A number of biologically important proteins or protein domains identified recently are fully or partially unstructured (unfolded). Methods that allow studies of the propensity of such proteins to fold naturally are valuable. The traditional biophysical approaches using alcohols to drive ␣-helix formation raise serious questions of the relevance of alcohol-induced structure to the biologically important conformations. Recently we illustrated the extraordinary capability of the naturally occurring solute, trimethylamine N-oxide (TMAO), to force two unfolded proteins to fold to native-like species with significant functional activity. In the present work we apply this technique to recombinant human glucocorticoid receptor fragments consisting of residues 1-500 and residues 77-262. CD and fluorescence spectroscopy showed that both were largely disordered in aqueous solution. TMAO induced a condensed structure in the large fragment, indicated by the substantial enhancement in intrinsic fluorescence and blue shift of fluorescent maxima. CD spectroscopy demonstrated that the TMAO-induced structure is different from the ␣-helix-rich conformation driven by trifluoroethanol (TFE). In contrast to TFE, the conformational transition of the 1-500 fragment induced by TMAO is cooperative, a condition characteristic of proteins with unique structures.An increasing number of biologically important proteins and protein domains have been found to be only partially structured or unstructured (unfolded) under physiological conditions (1). Notably, many of the nuclear transcription factors show disordered transactivation domains in aqueous solution (2). It is generally accepted that the structural uniqueness of proteins determines their biological function. Hence, the identification of unstructured proteins raises the question: what is the structural basis of the functional activity of such proteins/ domains? Whether they act being in unfolded state ("natively unfolded" proteins) or adopt structure upon specific interaction with target molecules is a crucial question. The induced-fit and acidic blob models of the function for such transcription factor proteins represent two opposite points of view (3). Hence, methods that allow studies of the propensity of proteins to fold naturally are valuable.Alcohols (trifluoroethanol, chloroethanol) have long been used to probe the propensity of unstructured protein/domain to form secondary structure (4 -6). Their use has in part been based on the assumption that alcohols might mimic the in vivo conditions under which the disordered domain interacts with a target molecule. It has long been known, however, that alcohols favor the ␣-helical conformation in peptides or proteins regardless of the type of the secondary structure the proteins/peptides form in the biologically relevant (native) conformation (7-9). Hence, until interacting partners of unstructured domains are identified, the current biophysical approaches using such alcohols to drive ␣-helix formation present serious difficulties in inte...

ObjectiveThe study was aimed at estimating the prevalence of type 2 diabetes mellitus and diabetic retinopathy in a rural population of South India.DesignA population-based cross-sectional study.Participants13 079 participants were enumerated.MethodsA multistage cluster sampling method was used. All eligible participants underwent comprehensive eye examination. The fundi of all patients were photographed using 45°, four-field stereoscopic digital photography, and an additional 30° seven-field stereo digital pairs were taken for participants with diabetic retinopathy. The diagnosis of diabetic retinopathy was based on Klein's classification.Main outcome measuresPrevalence of diabetes mellitus and diabetic retinopathy and associated risk factors.ResultsThe prevalence of diabetes in the rural Indian population was 10.4% (95% CI 10.39% to 10.42%); the prevalence of diabetic retinopathy, among patients with diabetes mellitus, was 10.3% (95% CI 8.53% to 11.97%). Statistically significant variables, on multivariate analysis, associated with increased risk of diabetic retinopathy were: gender (men at greater risk; OR 1.52; 95% CI 1.01 to 2.29), use of insulin (OR 3.59; 95% CI 1.41 to 9.14), longer duration of diabetes (15 years; OR 6.01; 95% CI 2.63 to 13.75), systolic hypertension (OR 2.14; 95% CI 1.20 to 3.82), and participants with poor glycemic control (OR 3.37; 95% CI 2.13 to 5.34).ConclusionsNearly 1 of 10 individuals in rural South India, above the age of 40 years, showed evidence of type 2 diabetes mellitus. Likewise, among participants with diabetes, the prevalence of diabetic retinopathy was around 10%; the strongest predictor being the duration of diabetes.

Studies of individual domains or subdomains of the proteins making up the nuclear receptor family have stressed their modular nature. Nevertheless, these receptors function as complete proteins. Studies of specific mutations suggest that in the holoreceptors, intramolecular domain-domain interactions are important for complete function, but there is little knowledge concerning these interactions. The important transcriptional transactivation function in the N-terminal part of the glucocorticoid receptor (GR) appears to have little inherent structure. To study its interactions with the DNA binding domain (DBD) of the GR, we have expressed the complete sequence from the N-terminal through the DBD of the human GR. Circular dichroism analyses of this highly purified, multidomain protein show that it has a considerable helical content. We hypothesized that binding of its DBD to the cognate glucocorticoid response element would confer additional structure upon the N-terminal domain. Circular dichroism and fluorescence emission studies suggest that additional helicity as well as tertiary structure occur in the two-domain protein upon DNA binding. In sum, our data suggest that interdomain interactions consequent to DNA binding imparts structure to the portion of the GR that contains a major transactivation domain.The major identified domains of the nuclear family of receptors are those for ligand binding, DNA binding (DBD), 1 and transactivation, with other functional areas mapped throughout the molecule (1-4). Although the ligand and DNA binding domains have modular structures and, in "domain swapping" experiments, a remarkable ability to carry out their function within the context of other proteins, intramolecular signaling is also important for the proper natural functions of these receptors. Recent experiments studying the effect of mutations on function emphasize the importance of this intramolecular signaling (5, 6). How and when information is exchanged between domains is largely unknown. This is due in part to the fact that no structures are yet available for multidomain proteins from the nuclear receptor family. One intriguing problem is the structural basis for the major transcriptional transactivation function (AF1, tau1) that mutagenesis experiments have defined in the human GR (7). By molecular genetics, AF1 is defined by amino acids 77-262. It appears to function by evoking physical interactions with the basal transcription mechanism, including Ada2 and TATA-binding protein (TBP), possibly through intermediary adapter protein (8, 9). But unlike the ligand binding domain and DBD, AF1 does not appear to function well out of its protein context. Studies of recombinant peptides from the GR containing AF1 have shown it to have little or no structure in simple buffer solutions (10). In the presence of the strong ␣-helix stabilizing agent trifluoroethanol, up to three ␣-helices could form at the C-terminal end of AF1, and functional mutagenesis has shown that the primary sequences at the C-terminal end of AF1 may...

PURPOSE.We report the 4-year incidence, progression, and risk factors of cataract subtypes in type 2 diabetes. METHODS.A total of 779 subjects completed baseline and 4-year follow-up.RESULTS. The incidences of nuclear opalescence (NO), nuclear color (NC), cortical cataract (CC), and posterior subcapsular cataract (PSC) were 70%, 55.2%, 25.7%, and 7.3%, respectively. One-step progressions of NO, NC, CC, and PSC were 14.3%, 16.1%, 8.8%, and 8.1%, respectively, and two-step or more progressions were 5.0%, 6.0%, 0.8%, and 6.0%, respectively. Incident NO was seen in patients 50 to 59 (odds ratio [OR] ¼ 3.3), NC in those 50 to 59 (OR ¼ 2.7) and 60 to 69 (OR ¼ 3.9), and CC in those 60 to 69 (OR ¼ 3.3) years old. A lower hemoglobin A 1c (HbA 1c ; OR ¼ 0.7), longer diabetes duration (OR ¼ 1.1), and hyperopia (OR ¼ 4.0) were associated with incident PSC. Women (OR ¼ 1.7) and patients with higher total cholesterol (OR ¼ 1.3) at baseline showed one-step NO progression. Patients 60 to 69 (OR ¼ 2.8) and ‡70 (OR ¼ 3.8) years old showed one-step NC progression, while those 60 to 69 years old showed one-step CC progression (OR ¼ 6.3). A lower HbA 1c (OR ¼ 0.3) was associated with one-step PSC progression. A higher low-density lipoprotein (OR ¼ 1.6) was associated with two-step or more NO progression. Patients 60 to 69 years old (OR ¼ 6.7) had a greater risk, while those with hyperopia at baseline (OR ¼ 0.2) had lower risk of two-step or more NC progression. Patients 40 to 49 years old constituted the reference group for all.CONCLUSIONS. The 4-year cumulative incidence of cataract is higher than that of progression. Greater age is a risk factor for incidence and progression of most types of cataract.

-methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, removes the mutagenic DNA adduct O 6 -alkylguanine, which is synthesized both endogenously and after exposure to alkylnitrosamines and alkylating antitumor drugs such as 2-chloroethyl-Nnitrosourea (CNU). The MGMT gene is highly regulated in mammalian cells and its overexpression, observed in many types of tumor cells, is often associated with cellular resistance to CNU. Dexamethasone, a synthetic glucocorticoid hormone, was found to increase MGMT expression in HeLa S3 cells, concomitant with their increased resistance to CNU. Two putative glucocorticoid responsive elements (GREs) were identi®ed in the human MGMT (hMGMT) promoter. Transient expression of the luciferase reporter gene driven by an hMGMT promoter fragment containing these GREs was activated by dexamethasone. DNase I footprinting assays demonstrated the binding of glucocorticoid receptor to these sequences. In vitro transcription experiment showed that these DNA sequences are functional in glucocorticoid receptor signal-mediated activation of transcription. These results suggest glucocorticoid-mediated induction of the MGMT gene contributes to high level expression of MGMT.