Disproportionately elevated fasting proinsulin levels in normoglycemic patients with thalassemia major are correlated to the degree of iron overload

OBJECTIVE: To analyze the secretion of the insulin precursor proinsulin in patients with beta-thalassemia and its possible relation to iron overload. METHODS: We assessed fasting proinsulin, insulin, C-peptide and glucose levels from 34 patients with beta-thalassemia and 33 healthy controls. The correlation to age, body mass index, hepatic iron concentration, serum ferritin and serum AST was analyzed. RESULTS: Fasting proinsulin (p < 0.002) and proinsulin-to-insulin ratio (p < 0.02) were significantly increased in patients with thalassemia irrespective of the degree of glucose tolerance. They correlated positively to serum ferritin, liver iron, patient age and serum AST (all p < 0.05). CONCLUSIONS: Disproportionately elevated proinsulin levels in thalassemic patients indicate early beta-cell dysfunction due to siderosis. An additional biological significance of hyperproinsulinemia and its possible ability to predict long-term iron toxicity in these patients remain to be clarified.     

Validation of a method for quantification of ketobemidone in human plasma with liquid chromatography-tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC-MS-MS) method for determination of the analgesic aminophenol ketobemidone in human plasma is presented. Two preparation methods for plasma samples containing ketobemidone were compared, liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Both methods showed good precision (n=10), 1.7% and 2.9%, respectively (0.04 micro M) and 1.1% and 2.5%, respectively (0.14 micro M). The accuracy was 98% and 103%, respectively (0.04 micro M) and 105% and 99%, respectively (0.14 micro M). Ketobemidone could be quantified at 0.43 nM, with a relative standard deviation of 17.5% (n=19) using LLE and 18.6% (n=10) using SPE. This level was an order of magnitude lower than earlier reported quantification limits. Quantitative data from plasma samples analyzed with LC-MS-MS were in good agreement with those obtained by gas chromatography with chemical ionization mass spectrometry (GC-CI/MS). This indicates that LC-MS-MS is a good alternative method to GC-MS as it is more sensitive and time-consuming derivatization can be avoided.     

Conjugative plasmid transfer in gram-positive bacteria.

Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.     

Use of tritium labeled amino acid conjugates of prostaglandins and thromboxanes as labeled ligands in prostanoid radioimmunoassay

Conjugates of prostaglandins and thromboxanes with tritium labeled amino acids were prepared and employed as labeled ligands in porstaglandin and thromboxane radioimmunoassays. Assays for PGF_2α, 15-keto-13, 14-dihydro-PGF_2α, TXB₂ and 15-keto-13, 14-dihydro-TXB₂ were evaluated in comparative studies using either these heterologous ligands or the corresponding homologus tritiated eicosanoid as tracers. Binding properties for the respective antibodies were found to be similar using either tracer.Three biological studies were also conducted, viz. study of the release of TXB₂ during collagen induced platelet aggregation, of 15-keto-13, 14-dihydro-TXB₂ during guinea pig pulmonary anaphylaxis, and of PGF_2α (measured as 15-keto-13, 14-dihydro-PGF_2α in peripheral plasma) during bovine luteolysis. The analyses gave comparable results using either the heterologous or the homologous assay.Thus, this type of labeled prostanoid conjugates may serve as a convenient alternative to homologous tracers in radioimmunoassay. Heterologous tracers may even in certain cases provide the only simple solution to the problem of preparing a labeled ligand of high specific activity.     

Microbial degradation of secondaryn-alkyl sulfates and secondary alkanols

Secondaryn-alkyl sulfates (and secondary alkanols) prove to be degraded aerobically by aPseudomonas species (Ps. B-2) to carbon dioxide, water and sulfate. The proposed mechanism for the biodegradation proceeds via the alcohol → ketone → hydroxyketone → dione → aldehyde + acid. Consequently, two fatty acids are formed which are subsequently degraded by β-oxidation. This pathway requires the presence of molecular oxygen for the hydroxylation of the ketone. Attempts to isolate bacteria growing on secondary alcohols anaerobically were unsuccessful.     

Cross-Reactions between the Cytotoxic T-Lymphocyte Responses of Human Immunodeficiency Virus-Infected African and European Patients

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.     

Hunting and consumption of rodents by children in the Lassa fever endemic area of Faranah,Guinea

As a consequence of the Ebola outbreak, human–animal contact has gained importance for zoonotic transmission surveillance. In Faranah (Upper Guinea), daily life is intertwined with rodents, such as the Natal multimammate mouse, Mastomys natalensis; a reservoir for Lassa virus (LASV). However, this contact is rarely perceived as a health risk by residents, although Lassa fever (LF) is known to be endemic to this region. Conversely, these observations remain a great concern for global health agendas. Drawing on ethnographic research involving interviews, focus group discussions, participant observations, and informal discussions over four months, we first identified factors that motivated children to hunt and consume rodents in Faranah villages, and thereafter, explored the knowledge of LF infection in children and their parents. Furthermore, we studied two dimensions of human-rodent encounters: 1) space-time of interaction and 2) factors that allowed the interaction to occur and their materiality. This approach allowed us to contextualize child-rodent contacts beyond domestic limits in the fallow fields, swamps, and at other times for this practice. A close look at these encounters provided information on rodent trapping, killing, and manipulation of cooking techniques and the risk these activities posed for the primary transmission of LASV. This research facilitated the understanding of children’s exposure to M. natalensis during hunting sessions and the importance of rodent hunting, which is a part of their boyish identity in rural areas. Determination of when, where, why, and how children, rodents, and environments interacted allowed us to understand the exposures and risks important for human and animal surveillance programs in the Lassa-endemic region.     

Label-free LC-MSMS analysis of vitreous from autoimmune uveitis reveals a significant decrease in secreted Wnt signalling inhibitors DKK3 and SFRP2

Equine recurrent uveitis is a severe and frequent blinding disease in horses which presents with auto-reactive invading T-cells, resulting in the destruction of the inner eye. Infiltration of inflammatory cells into the retina and vitreous is driven by currently unknown guidance cues, however surgical removal of the vitreous (vitrectomy) has proven therapeutically successful. Therefore, proteomic analyses of vitrectomy samples are likely to result in detection of proteins contributing to disease pathogenesis. Vitreous from healthy and ERU diseased horses were directly compared by quantitative mass spectrometry based on label-free quantification of peak intensities across samples. We found a significant upregulation of complement and coagulation cascades and downregulation of negative paracrine regulators of canonical Wnt signalling including the Wnt signalling inhibitors DKK3 and SFRP2. Based on immunohistochemistry, both proteins are expressed in equine retina and suggest localisation to retinal Müller glial cells (RMG), which may be the source cells for these proteins. Furthermore, retinal expression levels and patterns of DKK3 change in response to ERU. Since many other regulated proteins identified here are associated with RMG cells, these cells qualify as the prime responders to autoimmune triggers.