Sympathetic and adrenocorticoid influences on diet-induced thermogenesis and brown fat activity in the rat

Bilateral adrenalectomy markedly reduced body weight and energy gain and energetic efficiency of adult cafeteria-fed rats but enhanced the thermogenic response to food and stimulated brown fat activity. These changes were totally prevented by replacement of the animals with corticosterone (1 mg/rat/day). Unilateral denervation of the sympathetic nerves supplying the interscapular brown adipose tissue abolished the enhanced activity resulting from adrenalectomy and inhibited thermogenic activity in brown fat from cafeteria rats with intact adrenals, but had no effect in adrenalectomised animals treated with a high dose of corticosterone.     

Identification of a possible nucleotide binding site in CheW, a protein required for sensory transduction in bacterial chemotaxis

CheW is an essential component of the system which mediates chemotaxis in Salmonella typhimurium and Escherichia coli. Here we report the nucleotide sequence of the cheW gene as well as the purification and characterization of the CheW protein. The DNA sequence predicts a protein of 18,000 molecular weight. The pure protein exhibits an apparent molecular weight of 18,000 during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular sieve chromatography under nondenaturing conditions indicates a molecular weight of approximately 35,000, however. This result suggests that CheW is a homodimer. The predicted amino acid sequence between Thr-128 and Asp-160 fits a consensus exhibited by many proteins which bind purine nucleotides.     

Development of a single probe for documentation of chimerism following bone marrow transplantation.

Although numerous genetic markers are available for studying chimerism after bone marrow transplantation (BMT), there remains a need for a practical and highly informative method that is applicable in the early posttransplantation period. Using DNA restriction-fragment-length polymorphisms (RFLPs), we have evaluated the feasibility of developing a single synthetic oligonucleotide probe to study post-BMT chimerism. We have thus tested three candidate probes, termed O-3315-32, O-3315-80, and O-AY-29, that are homologous to tandemly repetitive sequences. Our results demonstrated donor-specific and recipient-specific fragments in 11 of 11 HLA-matched sibling pairs tested using probes O-3315-32 and O-3315-80. When probe O-AY-29 was used, 14 of 17 sibling pairs showed both donor and recipient markers, one had only a recipient marker, and two were identical. We showed that each of the three synthetic probes was effective in documenting donor marrow engraftment, mixed hematopoietic chimerism, the patient's pre-BMT phenotype (by using cultured skin fibroblasts obtained after BMT), and the origin of the malignant hematopoietic cells (i.e., of donor or recipient origin) in patients who developed recurrent hematologic malignancy following BMT. Compared with the use of cloned genomic probes, there are several important advantages to the use of synthetic oligonucleotide probes in studying post-BMT chimerism. Synthetic probes have absolute hybridization specificity and can be designed to suit the purposes of an individual study, since they have adjustable specificity that can be altered by changes in the length of the probe and by changes in the hybridization temperature. A single synthetic probe analogous to several highly polymorphic loci can have a polymorphism information content sufficiently high so that all but a small percentage of BMT patients could be followed easily; for example, if a probe were complementary to three highly polymorphic unlinked loci, it would discriminate approximately 98% of sibling donor/recipient pairs. This would be accomplished using only one restriction-endonuclease digestion and only one gel electrophoresis. Since other genetic markers, e.g., red blood cell antigens, immunoglobulin allotypes, and chromosome analysis, are not uniformly informative and, in some cases, cannot be used in the early posttransplantation period, the use of synthetic oligonucleotide probes for analysis of DNA RFLP is emerging as the method of choice for studies of post-BMT chimerism. This method will allow for the development of new knowledge that has not been possible with previous methods.     

Monoclonal antibodies to different sites on human transcobalamin II

Two IgG1K monoclonal antibodies to human transcobalamin II (TC II) were generated. These antibodies, 16.1 and 16.6, did not cross-react with the other two types of human cobalamin-binding proteins, intrinsic factor and R binder (TC I). Both antibodies cross-reacted with orangutan and simiang TC II but not with TC II from cynomolgus and howler monkeys, who are less closely related to humans. This finding suggests close structural similarity of human to ape TC II. The antibodies also did not react with TC II of lower mammals which included the horse, dog, guinea pig, and mouse; in particular, reaction did not occur with rabbit TC II, which has been considered structurally close to human TC II. Neither of the two antibodies was directed at the cobalamin-binding site of TC II. However, antibody 16.6 hindered TC II binding to cell receptor. This reactivity with the receptor-binding site should prove particularly useful in studies of that region of the TC II molecule.     

Pharmacological profile of a novel carbacyclin derivative with high metabolic stability and oral activity in the rat

A novel carbacyclin derivative (16S)-13,14-dehydro-16,20-dimethyl-3-oxa-18,18,19,19-tetradehydro- 6a- carbaprostaglandin-I2 (3-oxa-analogue) has been synthesized in order to find chemically and metabolically stable prostacyclin-mimetics with a potency equal or even superior to PGI2. The 3-oxa-analogue was found to be stabilized against beta-oxidation, a main metabolic degradation step also for chemically stable PGI2-analogues. The compound is orally available and displays a long duration of 4.5-48 h of antiaggregatory and hypotensive action. The 3-oxa-analogue inhibits ADP-induced platelet aggregation with an IC50 of 3.0 nM. Following intravenous application the 3-oxa-analogue lowers diastolic blood pressure in a dose dependent manner, the ED20 being 0.1-0.2 micrograms/kg after injection and less than or equal to 0.05 micrograms/kg/min after infusion respectively. In vivo platelet aggregation is inhibited after i.v. infusion of the 3-oxa-analogue with an IC50 of 0.037 micrograms/kg/min. As compared to Iloprost, the 3-oxa-analogue is 5-12 fold more potent with respect to in vivo hypotensive and anti-aggregatory effects. The results of the present studies indicate that the 3-oxa-analogue has a pharmacological profile comparable to prostacyclin (PGI2) and Iloprost. Due to the fact that the 3-oxa-analogue is chemically and metabolically stable, long term oral treatment can be achieved in clinical conditions in which PGI2 and Iloprost have already been shown to be therapeutically useful principles.     

Oxidative metabolism of dermatophytes

A method for preparing young, actively respiring dermatophyte mycelia was obtained through the use of concentrated spore inocula and short growth periods in static culture. These hyphal elements were uniform in appearance, and vacuoles were absent. Concentrated mycelial suspensions were obtained which could be transferred easily and accurately. Glucose stimulated oxygen uptake in young mycelia which had been grown in a medium with low carbohydrate content. The level of endogenous respiration was affected by exogenous glucose only when this substrate stimulated oxygen uptake by less than 14%. Low nicotinamide adenine dinucleotide phosphate (NADP) dehydrogenase activity was noted in microconidia which have a low endogenous Q_o2 value, whereas the activity of this enzyme was greater in macroconidia and mycelia which possess higher endogenous Q_o2 values. Microsporum gypseum oxidizes 50% of exogenous glucose and assimilates the remainder. A large percentage of this substrate was assimilated into nitrogenous substances.     

Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY

The protein (Escherichia coli CheY) that controls the direction of flagellar rotation during bacterial chemotaxis has been shown to be phosphorylated on the aspartate 57 residue. The residue phosphorylated is present within a conserved sequence in every member of a family of bacterial regulatory proteins. The phosphorylation is transient, with a much shorter half-life than that expected of a simple acyl phosphate intermediate, indicating that the sequence and conformation of the protein is designed to achieve a rapid hydrolysis. The CheY-phosphate linkage can be reductively cleaved by sodium borohydride. High-performance tandem mass-spectrometric analysis of proteolytic peptides derived from [3H]borohydride-reduced phosphorylated CheY protein was used to identify the position of phosphorylation. Mutants with altered aspartate 57 exhibited no chemotaxis. When aspartate 13, another conserved residue, was changed, greatly reduced chemotaxis was observed, suggesting an important role for aspartate 13. The rate-determining step of chemotactic signaling is governed by the kinetics of formation and hydrolysis of the CheY protein phosphoaspartate bond. The CheY protein apparently functions as a protein phosphatase that possesses a transient covalent intermediate. Transient phosphorylation of an aspartate residue is an effective mechanism for producing a biochemical signal with a short concentration-independent half-life. The duration of the signal can be controlled by small structural elements within the phosphorylated protein.