Use of nonoxynol-9 and changes in vaginal lactobacilli

The conformation of lens crystallins in vivo or in a highly concentrated solution is not well established. Most studies were carried out in dilute solutions in which protein-protein interaction is minimal. In order to see whether there is conformational change (tertiary and secondary structures) when crystallin solutions are brought to high concentrations, we have performed the following molecular spectroscopic measurements: circular dichroism (CD) and Fourier transform infrared (FTIR). Near-UV CD measurements showed a more than two-fold increase in CD intensity (molar ellipticity) for the total water-soluble (WS) protein from young calf lens nucleus in a highly concentrated solution (> 300 mg/ml in a 0.01-mm cell), when compared with a dilute solution (1000-fold dilution in a 10-mm cell). The individual crystallins in concentrated solutions also showed an increase in CD intensity, but of different magnitude: alpha-crystallin > beta-crystallin > gamma-crystallin. The increased CD indicates that lens crystallins are in a more compact structure in highly concentrated solutions; they likely undergo a transition from a mobile to an immobile state. Change in near-UV CD usually is caused by restricted mobility of aromatic side groups, particularly Trp. The transition involves not only a change in protein tertiary and/or quaternary structure, but also in protein backbone structure. The change of protein backbone structure was drawn from FTIR measurements. FTIR spectra, sensitive to the secondary structure in the amide I region, could be measured for a highly concentrated solution for which far-UV CD measurement is not feasible. The secondary structure that showed prominent change for alpha-crystallin in a highly concentrated solution was beta-conformation: increase in beta-turn with a concomitant decrease of alpha-helix structure.     

Activated rat macrophages produce a galectin-1-like protein that induces apoptosis of T cells: biochemical and functional characterization

Galectins, a family of closely related beta-galactoside-binding proteins, show specific immunomodulatory properties. We have recently identified the presence of a galectin-like protein in rat peritoneal macrophages by means of a cross-reactivity with a polyclonal Ab raised against a galectin purified from adult chicken liver. Galectin expression was up-regulated in inflammatory and activated macrophages, revealing a significant increase in phorbol ester- and formylmethionine oligopeptide-treated cells. In an attempt to further explore its functional significance, rat macrophage galectin was purified from activated macrophages by a single-step affinity chromatography on a lactosyl-Sepharose matrix. The eluted fraction was resolved as a single protein band of approximately 15,000 Da by SDS-PAGE that immunoreacted strongly with the anti-chicken galectin serum. Gel filtration studies revealed that the protein behaved like a dimer under native conditions, and saccharides bearing a beta-D-galactoside configuration were able to inhibit the hemagglutinating activity displayed by the purified galectin. In agreement with its isoelectric point of approximately 4.8, the amino acid analysis showed a definitive acidic pattern. Internal amino acid sequencing of selected peptides obtained by proteolytic cleavage revealed that this carbohydrate-binding protein shares all the absolutely preserved and critical residues found in other members of the mammalian galectin-1 subfamily. Finally, biochemical and ultrastructural evidence, obtained by genomic DNA fragmentation and transmission electron microscopy, are also provided to show its potential implications in the apoptotic program of T cells. This effect was quantified by using the terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling assay and was found to be associated to the specific carbohydrate-binding properties of galectin.     

Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod

We report here the isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3. The Drosophila Bub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. We further show that the localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, we demonstrate that the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways.     

Perturbation corrections for flat and thimble-type cylindrical standard ionization chambers for 60Co gamma rays: Monte Carlo calculations

The use of an ionization chamber for absorbed dose determinations in a medium requires one to take into account perturbation corrections due to the presence of the chamber cavity in the medium. Evaluation of these corrections for perturbation and their variation with depth in the medium has been performed for a flat cylindrical and a cylindrical (thimble-type) ionization chamber placed in a graphite phantom irradiated by a 60Co gamma beam using Monte Carlo calculations (EGS4 system with correlated sampling variance reduction technique). The results of these calculations agree with published experimental and theoretical data to better than 0.18%, with a statistical uncertainty of less than 0.17%.     

A VSG expression site-associated gene confers resistance to human serum in Trypanosoma rhodesiense

Twenty three isolates of Beauveria bassiana and 13 isolates of Metarhizium anisopliae were tested on third instar nymphs of Triatoma infestans, a serious vector of Chagas disease. Pathogenicity tests at saturated humidity showed that this insect is very susceptible to fungal infection. At lower relative humidity (50%), conditions expected in the vector microhabitat, virulence was significantly different among isolates. Cumulative mortality 15 days after treatment varied from 17.5 to 97.5%, and estimates of 50% survival time varied from 6 to 11 days. Maintaining lower relative humidity, four B. bassiana and two M. anisopliae isolates were selected for analysis of virulence at different conidial concentrations and temperatures. Lethal concentrations sufficient to kill 50% of insects (LC50) varied from 7.1 x 10(5) to 4.3 x 10(6) conidia/ml, for a B. bassiana isolate (CG 14) and a M. anisopliae isolate (CG 491) respectively. Most isolates, particularly B. bassiana isolates CG 24 and CG 306, proved to be more virulent at 25 and 30 degrees C, compared to 15 and 20 degrees C. The differential virulence at 50% humidity observed among some B. bassiana isolates was not correlated to phenetic groups in cluster analysis of RAPD markers. In fact, the B. bassiana isolates analyzed presented a high homogeneity (> 73% similarity).     

Functional and clinical relevance of CD44 variant isoform expression on B-cell chronic lymphocytic leukemia cells

BACKGROUND AND OBJECTIVE: Recent studies have shown that expression of adhesion molecules of the Ig superfamily, of integrins and of selectins allows definition of high vs low risk B-cell chronic lymphocytic leukemia (B-CLL). The proteoglycan CD44 is an adhesion molecule that may be expressed as a standard form of 85-95 KD or as several variant isoforms. The presence of certain CD44 variant (v) isoforms on neoplastic cells indicates poor prognosis in epithelial and lymphoid malignancies, as it is associated with tumor progression and metastasis. DESIGN AND METHODS: The expression of CD44 v3, 4, 5, 6, 7, 9 and 10 was analyzed in cells from 85 B-CLL patients. Indirect immunofluorescence and flow cytometry were used to identify CD44v. Functional studies were performed by analysis of adhesion to hyaluronate (HA), one CD44 ligand, and HA-induced Ca2+ influx. A variety of statistical methods were used to define phenotypic and functional differences between the various clones, to calculate survival curves, and for multivariate analyses. RESULTS: In 17/85 B-CLL (20%), one or more CD44v were detectable by indirect immunofluorescence, whereas in 68/85 cases (80%) this technique yielded negative results. However, moAb "mixes" against CD44v and patching of surface molecules on B-CLL cells have shown that all B-CLL clones express CD44v. This has been confirmed by Western blot in a number of cases. Thus, two groups of patients whose cells bear CD44v at high or low density, are distinguished. Functions of the two clonotypes were investigated, namely their adhesion to a CD44 ligand and hyaluronate (HA), and effect on HA-induced Ca2+ influx. Cells expressing high density CD44v adhere to HA-coated substrates more efficiently than cells with low density CD44v. In all clones, HA-signaling via CD44 yields Ca2+ influx. This indicates that CD44 mediates activatory signals following interaction with the ligand. INTERPRETATION AND CONCLUSIONS: The clinical relevance of these findings has been ascertained. The 17/85 cases whose cells bore high density CD44v had significantly worse prognostic features than those of patients with low density CD44v, namely more advanced disease stage, LDT < 12 months and therapy requirement. Moreover, the median survival in the former group of patients was < 5 years as opposed to > 12 years in the latter. Therefore, analysis of CD44v expression provides indications of biological and clinical relevance also in low grade lymphoproliferative disorders.     

Overfeeding macronutrients to critically ill adults: metabolic complications

Metabolic complications from overfeeding critically ill patients are serious and sometimes fatal. Nutrition care is best provided through repeated evaluation of patients' responses to feeding. Nutrition support may need to be modified over time to maintain metabolic stability and promote recovery. This article describes the etiology of 10 metabolic complications of overfeeding. Guidelines for recommending macronutrients are discussed, as are factors that could increase the risk of overfeeding. Patients who are very small, very large, or very old are particularly vulnerable to overfeeding. Overfeeding protein has led to azotemia, hypertonic dehydration, and metabolic acidosis. Excessive carbohydrate infusion has resulted in hyperglycemia, hypertriglyceridemia, and hepatic steatosis. High-fat infusions have caused hypertriglyceridemia and fat-overload syndrome. Hypercapnia and refeeding syndrome have also been caused by aggressive overfeeding. Dietitians can prevent or curtail the metabolic complications of overfeeding by identifying patients at risk, providing adequate assessment, coordinating interdisciplinary care plans, and delivering timely and appropriate monitoring and intervention. Dietitians need to document complications, interventions, and the outcomes of their clinical care to evaluate the appropriateness of existing nutrition guidelines.     

Consequences of retirement activities for distress and the sense of personal control

Mink were infected with Aleutian Mink Disease Parvovirus (AMDV) and sacrificed at monthly intervals after infection. During this time humoral immune responses and leucocyte numbers in blood, mesenteric lymph node, spleen and thymus were monitored. Serum hypergammaglobulinaemia was observed together with elevated antibody responses to AMDV NS1 and VP1/2 proteins. In blood, a highly significant increase in CD8+ lymphocytes was observed. However, (presumed)CD4+ cells defined as CD3+CD8- cells, and B lymphocytes remained relatively constant throughout the study. The (presumed)CD4+/CD8+ ratio decreased significantly from greater than 2 to less than 0.5 and MHC-II+ blood leucocytes increased significantly during infection, a large proportion of these being CD8+. Similar changes were observed in the mesenteric lymph node and spleen. Immunohistology of lymph nodes showed a massive expansion of the paracortical area due to increased numbers of CD8+ cells. The staining intensity of B lymphocytes in lymph nodes with a CD79a reactive monoclonal antibody was decreased in the late infection, indicating a possible greater number of plasma cells. Thymic involution was observed during the AMDV infection, although relative increases in CD3high (presumed)CD4+ and CD3highCD8+ single positive cells were observed. These increases were countered by a corresponding reduction in the CD3low(presumed)CD4+CD8+ double positive cell population. Immunohistology of the thymus in normal mink showed that most of the matured CD3+ T cells were present in the inner medulla, while only few CD3+ cells could be found in the outer cortex. In severely infected mink the thymic structural organisation vanished, and CD3+ cells were found throughout the organ.     

Nitrofurantoin-induced hepatotoxicity mediated by CD8+ T cells

Nitrofurantoin is a synthetic nitrofuran commonly used for the treatment and prophylaxis of urinary tract infections. We describe the case of a 75-yr-old woman who was taking nitrofurantoin as prophylaxis against recurrent urinary tract infections, and who subsequently developed pulmonary and hepatic toxicity. We postulate that a breakdown product of the drug or the drug itself complexed to an endogenous peptide is presented by the class I HLA antigen on the hepatocyte cell membrane, inducing cytotoxic T cell activation and subsequently, hepatocyte death.     

Intestinal adaptation is enhanced by epidermal growth factor independent of increased ileal epidermal growth factor receptor expression

BACKGROUND/PURPOSE: Intestinal adaptation after massive small bowel resection (SBR) is augmented by epidermal growth factor (EGF) via an unknown mechanism. We recently have observed that EGF increases the expression of EGF receptor mRNA and protein content in the remnant ileum after SBR. The purpose of this study was to determine whether the magnitude of EGF-induced receptor expression correlates with intestinal adaptation. METHODS: A 50% proximal SBR or sham operation (bowel transection with reanastomosis) was performed on male ICR mice. Animals from each group were then selected randomly to receive either human recombinant EGF (150 microg/kg/d) or saline by twice daily intraperitoneal injections. The remnant ileum was harvested at 1 week, and parameters of adaptation measured as changes in protein content. Ileal EGF receptor mRNA was quantitated using a ribonuclease protection assay. Changes in the expression ileal EGF receptor protein were determined by Western blot after immunoprecipitation. Comparisons of mean values between groups was performed using analysis of variance (ANOVA) and a P value of less than .05 was considered significant. Values are presented as mean +/- SEM. RESULTS: EGF was mitogenic to the ileum after sham operation as monitored by increases in ileal protein content (2.21 +/- 0.002 mg/cm Sham v 2.97 +/- 0.25 mg/cm Sham +/- EGF; P < .05). After SBR, adaptation resulted in increased ileal protein content (4.45 +/- 0.27 mg/cm), which was substantially boosted by EGF (5.98 +/- 0.39 mg/cm; P < .05). No differences were detected in ileal EGF receptor mRNA or protein expression between Sham or SBR groups that did not receive EGF. However, EGF significantly enhanced the expression of ileal EGF receptor mRNA to an equal extent after both sham and SBR (approximately threefold). The magnitude of this increase in EGF receptor protein (four- to sixfold) was similar in both EGF groups as shown by Western blotting. CONCLUSIONS: Changes in ileal EGF receptor expression are not mandatory for adaptation to occur. EGF upregulates the expression of mRNA and protein for its own intestinal receptor in vivo. Because EGF-induced receptor expression was comparable after both SBR and Sham operation, the beneficial effect of EGF during adaptation is likely caused by other factors in addition to increased receptor expression.