Biochemical analysis of mouse FKBP60, a novel member of the FKPB family.

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).     

Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.     

Determination of duodenogastric reflux by quantification of bile acids in the gastric juice

The existence of duodeno-gastric reflux was evaluated in a group of 15 healthy subjects, in fasting and for 24 hours. The assessment of duodeno-gastric reflux was made by quantitation of the bile acids (BA) present in the gastric juice. The individual free and conjugated BA were separated and quantified by means of thin-layer chromatography and in situ spectrofluorometry. In 7 of the subjects studied no BA were detected, and in the other 8 subjects the BA levels were below 6 mumol reflux/hour. There were no free BA detected in any of the subjects. The levels of BA in gastric juice increased progressively with age, but there were no differences between sex. The chromatographic technique used is highly sensitive for the analysis of BA in biological samples. The study of BA in the gastric juice provides a quantitative and reliable assessment of the degree of duodeno-gastric reflux.     

Atomic absorption spectrophotometry of rubidium levels in several biological media. Technical problems and preliminary results]

The mean of Rubidium rates in human plasma is found in the range of 0,1 mg/l with poor fluctuations; it is significantly less in woman than in man. There is a good linear correlation between rates in plasma and in red cell, rates which are in the same relation than potassium ones. Mean urinary clearance of rubidium is 16 ml/mm but there are large variations.     

Comparison of whole-cell protein electrophoretic profiles of Haemophilus influenzae: implementation of a microcomputer mainframe linked system and description of a new similarity coefficient

A microcomputer mainframe linked system is described which allows video camera data capture and storage of one-dimensional whole-cell protein electrophoresis gel images, processing of normalized traces to produce a similarity matrix, and analysis of the matrix using the commercial cluster analysis program CLUSTAN. A new similarity coefficient is introduced which takes into account both band position and intensity. Forty-five strains of Haemophilus influenzae, including the eight biotypes and six serotypes, were analyzed using this system. Results demonstrated groupings which are consistent with known genetic relationships.     

An improved purification method for cytosolic malate dehydrogenase from several sources

A new purification method for cytosolic malate dehydrogenases from several sources has been developed. The procedure, employing chromatographies on 5'AMP-Sepharose, DEAE-Sephacel and Blue-Sepharose, allows for a rapid isolation of the enzyme (approximately 40 hours), in large quantities, with good yields (45-54%). The specific activity of final preparations were around 1300 I.U./mg and were judged homogeneous by polyacrylamide gradient gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size exclusion chromatography and isoelectric focusing.     

Quantification of human spermatogenesis: germ cell degeneration during spermatocytogenesis and meiosis in testes from younger and older adult men

Germ cell degeneration during spermatocytogenesis and meiosis was investigated to explain the age-related decline in daily sperm production (DSP). Numbers of Types A-dark, A-pale, and B-spermatogonia, potential daily sperm production per g parenchyma (PDSP) based on type B-spermatogonia, early primary spermatocytes, and late primary spermatocytes, and DSP per g based on early spermatids were determined in 15 men aged 20 to 48 yr (mean +/- SEM, 33 +/- 2 yr) and 15 men aged 52 to 90 yr (65 +/- 3 yr). Testes obtained within 15 h of death (largely due to trauma or heart failure) were perfused vascularly with glutaraldehyde. The number of each cell type per g parenchyma was calculated as the product of the percentage of nuclei in the parenchyma times a correction factor for section thickness and nuclear diameter divided by the volume of a single nucleus of that cell type. Paired testicular weight was lower (p less than 0.01) in older men (33 +/- 3 g) than in the younger men (49 +/- 3 g). Younger and older men had similar numbers of A-dark, A-pale, and B-spermatogonia per g parenchyma. PDSP based on late primary spermatocytes and DSP based on early spermatids were lower (p less than 0.01) in older men than in younger men. In younger men, PDSP was similar (p greater than 0.05) between B-spermatogonia and late primary spermatocytes, whereas DSP measured at the spermatid level was abruptly lower than that estimated from younger cell types. Older men showed reduction in PDSP between early and late primary spermatocytes, with further reduction occurring in DSP at the spermatid level.(ABSTRACT TRUNCATED AT 250 WORDS)