Identification and characterization of P31m, a novel sperm protein in Cynomolgus monkey (Macaca fascicularis)

We have previously described a hamster sperm glycoprotein, P26h, which is implicated in the cascade of events occurring during the interaction between mature spermatozoa and the oocyte's zona pellucida. The P26h is acquired on the acrosomal cap of the spermatozoon during its maturation arising within the epididymis. Lately, using a polyclonal antiserum raised against P26h, a 34 kDa protein, P34H, has been identified on the acrosomal cap of the human spermatozoon. The cloning and sequencing of the cDNA encoding P34H has revealed a 65% similarity between the P34H and P26h amino acid sequences. Considering that P26h shows total immunocontraceptive properties in the hamster, it is of relevant importance to have an animal model phylogenetically closer to the human. Using the Cynomolgus monkey, we searched for a protein autologous to the human P34H. A 31 kDa protein, the P31m, localized on the acrosomal cap of the monkey spermatozoon has been identified by a Western blot analysis and by immunohistochemical techniques using an anti-hamster P26h antiserum. Northern blot analysis showed increasing high levels of the P31m mRNA through the epididymis and at lower levels in the testis. In situ hybridization showed the presence of the P31m mRNA in the principal cells of the epididymis. The cloning and sequencing of the cDNA encoding the P31m showed a high homology of 97% identity between the P31m and P34H nucleotidic sequences. This study clearly demonstrates that the monkey P31m is the homologous protein of the hamster P26h and of the human P34H. Mol. Reprod. Dev. 59: 431-441, 2001.     

The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1), EP(2), EP(3), EP(4), DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.     

Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p

Members of the double-stranded RNA (dsRNA) specific RNase III family are known to use a conserved dsRNA-binding domain (dsRBD) to distinguish RNA A-form helices from DNA B-form ones, however, the basis of this selectivity and its effect on cleavage specificity remain unknown. Here, we directly examine the molecular requirements for dsRNA recognition and cleavage by the budding yeast RNase III (Rnt1p), and compare it to both bacterial RNase III and fission yeast RNase III (Pac1). We synthesized substrates with either chemically modified nucleotides near the cleavage sites, or with different DNA/RNA combinations, and investigated their binding and cleavage by Rnt1p. Substitution for the ribonucleotide vicinal to the scissile phosphodiester linkage with 2'-deoxy-2'-fluoro-beta-d-ribose (2' F-RNA), a deoxyribonucleotide, or a 2'-O-methylribonucleotide permitted cleavage by Rnt1p, while the introduction of a 2', 5'-phosphodiester linkage permitted binding, but not cleavage. This indicates that the position of the phosphodiester link with respect to the nuclease domain, and not the 2'-OH group, is critical for cleavage by Rnt1p. Surprisingly, Rnt1p bound to a DNA helix capped with an NGNN tetraribonucleotide loop indicating that the binding of at least one member of the RNase III family is not restricted to RNA. The results also suggest that the dsRBD may accommodate B-form DNA duplexes. Interestingly, Rnt1p, but not Pac1 nor bacterial RNase III, cleaved the DNA strand of a DNA/RNA hybrid, indicating that A-form RNA helix is not essential for cleavage by Rnt1p. In contrast, RNA/DNA hybrids bound to, but were not cleaved by Rnt1p, underscoring the critical role for the nucleotide located at 3' end of the tetraloop and suggesting an asymmetrical mode of substrate recognition. In cell extracts, the native enzyme effectively cleaved the DNA/RNA hybrid, indicating much broader Rnt1p substrate specificity than previously thought. The discovery of this novel RNA-dependent deoxyribonuclease activity has potential implications in devising new antiviral strategies that target actively transcribed DNA.     

日光温室栽培对杏花及果实生长发育的影响

对日光温室内与露地栽培的金太阳杏的花期物候、花型及果实生长发育进行了系统观察与分析,结果表明,日光温室栽培比露地栽培的始花期提前33d,花期延长4d,不完全花比例上升33.25%;果枝上花的有效性顺序由露地时的中果枝>长果枝>短果枝>花束状果枝变为温室栽培的短果枝>花束状果枝>中果枝>长果枝;日光温室栽培使杏果实发育的第 、 阶段延长,第 阶段缩短,整个生育期延长15d。统计分析认为:日光温室栽培的第 阶段生长速率显著低于露地,第 阶段的累积生长量显著高于露地。较低的夜间温度是造成温室内杏果实第 阶段较长、生长较慢以及果个变大的原因。     

Structure-activity relationship studies on ortho-substituted cinnamic acids, a new class of selective EP(3) antagonists

A series of novel ortho-substituted cinnamic acids have been synthesized, and their binding activity and selectivity on the four prostaglandin E(2) receptors evaluated. Many of them are very potent and selective EP(3) antagonists (K(i) 3-10 nM), while compound 9 is a very good and selective EP(2) agonist (K(i) 8 nM). The biological profile of the EP(2) agonist 9 in vivo and the metabolic profile of selected EP(3) antagonists are also reported.     

Genomic affinities in Arachis section Arachis (Fabaceae): molecular and cytogenetic evidence

Section Arachis is the largest of nine sections in the genus Arachis and includes domesticated peanut, A. hypogaea L. Most species are diploids (x=10) with two tetraploids and a few aneuploids. Three genome types have been recognized in this section (A, B and D), but the genomes are not well characterized and relationships of several newly described species are uncertain. To clarify genomic relationships in section Arachis, cytogenetic information and molecular data from amplified fragment length polymorphism (AFLP) and the trnT-F plastid region were used to provide an additional insight into genome composition and species relationships. Cytogenetic information supports earlier observations on genome types of A. cruziana, A. herzogii, A. kempff-mercadoi and A. kuhlmannii but was inconclusive about the genome composition of A. benensis, A. hoehnei, A. ipaensis, A. palustris, A. praecox and A. williamsii. An AFLP dendrogram resolved species into four major clusters and showed A. hypogaea grouping closely with A. ipaensis and A. williamsii. Sequence data of the trnT-F region provided genome-specific information and showed for the first time that the B and D genomes are more closely related to each other than to the A genome. Integration of information from cytogenetics and biparentally and maternally inherited genomic regions show promise in understanding genome types and relationships in Arachis.     

Biased mutations and microsatellite variation

Mutation bias is one of the forces that may constrain the variation at microsatellite loci. Here, we study the dynamics of population statistics and the genetic distance between two populations under multiple stepwise mutations with linear bias and random drift. Expressions are derived for these statistics as functions of time, as well as at mutation-drift equilibrium. Applying these expressions to published data on humans and chimpanzees, the regression coefficient of mutation bias on allele size was estimated to be at least between - 0.0064 and -0.013. The assumption of mutational bias produces larger estimates of divergence times than are obtained in its absence; in particular, the time of split between African and non-African human populations is estimated to be between 183,000 and 222,000 years, assuming one-step mutations and no selection. With multistep mutations, the divergence time is estimated to be lower.