HLA class II DR-DQ amino acids and insulin-dependent diabetes mellitus: application of the haplotype method.

Insulin-dependent diabetes mellitus (IDDM) HLA class II DRB1-DQA1-DQB1 data from four populations (Norwegian, Sardinian, Mexican American, and Taiwanese) have been analyzed to detect the amino acids involved in the disease process. The combination of sites DRB1#67 and 86; DQA1#47; and DQB1#9, 26, 57, and 70 predicts the IDDM component in these four populations, when the results and criteria of the haplotype method for amino acids, developed in the companion paper in this issue of the Journal, are used. The following sites, either individually, or in various combinations, previously have been suggested as IDDM components: DRB1#57, 70, 71, and 86; DQA1#52; and DQB1#13, 45, and 57 (DQB1#13 and 45 correlates 100% with DQB1#9 and 26). We propose that DQA1#47 is a better predictor of IDDM than is the previously suggested DQA1#52, and we add DRB1#67 and DQB1#70 to the HLA DR-DQ IDDM amino acids. We do not claim to have identified all HLA DR-DQ amino acids-or highly correlated sites-involved in IDDM. The frequencies and predisposing/protective effects of the haplotypes defined by these seven sites have been compared, and the effects on IDDM are consistent across the populations. The strongest susceptible effects came from haplotypes DRB1 *0301/DQA1 *0501/ DQB1*0201 and DRB1*0401-5-7-8/DQA1*0301/ DQB1*0302. The number of strong protective haplotypes observed was larger than the number of susceptible ones; some of the predisposing haplotypes were present in only one or two populations. Although the sites under consideration do not necessarily have a functional involvement in IDDM, they should be highly associated with such sites and should prove to be useful in risk assessment.     

Comparing the Ability of Anthropometric Indicators in Identifying Metabolic Syndrome in HIV Patients

Background

Highly active antiretroviral therapy (HAART) can cause side effects in HIV patients, as the metabolic syndrome. Early identification of risk for development of cardiovascular diseases using available reliable and practical methods is fundamental. On this basis, the aim of this study was to compare the effectiveness of anthropometric indicators to identify metabolic syndrome in HIV patients on HAART.

Methods

It is a cross-sectional study. A number of 280 stable HIV patients were studied. It measured weight, height, waist circumference (WC), hip circumference (HP), thigh circumference (TC) and calculated body mass index (BMI), body adiposity index (BAI), waist to hip ratio (WHR) and waist to thigh ratio (WTR). There was also a performance of biochemical tests of lipid profile and fasting glucose. Systemic blood pressure was measured. The criteria proposed by the National Cholesterol Education Program III (NCEP-ATP III) to metabolic syndrome classification was used. Individuals were divided in groups with or without metabolic alterations and their anthropometric indicators were compared. Receiver operating characteristic (ROC) curves were designed for each anthropometric indicator using the metabolic syndrome classification to identify sensitivity and specificity.

Results

WC was a good tool to identify each metabolic disorder separately: total cholesterol (only females, p<0.05), triglycerides (only males, p<0.001), HDL cholesterol (p<0.05), LDL cholesterol (p<005) and fasting glycemic (p<005). WC also showed the best performance to identify metabolic syndrome in both genders (areas under the curve (AUCs): 0.79 and 0.76 for male and female, respectively), while BAI proved to be an inadequate indicator (AUCs: 0.63 and 0.67 for males and females), respectively, in this population.

Conclusions

The central adiposity measure (WC) had the best performance to identify metabolic syndrome, and it is a convenient, cheap and reliable tool that can be used in clinical practice routinely to prevent cardiovascular complications in HIV patients.     

A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase

 Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5′ end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence. Received: 27 April 1999 / Received last revision: 18 October 1999 / Accepted: 1 November 1999     

Two biologically active isomers of dihydroouabain isolated from a commercial preparation

Ouabain is a plant-derived cardiac glycoside that inhibits the catalytic activity of Na(+),K(+)-ATPase (sodium pump; NKA). Dihydroouabain, a derivative of ouabain with a reduced lactone ring, is commonly used as a sodium pump antagonist. It has been assumed that commercially available dihydroouabain is homogeneous. We now report that preparations of dihydroouabain contain two components each with a different potency for inhibition of sodium pump activity. We used reverse-phase HPLC chromatography, UV spectrophotometry, electrospray ionization-mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR) spectroscopy and two independent bioassays to characterize these compounds. The two dihydroouabain fractions (Dho-A and Dho-B) resolved by 3 min chromatographically, had UV absorbance maxima at 196 nm, and comprised 37% and 63% of the stock dihydroouabain, respectively. The molar potency of each component for inhibition of NKA from porcine cerebral cortex differed by 4. 4-fold (Dho-A, IC(50) = 7.13 +/- 0.8 microM; Dho-B, IC(50) = 1.63 +/- 0.12 microM). The relative potencies were 9% and 40% of those of ouabain, respectively. A similar pattern for phosphorylation of NKA was observed. Mass spectrometry (ESI-MS) and fragmentation patterns are consistent with Dho-A and Dho-B being isomers of identical molecular mass (587 Da) and each with six hydroxyl groups, a deoxyhexose sugar moiety and a lactone ring. Furthermore, NMR spectroscopy revealed structural differences between Dho-A and Dho-B by displaying noticeably different chemical shifts at only two groups of proton resonances assigned to H-21 and H-22. The ESI-MS and NMR results confirm the presence of the isomerism at C20 of the lactone ring. Our results demonstrate the existence of two molecular forms of dihydroouabain, each with a different biological potency. These findings underscore the importance of characterizing the purity of dihydroouabain commercial preparations. It also provides possible molecular models for investigating the metabolism of endogenous ouabain-like factors recently reported in mammals.     

The Mediterranean hydrologic budget from a Late Miocene global climate simulation

During the Late Miocene the Mediterranean experienced a period of extreme salinity fluctuations known as the Messinian Salinity Crisis (MSC). The causes of these high amplitude changes in salinity are not fully understood but are thought to be the result of restriction of flow between the Mediterranean and Atlantic, eustatic sea level change and climate. Results from a new Atmospheric General Circulation Model (AGCM) simulation of Late Miocene climate for the Mediterranean and adjacent regions are presented here. The model, HadAM3, was forced by a Late Miocene global palaeogeography, higher CO₂ concentrations and prescribed sea surface temperatures. The results show that fluvial freshwater fluxes to the Mediterranean in the Late Miocene were around 3 times greater than for the present day. Most of this water was derived from North African rivers, which fed the Eastern Mediterranean. This increase in runoff arises from a northward shift in the intertropical convergence zone caused by a reduced latitudinal gradient in global sea surface temperatures. The northwards drainage of the Late Miocene Chad Basin also contributes. Numerical models designed to explore Late Miocene salt precipitation regimes in the Mediterranean, which typically make use of river discharge fluxes within a few tens of percent of present-day values, may therefore be grossly underestimating these fluxes.Although the AGCM simulated Late Miocene river discharge is high, the model predicts a smaller net hydrologic budget (river discharge plus precipitation minus evaporation) than for present day. We discuss a possible mechanism by which this change in the hydrologic budget, coupled with a reduced connection between the Mediterranean and the global ocean, could cause the salinity fluctuations of the MSC.     

Influences of thermal environment on fish growth

Thermoregulation in ectothermic animals is influenced by the ability to effectively respond to thermal variations. While it is known that ectotherms are affected by thermal changes, it remains unknown whether physiological and/or metabolic traits are impacted by modifications to the thermal environment. Our research provides key evidence that fish ectotherms are highly influenced by thermal variability during development, which leads to important modifications at several metabolic levels (e.g., growth trajectories, microstructural alterations, muscle injuries, and molecular mechanisms). In Atlantic salmon (Salmo salar), a wide thermal range (Δ_T 6.4°C) during development (posthatch larvae to juveniles) was associated with increases in key thermal performance measures for survival and growth trajectory. Other metabolic traits were also significantly influenced, such as size, muscle cellularity, and molecular growth regulators possibly affected by adaptive processes. In contrast, a restricted thermal range (Δ_T 1.4°C) was detrimental to growth, survival, and cellular microstructure as muscle growth could not keep pace with increased metabolic demands. These findings provide a possible basic explanation for the effects of thermal environment during growth. In conclusion, our results highlight the key role of thermal range amplitude on survival and on interactions with major metabolism‐regulating processes that have positive adaptive effects for organisms.     

Nutrition of the Tamandua: I. Nutrient composition of termites (Nasutitermes spp.) and stomach contents from wild tamanduas (Tamandua tetradactyla)

Arboreal termites (Nasutitermes spp.) and stomach contents from tamanduas (Tamandua tetradactyla) were collected in central Venezuela during the mid part of the dry season (March) of 1993 and 1994. Nutritional analyses were performed on each caste (workers n = 3), soldiers (n = 5), and alates [n = (1]), on mixed caste samples (n = 1), and on stomach contents from live (n = 5) and roadkill (n = 5) tamanduas. The chemical analysis, expressed on a dry matter (DM) basis, of termite workers, which constituted the majority of the nest populations, showed the highest crude protein (CP) (67%) and the lowest DM (25%) and fat (2%) values. Ash content varied from a low of 4% in alates to a high of 7% in soldiers. The alates contained substantially higher DM (41%) and fat (40%), which was reflected in a higher caloric value (6.88 kcal/g) (gross energy) [GE], and relatively less CP (49%). Among the macrominerals, potassium (K) was consistently the highest, with an overall mean value of 0.54%, while the calcium (Ca) and phosphorus (P) levels showed overall means of 0.26% and 0.67%, respectively. Iron (Fe) was the highest among the trace minerals but highly variable (soldiers, 1,000 ppm; alates, 246 ppm; workers, 394 ppm). Differences in the concentrations of vitamin A and E were found among termites castes, with soldiers showing the highest values (20 and 85 μg/g for retinol and α-tocopherol, respectively). Acid detergent fiber (ADF) was lower in the alates (13%) and workers (27%) compared to the soldiers (35%). Alates' fat was more saturated (39%), while soldiers and workers had a much higher polyunsaturated fatty acid (PUFA) concentration. In general, similar nutrient profiles were found between the tamandua stomach contents and the overall mean composition of Nasutitermes spp. However, stomach contents had much higher ADF, ash, and Fe concentrations (31%, 14%, and 2,748 ppm) than termites (25%, 5%, and 652 ppm) but lower CP, fat, GE, and Ca values (51%, 11%, 4.58 kcal/g, and 0.11% vs. 58%, 15%, 6.01 kcal/g, and 0.26% in termites). The relatively low concentrations of Ca in both stomach contents and termites may be indicative of a low requirement in Myrmecophaga compared to other mammalian species. Diets consumed by free-ranging tamanduas contained on a DM basis 51% CP, 11% fat, 14% ash, 31% ADF, 4.58 kcal/g GE, 0.11% Ca, 0.41% P, 2.52 μg/g retinol, and 44.3 μg/g α-tocopherol. Duplication of these nutrient profiles might greatly benefit captive health and reproduction of this species. © 1996 Wiley-Liss, Inc.