Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.     

A Deletion Map of cyc1 Mutants and Its Correspondence to Mutationally Altered Iso-1-Cytochromes c of Yeast

Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants.     

The fine structure of the terminal segment of the bovine seminiferous tubule

Summary The intratesticular excurrent duct system of the bull is composed of rete testis, tubuli recti, and the terminal segment of the seminiferous tubules. Each terminal segment is surrounded by a vascular plexus and may be subdivided into a transitional region, middle portion, and terminal plug. The modified supporting cells of the middle portion and the terminal plug no longer display the typical Sertoli-Sertoli junctions seen in the transitional region and the seminiferous tubule proper. In the region of the terminal plug a distinct central lumen is generally not observed: spermatozoa and tubular fluid must pass through an intricate system of communicating clefts between the apices of the closely attached modified supporting cells. Vacuoles in the supranuclear region of the cells in the middle portion indicate strong transepithelial fluid transport. In analogy to the epithelium of rete testis and tubuli recti, the supporting cells of the terminal segment are capable of phagocytosing spermatozoa. The vascular plexus investing the terminal segment serves a dual purpose: it is a regulatory device for fluid and sperm transport, as well as an area of increased diapedesis for white blood cells.Supported by a grant from the Deutsche Forschungsgemeinschaft     

Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study)

Objective

to assess the levels and determinants of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) in a healthy Caucasian population.

Methods

population sample of 2884 men and 3201 women aged 35 to 75. IL-1β, IL-6 and TNF-α were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay.

Results

Spearman rank correlations between duplicate cytokine measurements (N = 80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48–3.90) pg/ml for IL-1β; 1.47 (0.71–3.53) pg/ml for IL-6; 2.89 (1.82–4.53) pg/ml for TNF-α and 1.3 (0.6–2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1β levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-α levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity.

Conclusion

Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.     

Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.     

Leprosy in a prison population: A new active search strategy and a prospective clinical analysis

BackgroundThis study evaluates an active search strategy for leprosy diagnosis based on responses to a Leprosy Suspicion Questionnaire (LSQ), and analyzing the clinical, immunoepidemiological and follow-up aspects for individuals living in a prison population.MethodsA cross-sectional study based on a questionnaire posing 14 questions about leprosy symptoms and signs that was distributed to 1,400 prisoners. This was followed by dermatoneurological examination, anti-PGL-I serology and RLEP-PCR. Those without leprosy were placed in the Non-leprosy Group (NLG, n = 1,216) and those diagnosed with clinical symptoms of leprosy were placed in the Leprosy Group (LG, n = 34).FindingsIn total, 896 LSQ were returned (64%), and 187 (20.9%) of the responses were deemed as positive for signs/symptoms, answering 2.7 questions on average. Clinically, 1,250 (89.3%) of the prisoners were evaluated resulting in the diagnosis of 34 new cases (LG), based on well-accepted clinical signs and symptoms, a new case detection rate of 2.7% within this population, while the NLG were comprised of 1,216 individuals. The confinement time medians were 39 months in the LG while it was 36 months in the NLG (p>0.05). The 31 leprosy cases who responded to the questionnaire (LSQ+) had an average of 1.5 responses. The symptoms “anesthetized skin area” and “pain in nerves” were most commonly mentioned in the LG while “tingling, numbness in the hands/feet”, “sensation of pricks and needles”, “pain in nerves” and “spots on the skin” responses were found in more than 30% of questionnaires in the NLG. Clinically, 88.2% had dysesthetic macular skin lesions and 97.1% presented some peripheral nerve impairment, 71.9% with some degree of disability. All cases were multibacillary, confirming a late diagnosis. Anti-PGL-I results in the LG were higher than in the NLG (p<0.0001), while the RLEP-PCR was positive in 11.8% of the patients.InterpretationOur findings within the penitentiary demonstrated a hidden prevalence of leprosy, although the individuals diagnosed were likely infected while living in their former communities and not as a result of exposure in the prison. The LSQ proved to be an important screening tool to help identify leprosy cases in prisons.     

An undecided coiled coil: the leucine zipper of Nek2 kinase exhibits atypical conformational exchange dynamics

Leucine zippers are oligomerization domains used in a wide range of proteins. Their structure is based on a highly conserved heptad repeat sequence in which two key positions are occupied by leucines. The leucine zipper of the cell cycle-regulated Nek2 kinase is important for its dimerization and activation. However, the sequence of this leucine zipper is most unusual in that leucines occupy only one of the two hydrophobic positions. The other position, depending on the register of the heptad repeat, is occupied by either acidic or basic residues. Using NMR spectroscopy, we show that this leucine zipper exists in two conformations of almost equal population that exchange with a rate of 17 s(-1). We propose that the two conformations correspond to the two possible registers of the heptad repeat. This hypothesis is supported by a cysteine mutant that locks the protein in one of the two conformations. NMR spectra of this mutant showed the predicted 2-fold reduction of peaks in the (15)N HSQC spectrum and the complete removal of cross peaks in exchange spectra. It is possible that interconversion of these two conformations may be triggered by external signals in a manner similar to that proposed recently for the microtubule binding domain of dynein and the HAMP domain. As a result, the leucine zipper of Nek2 kinase is the first example where the frameshift of coiled-coil heptad repeats has been directly observed experimentally.