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.     

Intra- and inter-specific effects of mast seeding on seed fates of two sympatric <Emphasis Type="Italic">Corylus</Emphasis> species

We released seeds of two sympatric tree species, Corylus mandshurica (seed with thinner seed hull, higher nutrition) and C. heterophylla (seeds with thicker seed hull, lower nutrition) in the masting year of C. mandshurica in 2008, and C. heterophylla in 2009, respectively, to investigate how seed masting of the two sympatric Corylus species affects seed removal and dispersal fitness of the two species differently at both intra- and inter-specific levels. At intra-specific level, the authors found mast seeding of both C. mandshurica and C. heterophylla significantly reduced seed removal, seed consumption, but increased seed dispersal distance and seed dispersal fitness of the released seeds. Mast seeding of C. mandshurica increased seed caching of C. mandshurica. At inter-specific level, the authors found mast seeding of C. mandshurica reduced seed removal of C. heterophylla, but mast seeding of C. heterophylla did not significantly reduce seed removal of C. mandshurica. Mast seeding of C. mandshurica reduced seed consumption of C. heterophylla, while mast seeding of C. heterophylla reduced seed consumption of C. mandshurica. We found mast seeding of C. mandshurica significantly reduced seed dispersal distance of C. heterophylla, while mast seeding of C. heterophylla significantly increased seed dispersal distance of C. mandshurica. We found that mast seeding of C. mandshurica significantly increased seed dispersal fitness of C. heterophylla, while mast seeding of C. heterophylla did not significantly increase seed dispersal fitness of C. mandshurica. More studies are needed to reveal the ecological consequences of mast seeding at inter-specific or community-level. Seed traits may attribute the differences of mast seeding at inter-specific level. Because seeds with thinner seed hull and higher nutrition were more harvested and eaten by rodents, mast seeding of C. mandshurica might have reduced seed removal and seed consumption, but increased dispersal fitness of C. heterophylla (seeds with thicker seed hull, lower nutrition). Therefore, synchrony among species is, or is not, selectively beneficial to the focus species depends on seed traits which determine gains from mast seeding at inter-specific level.     

Thermodynamic and sequential characteristics of phase separation and droplet formation for an intrinsically disordered region/protein ensemble

Liquid–liquid phase separation (LLPS) of some IDPs/IDRs can lead to the formation of the membraneless organelles in vitro and in vivo, which are essential for many biological processes in the cell. Here we select three different IDR segments of chaperon Swc5 and develop a polymeric slab model at the residue-level. By performing the molecular dynamics simulations, LLPS can be observed at low temperatures even without charge interactions and disappear at high temperatures. Both the sequence length and the charge pattern of the Swc5 segments can influence the critical temperature of LLPS. The results suggest that the effects of the electrostatic interactions on the LLPS behaviors can change significantly with the ratios and distributions of the charged residues, especially the sequence charge decoration (SCD) values. In addition, three different forms of swc conformation can be distinguished on the phase diagram, which is different from the conventional behavior of the free IDP/IDR. Both the packed form (the condensed-phase) and the dispersed form (the dilute-phase) of swc chains are found to be coexisted when LLPS occurs. They change to the fully-spread form at high temperatures. These findings will be helpful for the investigation of the IDP/IDR ensemble behaviors as well as the fundamental mechanism of the LLPS process in bio-systems.     

Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

Salmonella typhimurium histidinol dehydrogenase produces histidine from the amino alcohol histidinol by two sequential NAD-linked oxidations which form and oxidize a stable enzyme-bound histidinaldehyde intermediate. The enzyme was found to catalyze the exchange of 3H between histidinol and [4(R)-3H]NADH and between NAD and [4(S)-3H]NADH. The latter reaction proceeded at rates greater than kcat for the net reaction and was about 3-fold faster than the former. Histidine did not support an NAD/NADH exchange, demonstrating kinetic irreversibility in the second half-reaction. Specific activity measurements on [3H]histidinol produced during the histidinol/NADH exchange reaction showed that only a single hydrogen was exchanged between the two reactants, demonstrating that under the conditions employed this exchange reaction arises only from the reversal of the alcohol dehydrogenase step and not the aldehyde dehydrogenase reaction. The kinetics of the NAD/NADH exchange reaction demonstrated a hyperbolic dependence on the concentration of NAD and NADH when the two were present in a 1:2 molar ratio. The histidinol/NADH exchange showed severe inhibition by high NAD and NADH under the same conditions, indicating that histidinol cannot dissociate directly from the ternary enzyme-NAD-histidinol complex; in other words, the binding of substrate is ordered with histidinol leading. Binding studies indicated that [3H]histidinol bound to 1.7 sites on the dimeric enzyme (0.85 site/monomer) with a KD of 10 microM. No binding of [3H]NAD or [3H]NADH was detected. The nucleotides could, however, displace histidinol dehydrogenase from Cibacron Blue-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)