Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.     

A survey of histoplasmosis and blastomycosis in U.A.R. (Egyptian sector) a preliminary report

Summary A selected group of 525 individuals with pulmonary diseases, granulomas and other medical conditions was tested for histoplasmin and blastomycin dermal reactions. No positive results were observed. Few doubtful positive reactions were recorded (3 to histoplasmin and 7 to blastomycin). None of the patients with chronic cutaneous granulomas exhibited any reaction.Although the number of subjects studied is small, these preliminry findings suggest the probable absence of histoplasmosis and blastomycosis in Egypt.     

Chromosomal and histological changes in the reproductive organs of infertile men

Summary The chromosomal changes in the process of spermatogenesis in 27 infertile men have been examined. Normal chromosomal meiotic activity was found in 44% of cases, various chromosomal anomalies were seen in 18%, and no cells in meiosis were detected in 37% of cases.     

Phagocytic functions of microglial cells in the central nervous system and their importance in two neurodegenerative diseases: multiple sclerosis and Alzheimer’s disease

Microglial cells are the resident phagocytic cells of the central nervous system (CNS). They possess a wide range of receptors allowing them to identify and internalize numerous pathogens. We will discuss here the role of the most important receptors of microglia involved in non-opsonin-dependent phagocytosis (mannose receptor, β-glucan receptor, scavenger receptor) and that of receptors involved in the opsonin-dependent phagocytosis, namely the complement 3 (CR3) and the Fcγ receptors (FcγR). First, the molecular and cellular mechanisms induced when these receptors are conducting a phagocytic event are presented. In the second part, we will discuss the role these receptors may play in multiple sclerosis and Alzheimer’s disease, in the elimination by phagocytosis of myelin and beta amyloid peptide respectively. The first two authors contributed equally to this work.     

Circulating tumor cells: detection, molecular profiling and future prospects

Disseminated malignancy is responsible for the vast majority of cancer-related deaths. During this process, circulating tumor cells (CTC) are generated, spread from the primary tumor, colonize distant organs and lead to overt metastatic disease. CTC are essential for establishing metastasis; however, they are not sufficient as this process is highly inefficient and most will fail to grow in target sites. Several CTC die during migration while others remain dormant for several years and very few grow into macrometastases. CTC have been well documented in the bloodstream of cancer patients; however, the clinical relevance of this detection is still the subject of controversies and their biology is poorly understood. Indeed, available markers fail to distinguish between subgroups of CTC, and several current methods lack sensitivity, specificity or reproducibility in CTC characterization and detection. The advent of more precise technologies is renewing the interest in CTC biology. We will review herein recent findings on CTC biology, on the role of host-tumor interactions in CTC shedding and implantation, available methods of CTC detection and future perspectives for the molecular characterization of the CTC subset(s) responsible for the development of metastasis. Ultimately, understanding CTC biology and host-tumor 'complementarities' will help define metastasis-related biomarkers providing formidable and tailored novel therapeutic targets.     

CD36-deficient mice are resistant to alcohol- and high-carbohydrate-induced hepatic steatosis

CD36 is a scavenger receptor with multiple ligands and cellular functions, including facilitating cellular uptake of free fatty acids (FFAs). Chronic alcohol consumption increases hepatic CD36 expression, leading to the hypothesis that this promotes uptake of circulating FFAs, which then serve as a substrate for triglyceride (TG) synthesis and the development of alcoholic steatosis. We investigated this hypothesis in alcohol-fed wild-type and Cd36-deficient (Cd36^−/−) mice using low-fat/high-carbohydrate Lieber-DeCarli liquid diets, positing that Cd36^−/− mice would be resistant to alcoholic steatosis. Our data show that the livers of Cd36^−/− mice are resistant to the lipogenic effect of consuming high-carbohydrate liquid diets. These mice also do not further develop alcoholic steatosis when chronically fed alcohol. Surprisingly, we did not detect an effect of alcohol or CD36 deficiency on hepatic FFA uptake; however, the lower baseline levels of hepatic TG in Cd36^−/− mice fed a liquid diet were associated with decreased expression of genes in the de novo lipogenesis pathway and a lower rate of hepatic de novo lipogenesis. In conclusion, Cd36^−/− mice are resistant to hepatic steatosis when fed a high-carbohydrate liquid diet, and they are also resistant to alcoholic steatosis. These studies highlight an important role for CD36 in hepatic lipid homeostasis that is not associated with hepatic fatty acid uptake.     

Can Ascending Infection from Bladder Serve as the Portal of Entry for Primary Renal Zygomycosis?

Mucormycosis is an uncommon opportunistic infection by filamentous fungi that usually develops in immmunocompromised patients. Most individuals have an underlying systemic disease, such as diabetes mellitus, malignancy, uraemia, burns, renal transplant recipients and those on corticosteroid and immunosuppressive therapy. Many cases of primary renal zygomycosis with lungs serving as the portal of entry have been reported from this region. We describe two autopsy cases of renal zygomycosis where bladder appeared to be the portal of entry for the fungus.     

Synthesis of New Bis(pyrazolo[1,5-a]pyrimidines) Linked to Different Spacers as Potential MurB Inhibitors

An efficient protocol was adopted to efficiently prepare three new series of bis(pyrazolo[1,5-a]pyrimidines) linked to different spacers. The new bis(pyrazolo[1,5-a]pyrimidines) were prepared in 80–90 % yields by reacting the respective bis(enaminones) and 4-(4-substituted benzyl)-1H-pyrazole-3,5-diamines in pyridine at reflux temperature for 5–7 h. The new products showed a wide spectrum of antibacterial activity against six different bacterial strains. In general, propane- and butane-linked bis(pyrazolo[1,5-a]pyrimidines), which are attached to 3-(4-methyl- or 4-methoxybenzyl) units, had the best antibacterial activity with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values up to 2.5 and 5.1 μM, respectively. Additionally, the previous products demonstrated promising MurB inhibitory activity with IC₅₀ values up to 7.2 μM.     

The in vivo genotoxicity of cisplatin,isoflurane and halothane evaluated by alkaline comet assay in Swiss albino mice

The aim of this study was to evaluate the genotoxicity of repeated exposure to isoflurane or halothane and compare it with the genotoxicity of repeated exposure to cisplatin. We also determined the genotoxicity of combined treatment with inhalation anaesthetics and cisplatin on peripheral blood leucocytes (PBL), brain, liver and kidney cells of mice. The mice were divided into six groups as follows: control, cisplatin, isoflurane, cisplatin–isoflurane, halothane and cisplatin–halothane, and were exposed respectively for three consecutive days. The mice were treated with cisplatin or exposed to inhalation anaesthetic; the combined groups were exposed to inhalation anaesthetic after treatment with cisplatin. The alkaline comet assay was performed. All drugs had a strong genotoxicity (P < 0.05 vs. control group) in all of the observed cells. Isoflurane caused stronger DNA damage on the PBL and kidney cells, in contrast to halothane, which had stronger genotoxicity on brain and liver cells. The combination of cisplatin and isoflurane induced lower genotoxicity on PBL than isoflurane alone (P < 0.05). Halothane had the strongest effect on brain cells, but in the combined treatment with cisplatin, the effect decreased to the level of cisplatin alone. Halothane also induced the strongest DNA damage of the liver cells, while the combination with cisplatin increased its genotoxicity even more. The genotoxicity of cisplatin and isoflurane on kidney cells were nearly at the same level, but halothane caused a significantly lower effect. The combinations of inhalation anaesthetics with cisplatin had stronger effects on kidney cells than inhalation anaesthetics alone. The observed drugs and their combinations induced strong genotoxicity on all of the mentioned cells.