Improved Detection of Abnormal Glucose Tolerance in Africans: The Value of Combining Hemoglobin A1c With Glycated Albumin.

OBJECTIVE: In African-born Blacks living in America, we determined by BMI category 1) prevalence of abnormal glucose tolerance (Abnl-GT) and 2) diagnostic value and reproducibility of hemoglobin A1c (HbA1c), fructosamine, and glycated albumin (GA). RESEARCH DESIGN AND METHODS: Participants (n = 416; male, 66%; BMI 27.7 ± 4.5 kg/m2 [mean ± SD]) had an oral glucose tolerance test with HbA1c, GA, and fructosamine assayed. These glycemic markers were repeated 11 ± 7 days later. Abnl-GT diagnosis required 0 h ≥5.6 mmol/L (≥100 mg/dL) and/or 2 h ≥7.8 mmol/L (≥140 mg/dL). Thresholds for HbA1c, GA, and fructosamine were the values at the 75th percentile for the population (39 mmol/mol [5.7%], 14.2%, and 234 µmol/L, respectively). RESULTS: Abnl-GT prevalence in the nonobese was 34% versus 42% in the obese (P = 0.124). Reproducibility was excellent for HbA1c and GA (both κ ≥ 0.8), but moderate for fructosamine (κ = 0.6). Focusing on HbA1c and GA in the nonobese, we found as single tests the sensitivities of HbA1c and GA were 36% versus 37% (P = 0.529). Combining HbA1c and GA, sensitivity increased to 58% because GA identified 37% of Africans with Abnl-GT not detected by HbA1c (P value for both tests vs. HbA1c alone was <0.001). For the obese, sensitivities for HbA1c, GA, and the combined tests were 60%, 27%, and 67%, respectively. Combined test sensitivity did not differ from HbA1c alone (P = 0.25) because GA detected only 10% of obese Africans with Abnl-GT not detected by HbA1c. CONCLUSIONS: Adding GA to HbA1c improves detection of Abnl-GT in nonobese Africans.

Stress Measured by Allostatic Load Varies by Reason for Immigration, Age at Immigration, and Number of Children: The Africans in America Study.

Stress leads to physiologic dysfunction and cardiometabolic disease. Allostatic load score (ALS) measures stress-induced cardiovascular, metabolic, and inflammatory biomarkers. We estimated the odds of high ALS by reason for and age at immigration, duration of American residence, number of children, and socioeconomic status in 193 African immigrants (male: 65%, age 41 ± 10 y (mean ± Standard Deviation (SD)), range 22-65 y). ALS was calculated with High-ALS defined as ALS ≥ 3.0 and Low-ALS defined as ALS < 3.0. Oral glucose tolerance tests (OGTT) were performed, the cardiovascular disease (CVD) risk estimated, and TNF-α, an inflammatory cytokine, measured. Logistic regression was used to estimate odds of High-ALS. In the High- and Low-ALS groups, ALS were 4.0 ± 1.2 vs. 1.3 ± 0.7, diabetes prevalence: 14% vs. 4%, CVD risk: 23% vs. 8%, TNF-α levels: 15 ± 9 vs. 11 ± 6 pg/mL, respectively (all p ≤ 0.01). Immigrants were more likely to be in the High-ALS group if their reason for immigration was work or asylum/refugee (OR 2.18, p = 0.013), their age at immigration was ≥30 y (OR 3.28, p < 0.001), their duration of residence in United States was ≥10 y (OR 3.16, p = 0.001), or their number of children was ≥3 (OR 2.67, p = 0.019). Education, income, health insurance, marital status, and gender did not affect High-ALS odds. Factors adversely influencing allostatic load and cardiometabolic health in African immigrants were age at and reason for immigration, duration of residence in America, and number of children.

Is UV-Induced Electron-Driven Proton Transfer Active in a Chemically Modified A·T DNA Base Pair?

Transient electronic and vibrational absorption spectroscopies have been used to investigate whether UV-induced electron-driven proton transfer (EDPT) mechanisms are active in a chemically modified adenine-thymine (A·T) DNA base pair. To enhance the fraction of biologically relevant Watson-Crick (WC) hydrogen-bonding motifs and eliminate undesired Hoogsteen structures, a chemically modified derivative of A was synthesized, 8-(tert-butyl)-9-ethyladenine (8tBA). Equimolar solutions of 8tBA and silyl-protected T nucleosides in chloroform yield a mixture of WC pairs, reverse WC pairs, and residual monomers. Unlike previous transient absorption studies of WC guanine-cytosine (G·C) pairs, no clear spectroscopic or kinetic evidence was identified for the participation of EDPT in the excited-state relaxation dynamics of 8tBA·T pairs, although ultrafast (sub-100 fs) EDPT cannot be discounted. Monomer-like dynamics are proposed to dominate in 8tBA·T.

The C terminus of sigma(32) is not essential for degradation by FtsH.

A key step in the regulation of heat shock genes in Escherichia coli is the stress-dependent degradation of the heat shock promoter-specific sigma(32) subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including sigma(32), as degradation signals for AAA proteases. We investigated the role of the C terminus of sigma(32) in FtsH-dependent degradation by analysis of C-terminally truncated sigma(32) mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of sigma(32) did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of sigma(32) was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated sigma(32) mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of sigma(32) in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.

The heat shock response of Escherichia coli.

A large variety of stress conditions including physicochemical factors induce the synthesis of more than 20 heat shock proteins (HSPs). In E. coli, the heat shock response to temperature upshift from 30 to 42 degrees C consists of the rapid induction of these HSPs, followed by an adaptation period where the rate of HSP synthesis decreases to reach a new steady-state level. Major HSPs are molecular chaperones, including DnaK, DnaJ and GrpE, and GroEL and GroES, and proteases. They constitute the two major chaperone systems of E. coli (15-20% of total protein at 46 degrees C). They are important for cell survival, since they play a role in preventing aggregation and refolding proteins. The E. coli heat shock response is positively controlled at the transcriptional level by the product of the rpoH gene, the heat shock promoter-specific sigma32 subunit of RNA polymerase. Because of its rapid turn-over, the cellular concentration of sigma32 is very low under steady-state conditions (10-30 copies/cell at 30 degrees C) and is limiting for heat shock gene transcription. The heat shock response is induced as a consequence of a rapid increase in sigma32 levels and stimulation of sigma32 activity. The shut off of the response occurs as a consequence of declining sigma32 levels and inhibition of sigma32 activity. Stress-dependent changes in heat shock gene expression are mediated by the antagonistic action of sigma32 and negative modulators which act upon sigma32. These modulators are the DnaK chaperone system which inactivate sigma32 by direct association and mediate its degradation by proteases. Degradation of sigma32 is mediated mainly by FtsH (HflB), an ATP-dependent metallo-protease associated with the inner membrane. There is increasing evidence that the sequestration of the DnaK chaperone system through binding to misfolded proteins is a direct determinant of the modulation of the heat shock genes expression. A central open question is the identity of the binding sites within sigma32 for DnaK, DnaJ, FtsH and the RNA polymerase, and the functional interplay between these sites. We have studied the role of two distinct regions of sigma32 in its activity and stability control: region C and the C-terminal part. Both regions are involved in RNA polymerase binding.

Control of Azospirillum brasilense NifA activity by P(II): effect of replacing Tyr residues of the NifA N-terminal domain on NifA activity.

It was previously reported that the N-terminal domain of Azospirillum brasilense NifA was a negative regulator of the NifA activity and that the P(II) protein prevented this inhibition under nitrogen fixing conditions. Here, we show that a mutation of a single Tyr residue at position 18 of the N-terminal domain of NifA led to an active NifA protein that did not require P(II) for activation under nitrogen fixation conditions.

Role of region C in regulation of the heat shock gene-specific sigma factor of Escherichia coli, sigma32.

Expression of heat shock genes is controlled in Escherichia coli by the antagonistic action of the sigma32 subunit of RNA polymerase and the DnaK chaperone system, which inactivates sigma32 by stress-dependent association and mediates sigma32 degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among sigma32 homologs, termed region C, was proposed to play a role in sigma32 degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of sigma32, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the sigma32 protein did not affect sigma32 degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in sigma32 control by DnaK and FtsH. Instead, the sigma32 mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of sigma32 to RNA polymerase. Region C thus confers on sigma32 a competitive advantage over other sigma factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.

Modulation of NifA activity by PII in Azospirillum brasilense: evidence for a regulatory role of the NifA N-terminal domain.

Azospirillum brasilense NifA, which is synthesized under all physiological conditions, exists in an active or inactive from depending on the availability of ammonia. The activity also depends on the presence of PII, as NifA is inactive in a glnB mutant. To investigate further the mechanism that regulates NifA activity, several deletions of the nifA coding sequence covering the amino-terminal domain of NifA were constructed. The ability of these truncated NifA proteins to activate the nifH promoter in the absence or presence of ammonia was assayed in A. brasilense wild-type and mutant strains. Our results suggest that the N-terminal domain is not essential for NifA activity. This domain plays an inhibitory role which prevents NifA activity in the presence of ammonia. The truncated proteins were also able to restore nif gene expression to a glnB mutant, suggesting that PII is required to activate NifA by preventing the inhibitory effect of its N-terminal domain under conditions of nitrogen fixation. Low levels of nitrogenase activity in the presence of ammonia were also observed when the truncated gene was introduced into a strain devoid of the ADP-ribosylation control of nitrogenase. We propose a model for the regulation of NifA activity in A. brasilense.

Characterization of the ntrBC genes of Azospirillum brasilense Sp7: their involvement in the regulation of nitrogenase synthesis and activity.

A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, was cloned. The nucleotide sequence of a 3.8 kb subfragment was established. This led to the identification of two open reading frames, encoding polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the putative Azospirillum ntrC gene was shown to restore nitrogen fixation under free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulinodans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A brasilense. These mutants were prototrophic and Nif+. However, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusion was expressed in the mutants at about 60% of the level in the wild type. In the presence of ammonia, the fusion was similarly expressed (60% of the maximum) both in the wild type and mutants. Addition of ammonia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitrogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixation, although NtrC controls nifA expression to some extent. They are, however, required for the switch-off of nitrogenase activity.

Regulation of nitrogen fixation in Azospirillum brasilense Sp7: involvement of nifA, glnA and glnB gene products.

The expression of nifA-, niH- and nifB-lacZ fusions was examined in different mutants of Azospirillum brasilense. Mutations in nifA, glnA and glnB severely impaired the expression of nifH- and nifB-lacZ fusions. By contrast, a nifA-lacZ fusion was not affected in a nifA or a glnB background and was only partially impaired in glnA mutants. It is proposed that in A. brasilense, the PII protein and glutamine synthetase are involved in a post-translational modification of NifA.