Cryptic Plasmids in a Minicell-Producing Strain of Salmonella typhimurium

A minicell-producing strain of Salmonella typhimurium contains two cryptic plasmids. One has a molecular weight of 2.6 x 10(6) to 2.8 x 10(6), is present in multiple copies per cell, and segregates into minicells. The other has a molecular weight of 130 x 10(6), is present in few copies per cell, and probably does not segregate into minicells.     

Insulin therapy for inpatients with diabetes: Perceptions of resident physicians from disparate geographic training programs

Background: Improving diabetes management in hospitalized patients will require educational efforts for all practitioners, particularly resident physicians. Thus, a better understanding of residents' beliefs about diabetes in the hospital must be obtained.Objective: The purpose of this article was to compare and contrast perceptions of resident physicians from 2 geographically distinct training programs regarding management of inpatients with diabetes.Methods: Residents from training programs in the southwestern and southeastern United States were surveyed in 2006 and 2007 about their views on the importance of inpatient glucose control, their perceptions about desirable target glucose ranges, and the problems they encountered when trying to manage hyperglycemia in hospitalized patients.Results: Responses were obtained from 52 of 66 residents at site 1 and from 65 of 85 residents at site 2 (N = 117 total respondents; total response, 77%; mean age, 31 years; 48% men; 61% primary care). Combined analyses revealed that respondents believed that glucose control was “very important” in critically ill patients (96%), perioperative patients (82%), and noncritically ill patients (66%). Most residents indicated that they would target a therapeutic glucose range within published recommendations. Less than half felt “very comfortable” managing inpatient hyperglycemia, hypoglycemia, subcutaneous insulin, or insulin drips. Respondents were not very familiar with existing institutional policies or preprinted order sets for insulin therapy. The most commonly reported barrier to management of inpatient hyper-glycemia was lack of knowledge about appropriate insulin regimens and their use.Conclusions: Trainees from 2 very different educational programs shared common beliefs, knowledge deficits, and perceived barriers about inpatient glucose management. Our findings indicate that trainees were uncertain about how to use insulin therapy in the hospital. Future inpatient diabetes quality-improvement efforts should focus on development of uniform educational programs targeting the management of inpatient diabetes, particularly as it relates to insulin use.     

Enantioselective metabolism of primaquine by human CYP2D6

Given the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated. Six diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (Hss LDH) or plasmodial lactate dehydrogenase (Pf LDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro. All tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of Pf LDH; specificity between the residues involved in H-bonds of the Pf LDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of Pf LDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive. The presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than Pf LDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.     

In Vivo IFN-γ Secretion by NK Cells in Response to Salmonella Typhimurium Requires NLRC4 Inflammasomes

Natural killer (NK) cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-bacterial immunity. NK cell-produced interferon gamma (IFN-γ) contributes to the control of early exponential replication of bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c⁺ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18), independently of Toll-like receptor (TLR)-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.     

Renaturation of dextranase activity from culture supernatant fluids of Streptococcus sobrinus after sodium dodecylsulfate polyacrylamide gel electrophoresis

A rapid and reproducible method for the assay of individual fractions of the multicomponent dextranase activity of Streptococcus sobrinus after reduction/denaturation and electrophoresis in acrylamide gels is described. Multiple forms of dextranase, possible virulence factors in the formation of dental caries by oral Streptococci (S. mutans, S. sobrinus, S. sanguis, S. cricetus, and S. rattus), have been separated in sodium dodecylsulfate-polyacrylamide gels into which an indicator substrate, blue dextran, has been incorporated, and identified after renaturation to remove the reducing/denaturing agents of the Laemmli buffer system.     

Immunochemical heterogeneity of human plasma high density lipoproteins. Identification with apolipoprotein A-I- and A-II-specific monoclonal antibodies

Three mouse monoclonal antibodies specific for human apolipoprotein (apo) A-I and one specific for human apo-A-II were characterized with respect to their binding of high density lipoprotein (HDL) particles in solution. The apo-A-II-specific antibody bound 85% of 125I-HDL and 100% of soluble 125I-apo-A-II. However, none of the apo-A-I-specific antibodies bound greater than 60% of either HDL or soluble apo-A-I. Technical issues such as limiting amounts of antibody or antigen, radioiodination of the ligands, unavailability of the epitopes for reaction with antibody, selective binding of apo-A-I isoforms, and individual allotypic differences in apo-A-I were not responsible for the observed incomplete binding of all HDL and apo-A-I. The results suggested the existence of intrinsic immunochemical heterogeneity of apo-A-I both as organized on HDL as well as in free apo-A-I in solution. The validity of this observed heterogeneity was supported by demonstrating that (i) increased binding of HDL occurred when each of the apo-A-I antibodies was combined to form an oligoclonal antibody mixture, and (ii) 100% binding of HDL occurred when two apo-A-I antibodies were combined with the single apo-A-II antibody. To understand the basis for the heterogeneity of expression of apo-A-I epitopes on HDL, two hypotheses were examined. The first hypothesis that these apo-A-I antibodies distinguished apo-A-I molecules from different synthetic sources was not substantiated. Two of the antibodies bound epitopes on apo-A-I molecules in both thoracic duct lymph as an enriched source of intestinal HDL and the culture supernatants of the hepatic cell line Hep G2 as a source of hepatic HDL. The second hypothesis that the antibodies identified differences in the expression of apo-A-I on HDL subpopulations that were distinguished on the basis of size or net particle charge, i.e. organizational heterogeneity, appeared to provide the best available explanation for the immunochemical heterogeneity of apo-A-I in HDL. Relative differences in the expression of three distinct apo-A-I epitopes were demonstrated in HDL subpopulations obtained by either density gradient ultracentrifugation or chromatofocusing. In light of these studies, we conclude that there is intrinsic heterogeneity in the expression of intramolecular loci representing the apo-A-I epitopes identified by our monoclonal antibodies. Such heterogeneity must be considered in analysis of the biology and physiology of apo-A-I and lipoprotein particles bearing this chain.