Effects of paclitaxel before vitrification on the nuclear maturation and development of immature porcine oocytes.

BACKGROUND: Cryopreservation of porcine oocytes is difficult compared with other species and immature oocytes particularly so compared to the meiotic stage. OBJECTIVE: To evaluate the efficacy of a pretreatment with 1 micromole per L paclitaxel (PTX, 30 min exposure) before vitrification to promote the maturation of porcine immature oocytes. MATERIALS AND METHODS: Cumulus cell-enclosed oocytes (COs) aspirated from porcine ovaries were divided into three groups: i) non-pretreated with PTX and non-vitrified group (control group); ii) pretreated with PTX and vitrified group (PTX-V group); and iii) non-pretreated with PTX and vitrified group (nPTX-V group). RESULTS: The nuclear maturation rate up to the preovulatory stage was significantly lower (P < 0.05) in the nPTX-V group than in the control group, but was similar in the PTX-V and control groups. No significant differences were observed in viability assessed by a normal CO morphology and the embryonic development of oocytes activated by the parthenogenetic stimulation between the PTX-V and control groups, but not the non-PTX-V group. CONCLUSION: PTX may promote the maturation of vitrified porcine immature oocytes. Doi.org/10.54680/fr23510110812.

Changes in peripheral anti-Müllerian hormone concentration and their relationship with testicular structure in beef bull calves.

The aim of this study was to clarify the time-course of changes in anti-Müllerian hormone (AMH) and testosterone (T) concentrations in peripheral blood and to determine the relationships between blood AMH concentration and testicular development during the early postnatal and prepubertal periods in beef bull calves. A total of 17 Japanese Black bull calves were enrolled in this study. The wk in which the calf was born (within 6 d after birth) was defined as M 0. Blood samples were taken once in every mo from M 0 to M 6 from each bull calf, and plasma AMH and T concentrations were determined. Of the 17 calves, 10 were castrated at 6 mo of age (prepuberty) and the right testis was histologically examined. Plasma AMH concentration (means ± SE) at M 0, 1, and 2 were 123.5 ± 9.8, 189.6 ± 18.7, and 254.6 ± 14.1 ng/mL, respectively. From M 0 through M 2, plasma AMH concentration was significantly greater each mo than in the previous mo (P < 0.05); however, plasma AMH concentration significantly decreased over the last 3 mo of the study (P < 0.05). The average age at which plasma AMH concentration was the highest was 2.3 ± 0.1 mo of age. Plasma T concentration significantly increased from M 0 (0.18 ± 0.02 ng/mL) until M 6 (6.52 ± 1.41 ng/mL). Plasma AMH and T concentrations at M 4, 5, and 6 were significantly negatively correlated (P < 0.05). Linear regression did not reveal a significant relationship between Sertoli or Leydig cell numbers and plasma AMH or T concentrations, respectively. In conclusion, blood AMH concentration peaks at 2 mo of age and is negatively correlated with blood T concentration from 4 to 6 mo of age. Although prepubertal blood AMH or T concentrations did not reflect Sertoli or Leydig cell numbers at the end of the prepubertal period, blood AMH concentration may be indicative of abnormal Sertoli cells function.

The mouse ZFH-4 protein contains four homeodomains and twenty-two zinc fingers.

We isolated the mouse zfh-4 cDNA which is 12 kb long and capable of encoding a 3,550-amino acid protein containing four homeodomains and 22 zinc fingers including two pseudo zinc finger motifs. The mouse ZFH-4 is 51% homologous to the mouse ATBF1 and 23% to the Drosophila ZFH-2. The homeodomain and zinc finger regions are highly conserved between ZFH-4 and ATBF1 except that one zinc finger is missing in ZFH-4. Analysis of partial genomic sequences showed that the mouse zfh-4 and ATBF1 genes are similar in exon-intron organization. RT-PCR analysis of zfh-4 transcripts in adult mouse tissues showed that zfh-4 expression was low but reproducibly detectable in brain, heart, lung and muscle. In these mouse tissues, ATBF1 transcripts were poorly amplified by PCR under the conditions where zfh-4 transcripts were amplified, suggesting that the expression of zfh-4 mRNA is higher than that of ATBF1 mRNA. Other comparative analysis suggests functional similarities and dissimilarities between ZFH-4 and ATBF1.

Synthesis and characterization of orally active nonpeptide vasopressin V2 receptor antagonists.

The present study was undertaken to evaluate whether a novel series of 2,6-diaza-5-oxobicyclo[5.4.0]undeca-1(7),8,10-triene derivatives exhibited antagonistic activity for vasopressin V1 and V2 receptors. Most of these compounds were synthesized and showed a high affinity potential for V2 receptor and low to moderate affinity potential for V1 receptor. The most potent and V2-selective compound, N-[4-[2,6-diaza-6-[2-(4-methylpiperazinyl)-2-oxoethyl] -5- oxobicyclo[5.4.0]undeca-1(7),8,10-trien-2-yl]-carbonyl]pheny l][2-(4- methylphenyl)phenyl]-formamide (11b), exhibited IC50's of 2.9 nM for the V2 receptor and 200 nM for the V1 receptor, respectively. When administered orally to rat, 11b showed an approximately 18-fold increased urine volume in comparison with control rat.

WA8242A1, A2 and B, novel secretary phospholipase A2 inhibitors produced by Streptomyces violaceusniger. III. Structure elucidation and total synthesis of WA8242B.

The structure of WA8242B, a potent novel inhibitor against phospholipase A2, was fully characterized by spectroscopic methods and chemical degradation. The success of total synthesis of WA8242B confirmed the structure and allowed the pharmacological study of WA8242B. The structures of WA8242A1 and A2 were also described.

The physiological roles of membrane ergosterol as revealed by the phenotypes of syr1/erg3 null mutant of Saccharomyces cerevisiae.

Ergosterol is a major sterol component of fungal plasma membranes. The effects of disrupting the Saccharomyces cerevisiae SYR1/ERG3 gene, which encodes sterol C-5 desaturase, an enzyme of ergosterol biosynthesis pathway, were markedly different for different S. cerevisiae strains and growth temperatures. The null mutation of SYR1 (delta syr1) in strain RAY-3A had only a slight effect on the growth rate at 28 degrees C. However, at this temperature, the same mutation caused poor growth in strain KA-311A and no growth in strain W303-1A. The delta syr1 disruptant of these strains were able to grow at 37 degrees C, as well as their parental strains. Moreover, the growth of the delta syr1 disruptant of W303-1A and KA-311A strains were severely inhibited at 16 degrees C. These results indicated that ergosterol is essential for growth at low temperatures, and the effects of the gene disruption are variable by the genetic background. The growth defect at low temperatures appeared to be due to the defect of tryptophan uptake in the delta syr1 mutants. The delta syr1 mutants were sensitive to a wide variety of drugs, chemicals, and ions, suggesting that yeast ergosterol is important as permeability barrier against various chemical stresses.

PAF inhibitory activity of diketopiperazines: structure-activity relationships.

FR900452, a natural product isolated from the culture broth of Streptomyces phaeofaciens No. 7739, was found to inhibit PAF-induced rabbit platelet aggregation with an IC50 of 3.7 x 10(-7)M. FR900452, 1-methyl-3-[1-[5- methylthiomethyl-6-oxo-3-(2-oxo-3-cyclopenten-1-yli- dene)-2-piperazinyl]ethyl]-2-indoline, has an oxocylopentylidene group incorporated as a vinylogous amide in a diketopiperazine skeleton. This unique structure led us to synthesize diketopiperazine derivatives, 3-arylalkyl- 6-substituted-piperazine-2,5-diones. Their observed PAF inhibitory activity suggest that the D-D configuration of diketopiperazine is an important factor for anti-PAF activity and that the hydrophobic aromatic portion may play a specific role in the binding of the diketopiperazine to the PAF receptor.

WF-10129, a novel angiotensin converting enzyme inhibitor produced by a fungus, Doratomyces putredinis.

WF-10129 is an angiotensin converting enzyme (ACE) inhibitor produced by Doratomyces putredinis. IC50 of the compound is 1.4 X 10(-8) M for the ACE activity. WF-10129 was purified from cultured filtrate by successive ion exchange chromatography and HPLC. The chemical structure 1 was elucidated on the basis of spectroscopic and chemical evidence. The compound is a dipeptide composed of L-tyrosine and a novel amino acid. WF-10129 inhibits the pressor response of angiotensin I when administered intravenously at 0.3 mg/kg in rats.

Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor.

The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.

Inhibition of human leukocyte elastase, cathepsin G, chymotrypsin A alpha, and porcine pancreatic elastase with substituted isobenzofuranones and benzopyrandiones.

Several 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones, 3-(1-haloalkylidene)-1(3H)-isobenzofuranones, and 3-bromomethyl-1H-2-benzopyran-1-ones containing masked halo ketone functional groups were synthesized and tested as inhibitors of several serine proteases including human leukocyte (HL) elastase and cathepsin G. While many of the 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones were quite potent inhibitors of the enzymes tested, the alkylideneisobenzofuranones and benzopyran-1-ones inhibited poorly or not at all. The 3-halo-3-(1-haloalkyl)-1(3H)-isobenzofuranones decomposed rapidly upon addition to buffer to give the corresponding 3-alkyl-1H-2-benzopyran-1,4(3H)-diones. The pure benzopyran-1,4-diones were extremely potent inhibitors of HL elastase and chymotrypsin A alpha but did not inactivate porcine pancreatic elastase or cathepsin G. Enzymes inhibited by the isobenzofuranones and benzopyran-1,4-diones regained activity slowly upon standing or after dialysis (t1/2 = 5-16 h) and more rapidly in the presence of 0.5 M hydroxylamine, which indicated the presence of labile acyl moieties in the inhibited enzyme. These results are consistent with a scheme in which the active site serine of the protease reacts with the lactone carbonyl of these inhibitors to give a stable acyl enzyme and alkylation of another active site residue by the unmasked halo ketone functional group does not occur.

Studies on a new immunoactive peptide, FK-156. IV. Synthesis of FK-156 and its geometric isomer.

For the structural confirmation of FK-156, two possible structures, 1 and its geometric isomer 2, were synthesized. Di-Z-meso-diaminopimelic acid (4) was converted into 14 via a sequence of reactions involving, as key steps, an enzyme-mediated asymmetric hydrolysis (6 leads to 7), followed by carbobenzyloxylation using a copper chelate procedure (7 leads to 8). Condensation of 14 and the appropriately protected lactoyl dipeptide 17 and removal of the protecting groups of the resulting 18 afforded 1. Protection of 7 to 22, followed by coupling to glycine via an azide method, gave 25. Derivatization of 25 to 29 and condensation with 17 gave 30, which was deprotected to yield 2. Compound 1 proved to be identical in all respects with the natural product.