Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.     

Association between misalignment of circadian rhythm and obesity in Korean men: Sixth Korea National Health and Nutrition Examination Survey

ABSTRACT

Background: The disruption of circadian rhythm has been found to associate with obesity in vivo and in vitro. Sleep duration, eating habits, total feeding time, and nightshift work can also affect circadian rhythms. This study investigated the association between misalignment of circadian rhythm and obesity in Korean men, using a cross-sectional database.

Methods: This study used data from the Korean National Health and Nutrition Examination Survey (KNHANES), whose study population was 3,658 men aged 18 to 60 years. General and abdominal obesity was defined as a body mass index (BMI) ≥ 25 kg/m² and waist circumference ≥ 90 cm, respectively. Circadian rhythm factors were determined with a self-report questionnaire and included breakfast frequency, sleep duration, and work time. Frequency of breakfast was divided into regular breakfast (five to seven times a week) and irregular breakfast (less than five times a week). Sleep duration was divided into less than 7 hours, 7–9 hours, and over 9 hours. Working time was defined as day/evening, night shift, and other type. The adjusted odds ratios (aORs) and 95% confidence intervals (CIs) for general and abdominal obesity were calculated using multivariable logistic regression according to the number of factors that disturb the circadian rhythm.

Results: Participants with 1 (aOR 1.34, 95% Cl 1.10–1.61) and ≥2 (aOR 1.62, 95% Cl 1.29–2.05) factors disturbing circadian rhythms were associated with elevated risk for general obesity. Similarly, those with 1 (aOR 1.33, 95% Cl 1.09–1.63) and ≥2 (aOR 1.70, 95% Cl 1.32–2.20) factors had elevated risk for abdominal obesity.

Conclusions: Factors disturbing the circadian rhythm were associated with general and abdominal obesity. Additional studies are needed, and associations with metabolic diseases should be investigated.     

The Drosophila tissue polarity gene starry night encodes a member of the protocadherin family.

The tissue polarity genes control the polarity of hairs, bristles and ommatidia in the adult epidermis of Drosophila. We report here the identification of a new tissue polarity gene named starry night (stan). Mutations in this essential gene alter the polarity of cuticular structures in all regions of the adult body. The detailed polarity phenotype of stan on the wing suggested that it is most likely a component of the frizzled (fz) pathway. Consistent with this hypothesis, stan appears to be downstream of and required for fz function. We molecularly cloned stan and found that it encodes a huge protocadherin containing nine cadherin motifs, four EGF-like motifs, two laminin G motifs, and seven transmembrane domains. This suggests that Stan functions in signal reception, perhaps together with Fz.     

The enhancement of electrostriction caused by lowering the solvent dielectric constant leads to the decrease of activation energy in trypsin catalysis.

The contribution of electrostriction of the solvent to the stabilization of the negatively charged tetrahedral transition state of a trypsin-catalyzed reaction was probed by means of kinetic studies involving high-pressure and solvent dielectric constant. A good correlation was observed between the increased catalytic efficiency of trypsin and the decreased solvent dielectric constant. When the dielectric constant of the solvents was lowered by 4.68 units, the loss of activation energy and that of free energy of activation were 2.26 kJ/mol and 3.09 kJ/mol, respectively. The activation volume for k(cat) decreased significantly as the dielectric constant of the solvent decreased, indicating that the degree of electrostriction of the solvent around the charged tetrahedral transition state has been enhanced. These observations demonstrate that the increase in the catalytic efficiency of the trypsin reaction with decreasing dielectric constant resulted from the stabilization of electrostatic energy for the formation of an oxyanion hole, and this stabilization was caused by the increase of electrostricted water around the charged tetrahedral transition state. Therefore, we conclude that control of the solvent dielectric constant can stabilize the tetrahedral transition state, and this lowers the activation energy.     

Determination of rat brain buffering in vivo by 31P-NMR

Buffering capacity of most tissues is composed of both rapid and slow phases, the latter presumably due to active acid extrusion. To examine the time course of brain buffering the brain pH of Sprague-Dawley rats was measured using 31P-nuclear magnetic resonance. The effect on brain pH of 30- or 58-min exposures to 20% CO2 followed by 30- or 38-min recovery periods, respectively, was studied. Brain pH reached its lowest value after a 15-min exposure to elevated CO2, thereafter slowly and steadily increasing. During recovery brain pH rose rapidly in the first 5 min exceeding control brain pH by 0.08 pH units. Brain pH fell during the next 30 min despite increases in blood pH and decreases in blood CO2 tension. Calculated intrinsic brain buffering rose steadily threefold during the last 40 min of CO2 exposure and during the final 30 min of recovery. These data show that in rat brain there is a temporally late buffering process, most likely active acid extrusion, requiring greater than 30 min for full activation and at least 30 min for discontinuation.     

Human lymphoblastoid cell lines secreting antibodies with restricted HLA specificity

Peripheral B lymphocytes obtained from three healthy individuals who had been immunized against peripheral blood lymphocytes from appropriate HLA-incompatible donors were transformed by the use of Epstein-Barr virus. The transformed blastoid B cells were repeatedly subcultured by means of cluster picking, and the HLA antibody-producing cultures were identified by testing the culture supernatants by means of the cytotoxicity assay, using the corresponding donor cells. Thus far, four cell lines that secrete cytotoxic HLA antibodies (MP1, 3, 4, and 5) have been established. Specific immunoabsorption experiments revealed that the antibody activity is carried by lambda-type IgM for MP1, by kappa-type IgM for MP3 and MP5, and by both for MP4. Specificity analysis of a panel of HLA-pretyped cells indicated that MP1 detects DQw2, whereas MP5 recognizes B7. The specificity of MP3 was similar to a DQ specificity termed DC5 (probably equivalent to TA10) but not the same. In the case of MP4, both of the lambda-type and kappa-type antibodies appeared to be directed toward new HLA class 11 determinants.Abbreviations used in this paper HLA human major histocompatibility - EBV Epstein-Barr virus - B-LCL Blymphoblastoid cell line - NA not absorbed - PBS phosphate-buffered saline - SPA Sepharose protein A - NRS normal rabbit serum     

Absence of heat shock protein synthesis in isolated mitochondria and plastids from maize

Examination of the proteins synthesized by isolated mitochondria, chloroplasts, or proplastids from maize tissues showed that a heat treatment at 40 degrees C does not induce or enhance the synthesis of any protein when compared to preparations treated at the control temperature of 28 degrees C. These observations are consistent with the results obtained by labeling proteins in vivo under sterile conditions. In vivo labeling in the presence of cycloheximide during heat shock showed no heat shock protein synthesis. Labeling in the presence of chloramphenicol during heat shock showed a similar heat shock protein pattern as in the absence of the inhibitor. It is concluded that maize organelles do not synthesize heat shock proteins and that, if present, they may be due to bacterial contamination.