Bax protein, in tissues obtained from women in the secretory phase of the menstrual cycle. The Bcl-2 staining was more abundant in leiomyomas than in myometrium only in tissues obtained in the proliferative phase of the cycle. Bcl-2 was more abundant in leiomyomas from women of fertile age than in leiomyomas from menopausal women. No significant differences were observed for the Bcl-x or Bak proteins, whereas the Mcl-1 protein was significantly less abundant in secretory phase leiomyomas than in leiomyomas from menopausal women. Western blot analysis based on pools of tissue extracts from the different groups essentially confirmed the data obtained by immunohistochemistry. Bcl-2 family proteins are expressed in leiomyomas and myometrium in different phases related to and influenced by gonadal steroids. These proteins are suggested to interact with each other in the regulation of programmed cell death, apoptosis, but their specific role in growth control of uterine leiomyomas remains to be investigated.     

Transductional mapping of nine linked chromosomal genes in Bacillus thuringiensis

Summary We have previously described a phage (63) for generalized transduction in Bacillus thuringiensis and used it for mapping of four chromosomal antibiotic resistance markers, namely nalA-rifA-strA-spcA (Landén et al. 1981). From 63 we have now isolated a host range mutant called 64 which contains 52–56 megadalton of DNA. Phage 64 was found to be a more efficient transducing vector than 63. The host range of 64 is wide, with good growth on subspecies gelechiae, kurstaki, galleriae, thuringiensis and thompsoni, restriction on some derivatives of finitimus and ostrinae and no growth on alesti, israelensis and aizawai.Using 64 and a series of new mutants of subspecies gelechiae we have no added five new genes to the antibiotic resistance group described before. The gene order found was guaB-purB-metA-novA-(purA-nalA)-rifA-strA-spcA. Linkage was also demonstrated between hisA and lysA.     

Reducing Stock-Outs of Life Saving Malaria Commodities Using Mobile Phone Text-Messaging: SMS for Life Study in Kenya

Background

Health facility stock-outs of life saving malaria medicines are common across Africa. Innovative ways of addressing this problem are urgently required. We evaluated whether SMS based reporting of stocks of artemether-lumefantrine (AL) and rapid diagnostic tests (RDT) can result in reduction of stock-outs at peripheral facilities in Kenya.

Methods/Findings

All 87 public health facilities in five Kenyan districts were included in a 26 week project. Weekly facility stock counts of four AL packs and RDTs were sent via structured incentivized SMS communication process from health workers’ personal mobile phones to a web-based system accessed by district managers. The mean health facility response rate was 97% with a mean formatting error rate of 3%. Accuracy of stock count reports was 79% while accuracy of stock-out reports was 93%. District managers accessed the system 1,037 times at an average of eight times per week. The system was accessed in 82% of the study weeks. Comparing weeks 1 and 26, stock-out of one or more AL packs declined by 38 percentage-points. Total AL stock-out declined by 5 percentage-points and was eliminated by the end of the project. Stock-out declines of individual AL packs ranged from 14 to 32 percentage-points while decline in RDT stock-outs was 24 percentage-points. District managers responded to 44% of AL and 73% of RDT stock-out signals by redistributing commodities between facilities. In comparison with national trends, stock-out declines in study areas were greater, sharper and more sustained.

Conclusions

Use of simple SMS technology ensured high reporting rates of reasonably accurate, real-time facility stock data that were used by district managers to undertake corrective actions to reduce stock-outs. Future work on stock monitoring via SMS should focus on assessing response rates without use of incentives and demonstrating effectiveness of such interventions on a larger scale.     

Variability in benthic diazotrophy and cyanobacterial diversity in a tropical intertidal lagoon

Benthic nitrogen fixation has been estimated to contribute 15 Tg N year(-1) to the marine nitrogen budget. With benthic marine nitrogen fixation being largely overlooked in more recent surveys, a refocus on benthic diazotrophy was considered important. Variations in nitrogenase activity (acetylene reduction-gas chromatography) in a tropical lagoon in the western Indian Ocean (Zanzibar, Tanzania) were monitored over a 3-year period (2003-2005) and related to cyanobacterial and diazotrophic microbial diversity using a polyphasic approach. Different nitrogenase activity patterns were discerned, with the predominant pattern being high daytime activities combined with low nighttime activities. Analyses of the morphological and 16S rRNA gene diversity among cyanobacteria revealed filamentous nonheterocystous (Oscillatoriales) and unicellular (Chroococcales) representatives to be predominant. Analyses of the nifH gene diversity showed that the major phylotypes belonged to noncyanobacterial prokaryotes. However, as shown by cyanobacterial selective nifH-denaturing gradient gel electrophoresis analysis, cyanobacterial nifH gene sequences were present at all sites. Several nifH and 16S rRNA gene phylotypes were related to uncultured cyanobacteria or bacteria of geographically distant habitats, stressing the widespread occurrence of still poorly characterized microorganisms in tropical benthic marine communities.     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

A 12‐Week Aerobic Exercise Program Reduces Hepatic Fat Accumulation and Insulin Resistance in Obese,Hispanic Adolescents

The rise in obesity‐related morbidity in children and adolescents requires urgent prevention and treatment strategies. Currently, only limited data are available on the effects of exercise programs on insulin resistance, and visceral, hepatic, and intramyocellular fat accumulation. We hypothesized that a 12‐week controlled aerobic exercise program without weight loss reduces visceral, hepatic, and intramyocellular fat content and decreases insulin resistance in sedentary Hispanic adolescents. Twenty‐nine postpubertal (Tanner stage IV and V), Hispanic adolescents, 15 obese (7 boys, 8 girls; 15.6 ± 0.4 years; 33.7 ± 1.1 kg/m²; 38.3 ± 1.5% body fat) and 14 lean (10 boys, 4 girls; 15.1 ± 0.3 years; 20.6 ± 0.8 kg/m²; 18.9 ± 1.5% body fat), completed a 12‐week aerobic exercise program (4 × 30 min/week at ≥70% of peak oxygen consumption (VO₂peak)). Measurements of cardiovascular fitness, visceral, hepatic, and intramyocellular fat content (magnetic resonance imaging (MRI)/magnetic resonance spectroscopy (MRS)), and insulin resistance were obtained at baseline and postexercise. In both groups, fitness increased (obese: 13 ± 2%, lean: 16 ± 4%; both P < 0.01). In obese participants, intramyocellular fat remained unchanged, whereas hepatic fat content decreased from 8.9 ± 3.2 to 5.6 ± 1.8%; P < 0.05 and visceral fat content from 54.7 ± 6.0 to 49.6 ± 5.5 cm²; P < 0.05. Insulin resistance decreased indicated by decreased fasting insulin (21.8 ± 2.7 to 18.2 ± 2.4 µU/ml; P < 0.01) and homeostasis model assessment of insulin resistance (HOMA_IR) (4.9 ± 0.7 to 4.1 ± 0.6; P < 0.01). The decrease in visceral fat correlated with the decrease in fasting insulin (R² = 0.40; P < 0.05). No significant changes were observed in any parameter in lean participants except a small increase in lean body mass (LBM). Thus, a controlled aerobic exercise program, without weight loss, reduced hepatic and visceral fat accumulation, and decreased insulin resistance in obese adolescents.     

Rat liver foci study on coexposure with 50 Hz magnetic fields and known carcinogens

A study was performed to investigate possible interactions by magnetic fields (MF) with the processes of initiation and promotion of chemically induced preneoplastic lesions in rat liver. Male Sprague-Dawley rats were subjected to a 70% partial hepatectomy followed after 24 h by i.p. injection of diethylnitrosamine (DENA) as a tumour initiator. Starting one week after the DENA-treatment phenobarbital (PB) was given to promote growth of enzymatically altered foci of liver cells. MF was applied immediately after the partial hepatectomy and continued until sacrifice after 12 weeks of PB exposure. Homogenous horizontal AC magnetic fields with a frequency of 50 Hz and flux densities of 0.5 μT or 0.5 mT were used. The rats coexposed with MF and DENA plus PB did not gain weight as much as the rats exposed to the chemical agents only. The MF-exposure also resulted in a slight reduction in size and numbers of the focal lesions. The results suggest an interaction of MF with the processes of chemical carcinogenesis either as a result of stress or depending on effects on the proliferation of preneoplastic cells. © 1993 Wiley-Liss, Inc.