LADA patients treated with sitagliptin and insulin maintained β-cell function by comparison with insulin alone.

Immobilized metal ion affinity chromatography (IMAC) has been explored as a probe into the topography of histidyl residues of a protein molecule. An evaluation of the chromatographic behavior of selected model proteins-thioredoxin, ubiquitin, calmodulin, lysozyme, cytochrome c, and myoglobin on immobilized transition metal ions (Co2+, Ni2+, Cu2+, and Zn2 )-allows establishment of the following facets of the histidyl side chain distribution: (i) either interior or surface; (ii)-when localized on the surface, accessible or unaccessible for coordination; (iii) single or multiple; (iv) when multiple, either distant or vicinal. Moreover, proteins displaying single histidyl side chains on their surfaces may, in some instances, be resolved by IMAC; apparently, the microenvironments of histidyl residues are sufficiently diverse to result in different affinities for the immobilized metal ions. IMAC, previously introduced as an approach to the fractionation of proteins, has become also, upon closer examination, a facile probe into the topography of histidyl residues. This is possible because of the inherent versatility of IMAC; an appropriate metal ion (M2+) can be selected to suit the analytical purpose and a particular chromatographic protocol can be applied (isocratic pH, falling pH, and imidazole elution).The underlying principle of immobilized metal ion affinity chromatography (IMAC) of proteins is the coordination between the electron donor groupings on a protein surface (histidine, tryptophan, cysteine) and chelated (iminodiacetate; IDA) transition metal ions [IDA-M(II)]. This principle of immobilized metal ion affinity (IMA) has been presented by now in some detail (1-4).The practice of IMAC in the purification of proteins has had its empirical phase. There is now a need, from the body of data, to establish somewhat more detailed ground rules that would allow for the use of IMAC in a more predictive manner.Our present experience (4) clearly indicates that histidine functions as the predominant ligand in the IMAC of proteins based on metal ions belonging to the latter part of the first series of transition metal elements (and zinc). Therefore, topography of the protein surface with respect to the location of histidine residues becomes critical for better understanding and exploitation of an IMA event (1, 4-6).A facile probe into the topography of histidyl residues could be quite useful, especially if it could provide information about their surface disposition. This information is required for the rational exploitation of IMAC for protein isolation. In a simple case, one has only to seek information about the surface accessibility of a putative electron donor grouping (7). However, the evaluation of an IMA event in the case of proteins replete in histidine residues may require more comprehensive analysis (8).We now report that IMAC may be exploited as an analytical tool in addition to its use as a protein purification technique (9,10). IMAC can be used to ascertain several facets of the status of a histidyl...

OBJECTIVEThis study investigated the relationship between GAD autoantibody (GADA) titers and changing of b-cell function in patients with latent autoimmune diabetes in adults (LADA). RESEARCH DESIGN AND METHODSThis 3-year prospective study enrolled 95 subjects from 15 Chinese cities including 25 high-titer (GADA ‡180 units/mL) LADA patients, 42 low-titer (GADA <180 units/mL) LADA patients, and 28 type 2 diabetic patients, the latter two groups as controls of similar age, sex, and BMI. Clinical characteristics were determined annually, including glycosylated hemoglobin (HbA 1c ), fasting C-peptide (FCP), and 2-h postprandial C-peptide (PCP). RESULTSDespite similar initial FCP and PCP, FCP and PCP both decreased more in subjects with high GADA titer (FCP from mean 0.49 nmol/L at entry to 0.13 nmol/L at the third year; P < 0.05) than with low GADA titer (FCP from mean 0.48 to 0.38 nmol/L) and type 2 diabetes (FCP from mean 0.47 to 0.36 nmol/L); the latter two groups being similar. After 3 years, residual b-cell function (FCP >0.2 nmol/L) was detected in only 42% with an initial high GADA titer compared with 90% with a low GADA titer and 97% with type 2 diabetes (P < 0.01 for both). GADA positivity at the third year persisted more in subjects with initially high GADA (92%) than with low GADA (26%) titers (P < 0.01). CONCLUSIONSIn selected LADA patients, initial GADA titers identified subjects with different degrees of persistent autoimmunity and disease progression. LADA patients with a low GADA titer had metabolic phenotypes and loss of b-cell function similar to type 2 diabetic patients.

The seven-transmembrane CCR5 was recently found to double as a coreceptor for a genetically diverse family of human and nonhuman primate lentiviruses. Paradoxically, the main region of the envelope protein believed to be involved in CCR5 utilization was mapped to hypervariable region 3, or V3, of the envelope glycoprotein gp120. In this study, we addressed the question of whether functional convergence in CCR5 utilization is mediated by certain V3 residues that are highly conserved among HIV type 1 (HIV-1), HIV type 2, and simian immunodeficiency virus. Site-directed mutagenesis carried out on three such V3 residues revealed that the Arg-298 of HIV-1 gp120 has an important role in CCR5 utilization. In contrast, no effect was observed for the other residues we tested. The inability of Arg-298 mutants to use CCR5 was not attributed to global alteration of gp120 conformation. Neither the expression, processing, and incorporation of mutant envelope proteins into virions, nor CD4 binding were significantly affected by the mutations. This interpretation is further supported by the finding that alanine substitutions of five residues immediately adjacent to the arginine residue had no effect on CCR5 utilization. Taken together, our data strongly suggests that the highly conserved Arg-298 residue identified in the V3 of HIV-1 has a significant role in CCR5 utilization, and may represent an unusually conserved target for future anti-viral designs.