High-affinity folate binding in human liver membranes

High-affinity binding of³H-folate in Triton X-100 solubilized membranes of human liver displayed characteristics, e.g. apparent positive cooperativity, which are typical of specific folate binding. Ultrogel^® AcA 44 chromatography of solubilized membranes saturated with³H-folate revealed a major peak of 100 kDa and a minor peak of 25 kDa. The 100 kDa peak could represent a hydrophobic membrane associated molecular form of the protein. This notion was supported by the fact that the two peaks had identical molecular weights as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis with immunoblotting.     

Comparison of diagnostic methods for detection of low infestation levels ofVarroa jacobsoni in honey-bee (Apis mellifera) colonies

Thirty-five honey-bee colonies, originally free fromVarroa jacobsoni (Oudemans) were monitored approximately every third week for the presence of the mite during 16 months following an initial introduction of five to eight adultVarroa females in early July. Investigations of hive debris detected the presence ofV. jacobsoni in 22 colonies (63%) within three months of the mite introduction. During the first winter period (October–April), mites were found in the hive debris of 13 colonies (37%). In terms of detectingVarroa during the summer in colonies with sealed brood, investigations of hive debris were more effective than sampling of brood. Brood sampling was more effective than sampling of live bees. In colonies without sealed brood, investigations of hive debris or of live bee samples seemed approximately equally efficient. The highest correlation between sampling methods was found between daily mite downfall and mites per live bee (r=0.81) in colonies with sealed brood. During the winter, investigations of dead bees and hive debris were approximately equally efficient in detectingVarroa.     

Biphasic changes in leukocytes induced by strenuous exercise

Seven healthy male volunteers participated in short- (STR, 1.7 km), middle- (MTR, 4.8 km) and long- (LTR, 10.5 km) term runs at a speed close to their maximum. A prompt mobilization of white cells, and lymphocytes in particular, appeared following the exercise. The initial increase in the number of lymphocytes was succeeded by a significant decrease [(P less than 0.03) lymphopenial], which on average was 32%-39% of the pre-exercise values in all groups. A close correlation was found between the initial increase in plasma cortisol concentration after exercise and the subsequent lymphopenia. A modest enhancement in the number of granulocytes immediately after the exercise was accompanied by a comprehensive increase in polymorphonuclear (PMN) elastase concentration accounting for 78.6%, SEM 16.3%, 140.7%, SEM 31.8% and 241.3%, SEM 48.1% in the STR, MTR and LTR groups. No correlation was found between granulocyte number and the plasma PMN elastase concentration. A delayed granulocytosis was noted in all subjects, reaching a peak between 2 and 4 h after the exercise. The magnitude of the granulocytosis varied among subjects and peak values of the number of circulating granulocytes were found to be 5.7 x 10(9) cells.l-1, SEM 0.5, 6.7 x 10(9) cells.l-1, SEM 0.6 and 8.8 x 10(9) cells.l-1, SEM 0.5 in STR, MTR and LTR respectively, whereas the mean baseline value was 3.6 x 10(9) cells.l-1, SEM 0.4. The neutrophilic granulocytosis was not accompanied by a corresponding enhancement in PMN elastase concentration. The plasma cortisol concentration reached a peak 30 min after exercise and declined below the control level in 4 h. Neither the initial increase, nor the subsequent decrease in plasma cortisol concentration were found to be essential for the magnitude of the delayed leukocytosis.     

Familial porphyria cutanea tarda: hybridization analysis of the uroporphyrinogen decarboxylase locus

Familial porphyria cutanea tarda (PCT) results from a deficiency of uroporphyrinogen decarboxylase (URO-D) activity. Hybridization analysis of genomic DNA from unrelated normal individuals and PCT pedigree members failed to detect any major deletions, rearrangements or restriction fragment length polymorphisms at the URO-D locus.     

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.     

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.     

Carbonyl 13C NMR spectrum of basic pancreatic trypsin inhibitor: resonance assignments by selective amide hydrogen isotope labeling and detection of isotope effects on 13C nuclear shielding

The carbonyl region of the natural abundance 13C nuclear magnetic resonance (NMR) spectrum of basic pancreatic trypsin inhibitor is examined, and 65 of the 66 expected signals are characterized at varying pH and temperature. Assignments are reported for over two-thirds of the signals, including those of all buried backbone amide groups with slow proton exchange and all side-chain carbonyl groups. This is the first extensively assigned carbonyl spectrum for any protein. A method for carbonyl resonance assignments utilizing amide proton exchange and isotope effects on nuclear shielding is described in detail. The assignments are made by establishing kinetic correlation between effects of amide proton exchange observed in the carbonyl 13C region with development of isotope effects and in the amide proton region with disappearance of preassigned resonances. Several aspects of protein structure and dynamics in solution may be investigated by carbonyl 13C NMR spectroscopy. Some effects of side-chain primary amide group hydrolysis are described. The main interest is on information about intramolecular hydrogen-bond energies and changes in the protein due to amino acid replacements by chemical modification or genetic engineering.     

Characterization of a high molecular weight glycoprotein secreted by the peri-implantation bovine conceptus

Cow conceptuses were flushed from uteri on Day 17 of pregnancy and cultured with [3H]glucosamine and [14C]leucine. A high molecular weight glycoprotein (HMWG) having an Mr = 765,000 was isolated by a combination of anion-exchange and gel-filtration chromatography. Selective chemical and enzymatic degradations were performed. The HMWG was resistant to Pronase and peptide: N-glycanase F. Only endo-beta-galactosidase and harsh alkaline reducing conditions were successful in dissociating carbohydrate from the protein core, suggesting that carbohydrate chains are N-linked to Asn and contain beta-galactosidic linkages. The intact molecule could bind to an affinity column of Datura stramoniom lectin, suggesting the presence of beta(1-4)-linked oligomers of N-acetylglucosamine. The susceptibility of HMWG to endo-beta-galactosidase suggests that at least some of these oligomers are substituted with galactose to form N-acetyllactosamine. Binding of HMWG to lectin could be inhibited partially with N-acetyllactosamine or completely with a mixture of N, N'-diacetylchitobiose and N, N', N"-triacetylchitotriose. In summary, properties of the HMWG suggest it contains lactosaminoglycan components and is almost identical to an HMWG secreted by the Day 16 ovine conceptus. Thus, embryos of these two ruminant species secrete similar molecules during early pregnancy.     

Secretory proteins of the bovine conceptus alter endometrial prostaglandin and protein secretion in vitro

The conceptus is believed to produce factors that regulate endometrial function and prevent luteolysis during early pregnancy. Endometrial tissues were collected from cyclic (n = 8) and pregnant (n = 2) cows at Day 17 post-estrus and cultured for 24 and 48 h with bovine conceptus secretory proteins (bCSP) (0%, 10%, 100%), where the amount of protein produced by a bovine conceptus during 24 h of culture is 100%. Incorporation of [3H]leucine into secreted proteins was determined and examined qualitatively by trichloroacetic acid precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Levels of an intracellular endometrial inhibitor of prostaglandin synthesis were determined with a cotyledonary microsomal test system. Treatment with 10% and 100% bCSP reduced incorporation of [3H]leucine into secreted proteins. However, bCSP selectively induced two secreted proteins (13 and 10 kDa) from endometrium of cyclic cows. Prostaglandin F (PGF) secretion was decreased by bCSP treatment while prostaglandin E2 secretion was unaltered. An intracellular endometrial inhibitor of prostaglandin synthesis was induced by bCSP; synthesis of PGF by the cotyledonary prostaglandin-generating system was decreased when incubated with cytosol of endometrium treated with bCSP, but unaltered by cytosol from control tissues. In conclusion, products produced by the bovine conceptus are capable of regulating endometrial protein and prostaglandin biosynthesis in a fashion that could act to prevent luteolysis in vivo and provide endometrial secretory products for embryonic development.