Recombinant human preproinsulin expression, purification and reaction with insulin autoantibodies in sera from patients with insulin-dependent diabetes mellitus

A novel prokaryotic expression vector pGEX-6T was designed for high-level expression of recombinant fusion protein with a histidine-hexapeptide and glutathione-S-transferase at its N-terminus and the recombinant human preproinsulin at its C-terminus. Efficiency of expression was investigated in the Escherichia coli strain CAG456. The synthesized protein was sequestered in an insoluble form in inclusion bodies and was purified to homogeneity by one-step affinity chromatography based on the specific complex formation of the histidine-hexapeptide and a chelating matrix which was charged with Ni2+ ions. The antigenic nature of the purified recombinant preproinsulin fusion protein was evaluated by ELISA screening for insulin autoantibodies in selected sera from patients with recent-onset type 1 (insulin-dependent) diabetes mellitus classified by the existence of additional autoantibodies reactive against glutamic acid decarboxylase. 14% of the tested sera (n=43) conttained insulin autoantibodies which strongly recognized the recombinant human preproinsulin. Comparable measurements with both recombinant human preproinsulin and mature insulin suggested that the observed autoantigenicity of preproinsulin was mediated by the C-peptide or/and signal peptide

Insulin autoantibodies as determined by competitive radiobinding assay are positively correlated with impaired beta-cell function — The Ulm-Frankfurt population study

Out of a random population of 4208 non-diabetic pupils without a family history of Type I diabetes 44 (1.05%) individuals had islet cell antibody (ICA) levels greater or equal to 5 Juvenile Diabetes Foundation (JDF) units. 39 of these ICA-positives could be repeatedly tested for circulating insulin autoantibodies (CIAA) using a competitive radiobinding assay. The results were compared with the insulin responses in the intravenous glucose tolerance tests (IVGTT) and with HLA types. Six pupils were positive for CIAA. All of them had complement-fixing ICA, and 5 of them were HLA-DR4 positive. Three of the 6 showed a first-phase insulin response below the first percentile of normal controls. Our data indicate that in population-based studies CIAA can be considered as a high risk marker for impaired beta-cell function in non-diabetic ICA-positive individuals

The Role of Instructor Due Diligence in Volunteer Income Tax Assistance (VITA): Evidence From One University-Level Program

This paper reviews the tax preparation practice of one university-level Volunteer Income Tax Assistance (VITA) program to determine the role, if any, due diligence requirements for paid preparers should have on student preparers. The due diligence requirements as outlined in Circular 230 apply to paid preparers. The Volunteer Standards of Conduct are outlined in IRS publication 4012 and apply to VITA volunteers who are often students. This study analyzes the tax challenges faced by student prepares in a VITA program, and how the increased due diligence standards required of the IRS for paid preparers would be beneficial to the students participating in a VITA program. The study models tax return data to show the financial benefit application of due diligence standards would have for the Internal Revenue Service. The goal of this paper is to show the importance of higher due diligence standards, and we contribute to the literature by providing the first review of VITA tax preparation through the lens of paid-preparer due diligence requirements

Tracking the Trackers, Does Citizen Science Help or Hinder Rare Carnivore Research

Citizen science has gained a strong foothold in wildlife research in recent years. The quest for information about wildlife existing at the most extreme locations at the lowest densities requires more people than most research projects can pay for. Using volunteers for everything from wilderness monitoring to aquatic studies has become common practice in the face of budget short falls and efforts to gain public support. While reporting on citizen science used in small carnivore studies, I found a group of volunteers poised to help researchers learn about animals like wolverines, lynx, fishers, marten and wolves. Compelling people chasing captivating wildlife across some of the harshest winter climates on earth reveals much about the human spirit. Their stories will both inspire readers, and also question the sanity of the researchers and volunteers. But what limitations and problems come with using citizen scientists? A system based on paid wildlife technicians with years of education in wildlife research has largely shifted to volunteers with a few hours of training. Volunteers often overestimate their abilities, resulting in compromised data collection. Coordinating volunteers takes away field time from those organizing research projects. Finally, those working for free are often less reliable than paid researchers

No association between islet cell antibodies and coxsackie B, mumps, rubella and cytomegalovirus antibodies in non-diabetic individuals aged 7–19 years

Viral antibodies were tested in a cohort of 44 isletcell antibody-positive individuals age 7–19 years, and 44 of their islet cell antibody-negative age and sex-matched classmates selected from a population study of 4208 pupils who had been screened for islet cell antibodies. Anti-coxsackie B1-5 IgM responses were detected in 14 of 44 (32%) of the islet cell antibody-positive subjects and in 7 of 44 (16%) control subjects. This difference did not reach the level of statistical significance. None of the islet cell antibody-positive subjects had specific IgM antibodies to mumps, rubella, or cytomegalovirus. There was also no increase in the prevalence or the mean titres of anti-mumps-IgG or IgA and anti-cytomegalovirus-IgG in islet cell antibody-positive subjects compared to control subjects. These results do not suggest any association between islet cell antibodies, and possibly insulitis, with recent mumps, rubella or cytomegalo virus infection. Further studies are required to clarify the relationship between islet cell antibodies and coxsackie B virus infections

ARF6-Mediated Endosome Recycling Reverses Lipid Accumulation Defects in Niemann-Pick Type C Disease

In human Niemann-Pick Type C (NPC) disease, endosomal trafficking defects lead to an accumulation of free cholesterol and other lipids in late endosome/lysosome (LE/LY) compartments, a subsequent block in cholesterol esterification and significantly reduced cholesterol efflux out of the cell. Here we report that nucleotide cycling or cellular knockdown of the small GTP-binding protein, ARF6, markedly impacts cholesterol homeostasis. Unregulated ARF6 activation attenuates the NPC phenotype at least in part by decreasing cholesterol accumulation and restoring normal sphingolipid trafficking. These effects depend on ARF6-stimulated cholesterol efflux out of the endosomal recycling compartment, a major cell repository for free cholesterol. We also show that fibroblasts derived from different NPC patients displayed varying levels of ARF6 that is GTP-bound, which correlate with their response to sustained ARF6 activation. These studies support emerging evidence that early endocytic defects impact NPC disease and suggest that such heterogeneity in NPC disease could result in diverse responses to therapeutic interventions aimed at modulating the trafficking of lipids

IMPLEMENTAÇÃO DE UM DRIVER DE HARD DISK PARA O SISTEMA OPERACIONAL IFX

Arquivo em PD

DRIVER DE TECLADO PARA O SISTEMA OPERACIONAL IFX

Arquivo em PD

Insulinooporność a stężenie androgenów jajnikowych i nadnerczowych w surowicy otyłych kobiet bez chorób towarzyszących i z zespołem policystycznych jajników

The aim of the present study was to evaluate serum concentrations of adrenal and ovarian androgens and sex hormone – binding globulin in obese women without additional diseases and in obese women with polycystic ovary syndrome with and without insulin resistance. Material and methods: The study group involved 73 obese women (39 with PCOS - A and 34 obese without additional diseases – B). The serum concentration of glucose and insulin were measured and the study group was divided on the basis of HOMA index into two subgroups: A I – PCO without insulin resistance (n = 18, mean age 27,2 ± 5,9 yr ; BMI 33,2 ± 3,5 kg/m2); AII - PCO with insulin resistance (n = 21, mean age 27,5 ± 7,1 yr; BMI 37,6 ± 6,5 kg/m2); B I – obese without insulin resistance (n = 8, age 33,5 ± 7,5 yr; BMI 35,2 ± 4,8 kg/m2); B II – obese with insulin resistance (n = 24, age 30,3 ± 5,2 yr; BMI 36,4 ± 5,8 kg/m2). Body mass and height were measured and body mass index was calculated with formula. Body composition was measured using bioimpedance method. The serum concentrations of FSH, LH, total and free testosterone, androstendione, DHEAS, SHBG and insulin were determined by RIA method and glucose was determined by enzymatic procedure. Results: We observed significantly higher body mass, fat mass and BMI in AII subgroup when compared to AI, BI and BII subgroups. Only serum concentration of free testosterone was significantly higher in AII subgroup when compared to AI subgroup. We observed a positive correlation between serum concentrations of insulin and free testosterone in both groups A and B, moreover we observed positive correlations between serum concentrations of insulin and both DHEAS and LH in group B. Conclusions: It seems that insulin resistance plays a key role in the development of hyperandrogenism in obese women. However mechanisms leading to hyperandrogenism in PCOS are still unrevealed and seem to be more complex.Celem prezentowanej pracy była ocena stężenia w surowicy androgenów nadnerczowych i jajnikowych oraz globuliny wiążącej hormony płciowe w 2 grupach kobiet: otyłych bez chorób towarzyszących i otyłych z zespołem PCO z uwzględnieniem występowania u nich stanu insulinooporności.Materiał i metody: Badaniom poddano grupę 73 otyłych kobiet (39 z zespołem PCO - A i 34 z otyłością prostą bez chorób towarzyszących – B). Po wykonaniu oznaczeń stężenia w surowicy glukozy i insuliny na czczo, na podstawie wskaźnika HOMA badane grupy podzielono na podgrupy: A I – PCOS insulinowrażliwe (n = 18, wiek 27,2 ± 5,9 lat; BMI 33,2 ± 3,5 kg/m2); AII - PCOS insulinooporne (n = 21, wiek 27,5 ± 7,1 lat; BMI 37,6 ± 6,5 kg/m2); B I – otyłe insulinowrażliwe (n = 8, wiek 33,5 ± 7,5 lat; BMI 35,2 ± 4,8 kg/m2); B II – otyłe insulinooporne (n = 24, wiek 30,3 ± 5,2 lat; BMI 36,4 ± 5,8 kg/m2). Zmierzono masę ciała i wzrost, wskaźnik masy ciała wyliczono ze wzoru. Oceny składu ciała dokonano metodą bioimpedancji przy użyciu aparatu Bodystat. Metodą radioimmunologiczną (RIA) oznaczono stężenia: FSH, LH, testosteronu całkowitego i wolnego, androstendionu, DHEAS, SHBG oraz insuliny a metodą kolorymetryczną stężenia glukozy.Wyniki: Podgrupa AII charakteryzowała się istotnie wyższą masą ciała i masą tłuszczu oraz BMI w porównaniu z pozostałymi podgrupami. Tylko stężenie testosteronu wolnego było wyższe w podgrupie AII w porównaniu z podgrupą AI. W obu grupach A i B zaobserwowaliśmy dodatnie korelacje między stężeniami insuliny i testosteronu wolnego, ponadto w grupie B występowały dodatnie korelacje między stężeniami insuliny a DHEAS i LH. Wnioski: Wydaje się, że insulinooporność odgrywa kluczową rolę w rozwoju hiperandrogenizmu u otyłych kobiet ale w zespole PCO mechanizmy prowadzące do hiperandrogenizacji są o wiele bardziej złożone