Kinesin-related proteins in the mammalian testes: candidate motors for meiosis and morphogenesis.

The kinesin superfamily of molecular motors comprises proteins that participate in a wide variety of motile events within the cell. Members of this family share a highly homologous head domain responsible for force generation attached to a divergent tail domain thought to couple the motor domain to its target cargo. Many kinesin-related proteins (KRPs) participate in spindle morphogenesis and chromosome movement in cell division. Genetic analysis of mitotic KRPs in yeast and Drosophila, as well as biochemical experiments in other species, have suggested models for the function of KRPs in cell division, including both mitosis and meiosis. Although many mitotic KRPs have been identified, the relationship between mitotic motors and meiotic function is not clearly understood. We have used sequence similarity between mitotic KRPs to identify candidates for meiotic and/or mitotic motors in a vertebrate. We have identified a group of kinesin-related proteins from rat testes (termed here testes KRP1 through KRP6) that includes new members of the bimC and KIF2 subfamilies as well as proteins that may define new kinesin subfamilies. Five of the six testes KRPs identified are expressed primarily in testes. Three of these are expressed in a region of the seminiferous epithelia (SE) rich in meiotically active cells. Further characterization of one of these KRPs, KRP2, showed it to be a promising candidate for a motor in meiosis: it is localized to a meiotically active region of the SE and is homologous to motor proteins associated with the mitotic apparatus. Testes-specific genes provide the necessary probes to investigate whether the motor proteins that function in mammalian meiosis overlap with those of mitosis and whether motor proteins exist with functions unique to meiosis. Our search for meiotic motors in a vertebrate testes has successfully identified proteins with properties consistent with those of meiotic motors in addition to uncovering proteins that may function in other unique motile events of the SE.     

人前列腺生长因子的分离纯化及生物活性鉴定

从人良性增生前列腺组织中经硫酸铵沉淀和肝素－琼脂糖凝胶层析纯化出人前列腺生长因子（ｈＰＧＦ）,纯化倍数约１０００倍,ＳＤＳ－ＰＡＧＥ和等电聚焦电泳示分子量约为１７ｋＤ、等电点同标准ｂＦＧＦ,利用分离培养的人前列腺间质成纤维细胞进行活性鉴定,发现以１．３～１．７ｍｏｌ／ＬＮａＣｌ洗脱部分为ｈＰＧＦ,活性最高,对间质成纤维细胞有显著刺激增殖作用。     

外源钙调蛋白对植物细胞分裂增殖作用的研究

外源钙调蛋白（Ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）对胡萝卜悬浮细胞增殖具明显促进作用,不同浓度ＣａＭ的促进程度不同,７ｕｇ／ｍｌ时促进作用最大。ＣａＭ抑制剂ＴＦＰ（Ｔｒｉｆｌｕｏｐｅｒ－ａｚｉｎｅ）则明显抑制该悬浮细胞的增殖,ＴＦＰ浓度越高则抑制作用越强。另外,外源ＣａＭ可以加快珍珠梅花粉第二次有丝分裂,改变生殖细胞有丝分裂各期花粉管的比例,说明外源ＣａＭ对植物体细胞和性细胞的增殖和分裂均有促进作用。     

Magnesium Adenosine 5[prime]-Triphosphate-Energized Transport of Glutathione-S-Conjugates by Plant Vacuolar Membrane Vesicles

By characterization of the uptake of glutathione-S-conjugates, principally dinitrophenyl-S-glutathione (DNP-GS), by vacuolar membrane vesicles, we demonstrate that a subset of energy-dependent transport processes in plants are not H+-coupled but instead are directly energized by MgATP. The most salient features of this transport pathway are: (a) its specific, obligate requirement for MgATP as energy source; (b) the necessity for hydrolysis of the [gamma]-phosphate of MgATP for uptake; (c) the insensitivity of uptake to uncouplers of the transtonoplast H+ gradient (carbonylcyanide 4-trifluoromethoxyphenylhydrazone, gramicidin-D, and NH4Cl); (d) its pronounced sensitivity to vanadate and partial inhibition by vinblastine and verapamil; (e) the lack of chemical modification of DNP-GS either during or after transport; (f) the capacity of S-conjugates of chloroacetanilide herbicides, such as metolachlor-GS, but not free herbicide, to inhibit uptake; and (g) the ability of vacuolar membrane vesicles purified from a broad range of plant species, including Arabidopsis, Beta, Vigna, and Zea, to mediate MgATP-dependent, H+-electrochemical potential difference-independent DNP-GS uptake. On the basis of these findings it is proposed that the transport of DNP-GS across the vacuolar membrane of plant cells is catalyzed by a glutathione-conjugate transporter that directly employs MgATP rather than the energy contained in the transtonoplast H+-electrochemical potential difference to drive uptake. The broad distribution of the vacuolar DNP-GS transporter and its inhibition by metolachlor-GS are consistent with the notion that it plays a general role in the vacuolar sequestration of glutathione-conjugable cytotoxic agents.     

Phosphoryl amino acids: Common origin for nucleic acids and protein

A series of compounds (DAP-AA) composed of an amino acid (AA) and a dialkyl phosphoryl group (DAP) is the basic elements of life chemistry. Self-catalysis of DAP-AA gives the self-assembly oligopeptides, even in aqueous medium at 38°C. The oligo-nucleotides could also be assembled from nucleosides' phosphorylation by DAP-AA. DAP-AA acts as the energy source as well as the phosphoryl donor for the synthesis of nuclic Acids and protein. A general expression for the self assembly system is proposed.     

Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01

The single-strand origin (SSO) of the rolling-circle (RC), broad-host-range lactococcal plasmid pWVO1 was functionally characterized. The activity of this SSO in the conversion of single-stranded DNA to double-stranded DNA was tested both in vivo and in vitro. In addition, the effect of this SSO on plasmid maintenance was determined. The functional pWVO1 SSO comprises a 250 by region, containing two inverted repeats (IRs). The activity of each IR was tested, separately and in combination, in a plasmid derivative that was otherwise completely devoid of structures that might function as SSO. One of the IRs (IR 1) showed some homology with other previously described SSOs of the SSO_A type, as well as with the conversion signal of the Escherichia coli phage X174. This IR was shown to have a partial, RNA polymerise-independent activity in complementary strand synthesis, both in vivo and in vitro. The second IR, which had no activity of its own, was required for full SSO activity, both in vivo and in vitro. The conversion of single-stranded DNA to the double-stranded form by the complete SSO was only partly sensitive to inhibition by rifampicin, indicating the existence of an RNA polymerase-independent pathway for this event. The results suggest that the pWVO1 SSO can be activated by two different routes: an RNA polymerise-dependent one (requiring the entire SSO), and an RNA polymerase-independent one (requiring only IR I).     

Analysis of randomly isolated cDNAs from developing endosperm of rice (Oryza sativa L.): evaluation of expressed sequence tags,and expression levels of mRNAs

Using a cDNA library prepared from poly(A)⁺ RNA from 10-day-old rice endosperm, partial nucleotide sequences of randomly isolated clones were analyzed. A total of 153 (30.6%) out of 500 cDNA clones showed high amino acid identity to previously identified genes. There was significant redundancy in cDNAs encoding prolamine and glutelin. About 21.0% of the cDNA clones were found to code for seed storage protein genes. Consequently, 37 independent genes were identified. Using cDNA clones encoding glutelin, prolamine, seed allergen, -1,4-glucan branching enzyme, glycine-rich RNA binding protein, metallothionein, non-specific lipid-transfer protein and ubiquitin conjugating enzyme the accumulation of mRNA during rice seed development was compared. Genes associated with seed storage protein and starch biosynthesis were expressed according to expected developmental stages. Glycinerich RNA binding protein genes as well as metallothionein-like protein genes were highly expressed in developing seeds, but low in leaves of whole plants.     

Plant regeneration from protoplasts isolated from cotyledons of Sesbania bispinosa

Protoplasts were isolated from cotyledons of Sesbania bispinosa (Jacq.) W.F. Wight. In a liquid-over-agar culture system with Murashige and Skoog (MS) medium supplemented with 1 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d, 2 mg l^-1 benzyladenine (BA), 1 mg l^-1 glutamine and 0.5 and formed callus. The first division occurred after 3–4 days. Callus formed from the protoplasts differentiated shoots by organogenesis on MS medium with 1 mg l^-1 indolebutyric acid (IBA) and 1 mg l^-1 BA. These shoots developed into complete plantlets when excised and cultured on MS medium with 0.5 mg l^-1 IBA.     

Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad.

Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.