Effects of different methods of ovarian stimulation in adult rats

The in vitro fecundation programs normally use different ovarian stimulation agents. The purpose of the present study was to compare the effects of some of these agents on indices of ovulation and serum levels of estradiol, progesterone, and FSH. Treatments studied consisted of clomiphene citrate, PMSG alone and in combination with clomiphene citrate, pure human FSH and epimestrol. The data obtained show PMSG alone and PMSG plus clomiphene citrate to be the most effective treatments, in terms of number of oocytes harvested. No differences were noted between serum levels of oestradiol, progesterone and FSH.     

Vanadate inhibits 2,3-bisphosphoglycerate dependent phosphoglycerate mutases but does not affect the 2,3-bisphosphoglycerate independent phosphoglycerate mutases

There are two types of phosphoglycerate mutases. The 2,3-bisphosphoglycerate dependent phosphoglycerate mutases are inhibited by vanadate. In contrast, the 2,3-bisphosphoglycerate independent mutases are not affected. The effect of vanadate varies with pH, and can be reversed by dilution, EDTA and norepinephrine. The differential effect of vanadate on the two types of phosphoglycerate mutases supplies a novel way to easily differentiate both types of enzymes. In addition, it may contribute to the clarification of the mechanism of action of the 2,3-bisphosphoglycerate independent phosphoglycerate mutases.     

Differential protein expression in endothelial cells exposed to serum from patients with acute graft-vs-host disease,depending on steroid response

Graft-versus-host disease (GVHD) is a complication of allogeneic haematopoietic cell transplantation. Endothelial injury is crucial as pathophysiological substrate for GVHD. GVHD first-line treatment is high-dose corticosteroids, although some patients are steroid-refractory. Through the present study, we compared the endothelial proteomic profiles in response to serum from steroid-refractory acute GVHD (SR-aGVHD) and steroid-sensitive acute GVHD (SS-aGVHD) patients. Blood samples from SR-aGVHD (n = 4) and SS-aGVHD (n = 8) patients were collected at aGVHD diagnosis. Endothelial cell cultures were exposed (48 h) to patients' serum. Protein extraction and proteomic analysis were performed. Differences were statistically evaluated by multivariate analysis. Forty-four proteins contributed to separate all samples into the two study groups, among which 15 participated significantly (p < 0.05), 10 exhibiting a fold change >1.2. Differentially expressed proteins were mainly associated with oxidative phosphorylation (Cytochrome C oxidase subunit 6B1, CX6B1), inflammation and angiogenesis (Apolipoprotein D, APOD), cell survival (Rapamycin-insensitive companion of mTOR, RICTR), and oxidative stress (Riboflavin kinase, RIFK). This pilot study used a novel approach to distinguish the aGVHD response to steroid treatment. The proteins differentially expressed could constitute potential biomarkers for steroid-treatment response. These findings signify a step forward to identify the mechanisms of response to steroids, of high clinical relevance considering the SR-aGVHD elevated mortality.     

Effect of denervation on the distribution and developmental transition of phosphoglycerate mutase and creatine phosphokinase isozymes in rat muscles of different fiber-type composition

Phosphoglycerate mutase (PGM) and creatine phosphokinase (CK) occur as three isozymes (types MM, MB and BB) in mammals and these exhibit similar transitions during skeletal muscle development. To study the influence of innervation on this transition and on the maintenance of the isozyme phenotype in mature muscle, we have determined the changes produced by sciatic neurectomy in neonatal and adult rat hindlimb muscles. In 40-day-old rats, denervation decreased both PGM and CK activity, the effect being more pronounced in the fast-twitch extensorum digitorum longus (EDL) and gastrocnemius muscles than in the slow-twitch soleus muscle. It also produced a progressive increase in the proportion of MB- and BB-PGM isozymes in EDL and gastrocnemius but not in soleus, and an increase of MB- and BB-CK isozymes in all three muscles. In 5-day-old rats, denervation prevented the developmental increase of PGM and CK activity in all three muscles. Denervation also prevented the normal decrease in the relative amounts of the MB and BB isozymes of both enzymes which occur during postnatal muscle development. These results can be explained by the different effects of denervation upon slow and fast muscles, and by the distinct distribution of PGM and CK isozymes in rat type I and II muscle fibers.     

Stable Expression of a Synthetic Gene for the Human Motilin Receptor: Use in an Aequorin-Based Receptor Activation Assay

A synthetic gene for the human motilin receptor containing 33 unique restriction sites was designed and stably coexpressed in HEK293 cells with the bioluminescent Ca(2+) indicator protein aequorin. The dose-dependent response of the receptor to motilin was demonstrated using transient transfections, and a stable cell line was selected. [(125)I]Motilin binding was used to estimate receptor expression level for the stable cell line, and titration of a membrane preparation indicated a K(d) value of 0.8 nM. The same cell line was used to evaluate a panel of erythromycin-derived agonists and provided EC(50) values for receptor activation that agree closely with data obtained in contractility assays. The peptide antagonist ANQ11125 (Phe3Leu13 motilin 1-14) inhibited motilin induced response with a K(i) value of 10 nM. The system is well-suited for the screening of compound libraries and receptor mutagenesis studies.     

Metabolism of glycerate 2,3-P2--XI. Essential amino acids of pig phosphoglycerate mutase isozymes

Phosphoglycerate mutase isozymes (types M, B and MB) from pig tissues are inactivated upon treatment with reagents specific for histidyl, arginyl and lysyl residues. Their mutase, 2,3-bisphosphoglycerate synthase and 2,3-bisphosphoglycerate phosphatase activities are concurrently lost, although some differences exist in the rate of inactivation. No significant differences are observed between the isozymes. The reversion of the modifying reactions reactivates the three enzymatic activities. Substrates and cofactors protect against inactivation, the protective effects varying with the modifying reagent. Titration with pCMB shows the existence of two essential thiol groups per subunit type M. These results provide evidence of the intrinsic character of the three enzymatic activities, favor their location at the same active site and suggest the existence of separate binding sites for monophosphoglycerates and bisphophoglycerates. Both type M and B subunit from pig phosphoglycerate mutase are similar to type M subunit from rabbit and to the enzyme from yeast.     

PSI protein classifier: A new program automating PSI-BLAST search results

A new program, PSI Protein Classifier, generalizing the results of both successive and independent iterations of the PSI-BLAST program was developed. The technical opportunities of the program are described and illustrated by two examples. An iterative screening of the amino acid sequence database detected potential evolutionary relationships between GH5, GH13, GH27, GH31, GH36, GH66, GH101 and GH114 families of glycoside hydrolases. Analysis of the statistically significant sequence similarity (E-value analysis) allowed us to divide the family GH31 into 38 subfamilies.     

Disaccharidase activities after jejunoileal bypass in rat

Digestive enzymatic activities (maltase, lactase and sucrase) have been determined in the intestinal mucosa of rats subjected to a jejunoileal bypass of 45 cm. The weight and protein content of the mucosa (mg/cm) were significantly decreased in the bypassed segment and significantly increased in the unbypassed segment, as compared to control rats. Maltase, lactase and sucrase specific (U/g protein) and total activity (U/cm intestine) were significantly decreased in the bypassed jejunum, compared to sham-operated rats. In the ileum, maltase specific and total activities increased in bypassed animals while the lactase and sucrase activities remained unchanged.     

Nuclear location of phosphoglycerate mutase BB isozyme in rat tissues.

We have previously reported (Ure?a et al. Eur. J. Cell Biol. 1990) that in skeletal muscle, type MM phosphoglycerate mutase isozyme is present in the nucleus as well as in the cytosol. To determine whether type BB phosphoglycerate mutase isozyme is also present in nucleus, the subcellular location of this isozyme was studied in different rat tissues by cell fractionation and immunogold techniques. With the aid of high affinity-purified anti-phosphoglycerate mutase BB isozyme antibodies, the isozyme was located in the nucleus of neuronal, astroglial and liver cells but not in the nucleus of oligodendroglial and endothelial cells. Biochemical studies on purified nuclear fractions also demonstrated the presence of phosphoglycerate mutase activity in the nucleus. Both immunocytochemical and biochemical techniques showed that nuclear phosphoglycerate mutase-specific activity depended on the type of cell.     

Location of phosphoglycerate mutase in rat skeletal muscle. An immunocytochemical and biochemical study

The subcellular distribution of phosphoglycerate mutase was studied by immunogold techniques. With the aid of highly affinity-purified anti-phosphoglycerate mutase antibodies, the enzyme was found in both cytosol and nucleus of rat skeletal muscle. No evidence of interaction with contractile proteins was observed in cytosol. Nuclear location was also confirmed biochemically using purified nuclear preparations from rat skeletal muscle. Only one immunoreactive nuclear band was observed by Western blot experiments and corresponded to that of phosphoglycerate mutase mobility. Activity measurements from nuclear extracts showed that 25% of total specific activity is found in the nuclei.