Localization of G-proteins in macrophages and E. coli during phagocytosis

Heterotrimeric GTP-binding proteins (G-proteins) have been shown to play an important role in cellular signalling. However, G-protein involvement in the intracellular spreading of bacterial pathogens is still poorly understood. In this study, antibodies, that recognize G-protein alpha-subunits (anti-G alpha), were used to investigate the localization of G-proteins in the macrophage-like cell line P388D1 and E. coli, also in their L-forms, during phagocytosis. In E. coli, anti-G alpha-binding sites were detected preferably in the cell wall and septa of the whole bacterial forms as well as in the cytoplasm of L-forms. Western blotting of bacterial lysates demonstrated protein bands with positive immunoreaction to antibodies against Gs alpha, Gi alpha, and Gcommon alpha with a higher affinity to the antibody against Gs alpha. Immunoreaction with the anti-Gs alpha-antibody was markedly higher in pathogenic strains of E. coli. Because of the conserved structure in all GTP-binding proteins which seem to derive from a single primordial protein involved in signal transduction mechanisms, it is reasonable to assume that some anti-Ga-positive proteins in E. coli might be related to G-proteins of higher organisms. A putative candidate for bacterial G-proteins seems to be a 36 kDa protein. Enhancement in G-protein immunostaining in the cytoplasm of macrophages around the internalized bacteria testifies to the involvement of G-proteins in mediation of endocytosis responses of phagocytes.     

Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site

We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes.     

Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL-11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.     

Correlation between pharmacokinetic parameters of rifampicin and its biologically active metabolite as related to estimation of the relative bioavailability of the antibiotic

In the bioavailability studies with drugs biotransformed to biologically active metabolities only the concentrations of the parent drug (PD) are usually taken into account even when the pharmacokinetic data on the metabolite(s) (M) are available. However, such data may be useful as an alternative source for the bioavailability determination. Moreover, the clinical outcomes often depend on both the PD and M concentrations. The aim of the study performed with two rifampicin formulations, tablets and dragee, was to correlate the pharmacokinetic parameters of the PD and 25-O-deacetylrifampicin, a microbiologically active M of rifampicin, and to examine whether the bioavailability parameters based on the PD and M concentrations were compatible. The serum concentrations of the PD and M were determined in 8 healthy volunteers by HPLC. Despite different patterns of the PD and M pharmacokinetic profiles the PD peak concentration (Cmax) and especially the AUC correlated with Cmax or the AUC of the M (r = 0.76 and 0.92 respectively). Moreover, the extent of the absorption expressed as the AUC ratio for the PD correlated with the AUC ratio for the M (r = 0.86). However, neither the time to reach the maximum (tmax), nor the Cmax/AUC ratio, a measure of the absorption rate, based on the PD pharmacokinetic data correlated with the respective parameters calculated with using the M concentrations. Thus, only the estimates of the extent of the absorption and not of the absorption rate based on the PD and M data may be considered as compatible.     

Tyrosine phosphorylation of the Kv1.3 potassium channel

Kv1.3, a voltage-dependent potassium channel cloned from mammalian brain and T lymphocytes, contains multiple tyrosine residues that are putative targets for tyrosine kinases. We have examined the tyrosine phosphorylation of Kv1.3, expressed transiently in human embryonic kidney (or HEK) 293 cells, by endogenous and coexpressed tyrosine kinases. Tyrosine phosphorylation is measured by a strategy of immunoprecipitation followed by. Western blot analysis, using antibodies that specifically recognize Kv1.3 and phosphotyrosine. Coexpression of the constitutively active tyrosine kinase v-src, together with Kv1.3, causes a large increase in the tyrosine phosphorylation of the channel protein. This phosphorylation of Kv1.3 can be reversed by treatment with alkaline phosphatase before Western blot analysis. Coexpression with a receptor tyrosine kinase, the human epidermal growth factor receptor, also causes an increase in tyrosine phosphorylation of Kv1.3. The effects of endogenous tyrosine kinases were examined by treating Kv1.3-transfected cells with the specific membrane-permeant tyrosine phosphatase inhibitor pervanadate. Pervanadate treatment causes a time- and concentration-dependent increase in the tyrosine phosphorylation of Kv1.3. This increased tyrosine phosphorylation of Kv1.3 is accompanied by a time-dependent decrease in Kv1.3 current, measured by patch-clamp analysis with cell-attached membrane patches. The pervanadate-induced suppression of current and much of the channel tyrosine phosphorylation are eliminated by mutation of a specific tyrosine residue, at position 449 of Kv1.3, to phenylalanine. Thus, there is a continual phosphorylation and dephosphorylation of Kv1.3 by endogenous kinases and phosphatases, and perturbation of this constitutive phosphorylation/dephosphorylation cycle can profoundly influence channel activity.     

Prevalence of malaria in Dakar, Senegal. Comparative study of the plasmodial indices in pregnant and non-pregnant women

One hundred ASA I orthopaedic surgical patients (four randomized groups) were anaesthetized using continuous propofol and intermittent fentanyl (TIVA), with controlled ventilation via a tracheal tube in groups 1 and 2, and a laryngeal mask airway (LMA) in groups 3 and 4. Neuromuscular blockers were used in groups 1 and 3 only. There were no significant differences between groups in total anaesthetic requirements, as assessed by cardiovascular variables and movement. Coughing interfered with surgery and made controlled ventilation difficult to manage. In contrast, movement not associated with coughing did not impair surgery or ventilation. Patients in group 2 (tracheal tube, no neuromuscular blocker) required more interventions for coughing than the other groups, while patients in group 4 (LMA, no neuromuscular blocker) needed more boluses for movement than groups 1 and 3. Groups 1 and 2 (tracheal tube) had significantly higher heart rates and mean arterial pressures than groups 3 and 4 for varying periods up to 5 min after insertion of the airway management device. There was no correlation between mean arterial pressure and plasma concentrations of catecholamines related to insertion of either the tracheal tube or LMA. The LMA was found to be a highly effective device for controlled ventilation in TIVA and easier to manage than the tracheal tube in the absence of neuromuscular blockers.     

The choice of the method for the surgical treatment of bladder exstrophy

129 children with exstrophy of the bladder underwent primary surgical reconstruction according to G. A. Bairov. Morphofunctional findings in these children gave grounds for determination of three degrees of the bladder congenital defects. 39 patients had defect of the 1st degree, 53 of the 2nd and 37 of the 3d degree. The results of plastic reconstruction of the bladder with local tissue support the validity of such procedure only for patients with the congenital defect degree I. For degree II the benefit is relative. In degree III plastic surgery in contraindicated.     

Efficacy of silicone punctal plugs as adjuncts to topical pharmacotherapy of glaucoma--a pilot study. Punctal Plugs in Glaucoma Study Group

BACKGROUND: The concept of enhancing the ocular hypotensive effects of topical antiglaucoma medications by impeding lacrimal drainage of medication has been insufficiently studied. This investigation sought to evaluate the effect of bilateral inferior punctal occlusion using silicone punctal plugs on the ocular hypotensive effect of topically applied timolol. METHODS: A randomized, double-masked, cross-over clinical trial was conducted, comparing the ocular hypotensive effect of timolol maleate 0.25 percent, both with and without occlusion of the inferior punctum with the Freeman silicone punctal plug. Following a 2-week washout of topical medication, 17 subjects with early primary open-angle glaucoma or ocular hypertension received one drop of timolol 0.25 percent in each eye with or without punctal plugs in place. Blood pressure, resting pulse rate, and intraocular pressure were measured both before timolol instillation and at intervals of 1, 2, 4, 8, and 12 hours following drop instillation. Following a 2-week washout period, the subjects were evaluated with the alternative treatment. RESULTS: There was no statistically significant difference (p = 0.648) in IOP levels between treatment groups. CONCLUSIONS: This pilot suggests that need for a longer-term study with larger numbers of subjects to evaluate the potential role of silicone punctal plugs to enhance the ocular bioavailability of topically applied antiglaucoma medications.     

Isolation of bacterial endotoxins from Pseudomonas putida 36 and Escherichia coli (vulgaris)

A prospective analysis of 69 patients who had been treated for nasopharyngeal carcinoma (NPC) by external radiotherapy was carried out. Biopsies from the posterior nasopharynx were performed and analyzed by in situ hybridization using an antisense Epstein-Barr Early RNA (EBER) radio-labelled riboprobe. None of the patients had evidence of disease in the nasopharynx. One patient was found to have nasopharyngeal carcinoma detected only by in situ hybridization. In the subsequent 18-month follow-up of these clinically- and biopsy-negative patients, only one patient developed relapse in the nasopharynx. In situ hybridization is a valuable tool for the detection of NPC and should be routinely available in histopathology laboratories where NPC is regularly diagnosed.