Periodontal disease in gestational and type 1 diabetes mellitus pregnant women

Objective: The present study evaluated the relationship between periodontal disease and its clinical variables in Brazilian non-diabetic pregnant women (C), gestational diabetes mellitus (GDM), or type 1 diabetes mellitus (T1DM). Subjects and methods: A periodontal exam was performed in one hundred and sixty-one pregnant women (GDM:80; T1DM:31; C:50) by a single-blinded calibrated examiner who recorded plaque index (PI), gingival index (GI), bleeding index (BI), gingival margin location (GM), probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP), and tooth mobility index (MI). The medical variables were age, pregestational body mass index (pre-BMI), fasting plasma glucose (FPG), and glycated hemoglobin (HbA(1c)). Results: The GI, GM, PD, CAL, BOP, and MI were significantly higher (P < 0.01) among GDM and T1DM than for C. The PI was higher in GDM and similar between C and T1DM. The Adjusted Final Model for medical variables to evaluate the effects of groups on periodontal parameters confirmed these results. Conclusions: The presence of periodontal disease was significantly higher in Brazilian diabetic pregnancies (GDM and T1DM) when compared to non-diabetic pregnant women (C). The degree of periodontal disease was similar between the GDM and T1DM groups. Age, pregestational BMI, and HbA(1c) were factors related to CAL development in these two types of diabetes mellitus.

RNA trafficking signals in human immunodeficiency virus type 1

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.

Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.

Nonspecific down-regulation of CD8+ T-Cell responses in mice expressing human Papillomavirus Type 16 E7 oncoprotein from the keratin-14 promoter

The E7 oncoprotein of human papillomavirus 16 (HPV16) transforms basal and suprabasal cervical epithelial cells and is a tumor-specific antigen in cervical carcinoma, to which immunotherapeutic strategies aimed at cytotoxic T-lymphocyte (CTL) induction are currently directed. By quantifying major histocompatibility complex class I tetramer-binding T cells and CTL in mice expressing an HPV16 E7 transgene from the keratin-l l (K14) promoter in basal and suprabasal keratinocytes and in thymic cortical epithelium, we show that antigen responsiveness of both E7- and non-E7-specific CD8(+) cells is down-regulation compared to non-E7 transgenic control mice. We show that the effect is specific for E7, and not another transgene, expressed from the K14 promoter, Down-regulation did not involve deletion of CD8(+) T cells of high affinity or high avidity, and T-cell receptor (TCR) VP-chain usage and TCR receptor density were similar in antigen-responsive cells from E7 transgenic and non-E7 transgenic mice. These data indicate that E7 expressed chronically from the K14 promoter nonspecifically down-regulates CD8+ T-cell responses. The in vitro data correlated with the failure of immunized E7 transgenic mice to control the growth of an E7-expressing tumor challenge, We have previously shown that E7-directed CTL down-regulation correlates with E7 expression in peripheral but not thymic epithelium (T, Dean et al., J, Virol. 73:6166-6170, 1999), The findings have implications for the immunological consequences of E7-expressing tumor development and E7-directed immunization strategies. Generically, the findings illustrate a T-cell immunomodulatory function for a virally encoded human oncoprotein.

Subtropical drosophilids in Australia can be characterized by adult distribution across vegetation type and by height above forest floor

Understanding the pattern in which adult drosophilids of different species are distributed across and within different vegetation types is necessary for accurate interpretation of their local ecology and diversity. Such studies have been conducted mainly in temperate regions, and there is no basis for extrapolating their conclusions to tropical areas. This study describes the vertical distribution (0-20 m) of drosophilids attracted to banana baits in five different vegetation types in subtropical eastern Australia including open woodland, and rain-forest types. The distribution of most of the 15 common species could be characterized three-dimensionally by vegetation type and height above forest floor. Only one species, Scaptodrosophila lativittata, was common in all vegetation types and it was a canopy species in rain forests and a ground-level species in open woodland. Vertical distribution of some species clearly matched that of their larval hosts, but it did not in others. For example, the fungivore Leucophenga scutellata was mostly trapped well above the forest floor, yet it breeds at ground level, suggesting behavioural mode can influence vertical distributions. We conclude that the vertical dimension, although still poorly understood in relation to drosophilid habitats, needs to be taken into account when conducting and interpreting studies aimed at understanding drosophilid populations and communities in the subtropics.

Development of a selective photoactivatable antagonist for corticotropin-releasing factor receptor, type 2 (CRF2)

A novel photoactivatable analog of antisauvagine-30 (aSvg-30), a specific antagonist for corticotropin-releasing factor (CRF) receptor, type 2 (CRF2), has been synthesized and characterized. The N-terminal amino-acid D-Phe in aSvg-30 [D-Phe11,His12] Svg((11-40)) was replaced by a phenyldiazirine, the 4-(1-azi-2,2,2-trifluoroethyl) benzoyl (ATB) residue. The photoactivatable aSvg-30 analog ATB-[ His12] Svg was tested for its ability to displace [I-125-Tyr0] oCRF or [I-125-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 ( rCRF(1)) or mouse CRF receptor, type 2beta (mCRF(2beta)). Furthermore, the ability of ATB- [His12] Svg((12-40)) to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRF(1) (HEK-rCRF(1) cells) or mCRF(2beta) (HEK-mCRF(2beta) cells) was determined. Unlike astressin and photo astressin, ATB- [His12]Svg((12-40)) showed high selective binding to mCRF(2beta) (K-i = 3.1 +/- 0.2 nM) but not the rCRF(1) receptor (K-i = 142. 5 +/- 22.3 nM) and decreased Svg-stimulated cAMP activity in mCRF(2beta)-expressing cells in a similar fashion as aSvg-30. A66-kDa protein was identified by SDS/PAGE, when the radioactively iodinated analog of ATB- [His12]Svg((12-40)) was covalently linked to mCRF(2beta) receptor. The specificity of the photoactivatable I-125-labeled CRF2beta antagonist was demonstrated with SDS/PAGE by the finding that this analog could be displaced from the receptor by antisauvagine-30, but not other unrelated peptides such as vasoactive intestinal peptide (VIP).

Myocardial infarction and type II diabetes - preferential treatment for high risk patients?

Objectives: To compare variability of blood glucose concentration in patients with type II diabetes with (cases) and without (controls) myocardial infarction. A secondary objective was identification of predictive factors for higher blood glucose on discharge from hospital. Design: A retrospective matched case-control study. Participants: Medical notes of 101 type II diabetic patients admitted with a myocardial infarction (MI) and 101 type II diabetic patients (controls) matched on gender and age with no MI were reviewed. Blood glucose concentrations over two consecutive 48-h periods were collected. Demographic data and therapy on admission/discharge were also collected. Results: Patient characteristics were comparable on recruitment excluding family history of cardiovascular disease (P =0.003), dyslipidaemia (P =0.004) and previous history of MI (P =0.007). Variability of blood glucose in cases was greater over the first 48 h compared with the second 48 h (P =0.03), and greater when compared with controls over the first 48 h (P =0.01). Cases with blood glucose on discharge >8.2 mmol / L (n =45) were less likely to have a history of previous MI (P =0.04), ischaemic heart disease (P =0.03) or hypertension (P =0.02). Conclusions: Type II diabetics with an MI have higher and more variable blood glucose concentrations during the first 48 h of admission. Only cardiovascular 'high risk' patients had target blood glucose set on discharge. The desirability of all MI patients with diabetes, having standardized-glucose infusions to reduce variability of blood glucose, should be evaluated in a randomized controlled trial.

Effect of Rosiglitazone on Insulin Sensitivity and Body Composition in Type 2 Diabetic Patients

Objective: To investigate the effects of rosiglitazone (RSG) on insulin sensitivity and regional adiposity (including intrahepatic fat) in patients with type 2 diabetes. Research Methods and Procedures: We examined the effect of RSG (8 mg/day, 2 divided doses) compared with placebo on insulin sensitivity and body composition in 33 type 2 diabetic patients. Measurements of insulin sensitivity (euglycemic hyperinsulinemic clamp), body fat (abdominal magnetic resonance imaging and DXA), and liver fat (magnetic resonance spectroscopy) were taken at baseline and repeated after 16 weeks of treatment. Results: There was a significant improvement in glycemic control (glycosylated hemoglobin -0.7 +/- 0.7%, p less than or equal to 0.05) and an 86% increase in insulin sensitivity in the RSG group (glucose-disposal rate change from baseline: 17.5 +/- 14.5 mumol glucose/min/kg free fat mass, P < 0.05), but no significant change in the placebo group compared with baseline. Total body weight and fat mass increased (p &LE; 0.05) with RSG (2.1 +/- 2.0 kg and 1.4 +/- 1.6 kg, respectively) with 95% of the increase in adiposity occurring in nonabdominal regions. In the abdominal region, RSG increased subcutaneous fat area by 8% (25.0 +/- 28.7 cm(2), p = 0.02), did not alter intra-abdominal fat area, and reduced intrahepatic fat levels by 45% (-6.7 +/- 9.7%, concentration relative to water). Discussion: Our data indicate that RSG greatly improves insulin sensitivity in patients with type 2 diabetes and is associated with an increase in adiposity in subcutaneous but not visceral body regions.

Prediction of Golgi Type II membrane proteins based on their transmembrane domains

Motivation: A major issue in cell biology today is how distinct intracellular regions of the cell, like the Golgi Apparatus, maintain their unique composition of proteins and lipids. The cell differentially separates Golgi resident proteins from proteins that move through the organelle to other subcellular destinations. We set out to determine if we could distinguish these two types of transmembrane proteins using computational approaches. Results: A new method has been developed to predict Golgi membrane proteins based on their transmembrane domains. To establish the prediction procedure, we took the hydrophobicity values and frequencies of different residues within the transmembrane domains into consideration. A simple linear discriminant function was developed with a small number of parameters derived from a dataset of Type II transmembrane proteins of known localization. This can discriminate between proteins destined for Golgi apparatus or other locations (post-Golgi) with a success rate of 89.3% or 85.2%, respectively on our redundancy-reduced data sets.

Regulation of the mouse Nas1 promoter by vitamin D and thyroid hormone

The renal sodium-sulfate cotransporter, NaSi-1, a protein implicated to control serum sulfate levels, has been shown to be regulated in vivo by 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) and tri-iodothyronine (T-3). Recently, we cloned the mouse NaSi-1 gene (Nas1) and in the present study identified a 1,25-(OH)(2)D-3- and T-3-responsive element located within the Nas1 promoter. Mutational analysis of the Nas1 promoter resulted in identification of a direct repeat 6-type vitamin-D-responsive element (DR6 VDRE) at -525 to -508 and an imperfect inverted repeat 0-type T-3-responsive element (IR0 T3RE) at -436 to -425 which conferred 1,25(OH)(2)D-3 and T3 responsiveness, respectively. In summary, we have identified responsive elements that mediate the enhanced transcription of Nas1 by 1,25-(OH)(2)D-3 and T-3, and these mechanisms may provide important clues to the physiological control of sulfate homeostasis.

Detection of Influenza type A virus by LightCycler RT-PCR

Using the Roche LightCycler we developed a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the Influenza A LightCycler RT-PCR (FA-LC-RTPCR) for the rapid detection of Influenza A. The assay was used to examine 178 nasopharyngeal aspirate (NPA) samples, from patients with clinically recognised respiratory tract infection, for the presence of Influenza A RNA. The results were then compared to a testing algorithm combining direct immunofluorescent assy (DFA) and a culture augmented DFA (CA-DFA) assay. In total, 76 (43%) specimens were positive and 98 (55%) specimens were negative by both the FA-LC-RTPCR and the DFA and CA-DFA algorithm. In addition, the FA-LC-RTPCR detected a further 4 (2%) positive specimens, which were confirmed by a conventional RT-PCR method. The high level of sensitivity and specificity, combined with the rapid turnaround time for results, makes the LC-RT-PCR assay suitable for the detection of Influenza A in clinical specimens.

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT(1)-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT(1)-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT(1)-R in caveolae, AT(1)-R. caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT(1)-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT(1)-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT(1)-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT(1)-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT(1)-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT(1)-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT(1)-R through the exocytic pathway, but this does not result in AT(1)-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT(1)-R.