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。     

广州抽水蓄能电站蓄水库蓄水初期浮游生物调查

对我国首座核电站配套工程的抽水蓄能电站水库蓄水初期的浮游生物进行了调查。蓄水水库由上下两水库组成。两水库的浮游生物种类组成显著不同。上水库浮游植物共记录到３０种（属）,浮游动物１２种（属）;下水库浮游植物１９种（属）;浮游动物２５种（属）。上下两水库浮游生物数量均较贫乏,表明浮游生物群落尚处于初期发育阶段,但在局部浅水处已出现小规模的蓝藻水花,认为是水库蓄水初期必然的结果     

Production and cytogenetics of intergeneric hybrids between Brassica napus and Orychophragmus violaceus

The intergeneric hybrid between Brassica napus and Orychophragmus violaceus was obtained by means of embryo culture technique with the latter as the pollen parent. The hybrid was morphologically intermediate between its parents, but could produce a lot of seeds when selfed. Somatic separation of the genomes from the two parental species was observed during the mitotic divisions of some of the hybrid cells. Thus, the hybrid became the mixoploid in nature, consisting of haploid and diploid cells of B. napus, and a nuclear — cytoplasmic hybrid, with the cytoplasm of B. napus and the nuclei of O. violaceus, and the hybrid cells. Pollen mother cells with 19, 12 and 6 bivalents, respectively, were produced by the hybrid. From the selfed progeny of the hybrid, mainly two kinds of plants, B. napus and the hybrid, were found. The hybrid plants of the selfed progeny again produced two kinds of plants, B. napus and the hybrid.     

Structure and properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) produced by Alcaligenes latus

Summary Random copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) with a wide range of compositions varying from 0 to 83 mol% 4HB were produced by Alcaligenes latus from the mixed carbon substrates of 3-hydroxybutyric and 4-hydroxybutyric acids. The structure and physical properties of P(3HB-co-4HB) were characterized by¹H and¹³C NMR spectroscopy, gel-permeation chromatography, and differential scanning calorimetry. The isothermal radial growth rates of spherulites of P(3HB-co-4HB) were much slower than the rate of P(3HB) homopolymer. The enzymatic degradation rates of P(3HB-co-4HB) films by a PHB depolymerase were strongly influenced by the copolymer composition.     

The protein conformation and a zinc-binding domain of an autoantigen from mouse seminal vesicle.

The protein conformation of a mouse seminal vesicle autoantigen was studied by circular dichroism spectroscopy. At pH 7.4, the spectrum in the UV region appears as one negative band at 217 nm and one positive band at 200 nm. This together with the predicted secondary structures indicates no helices but a mixture of beta form, beta turn, and unordered form in the protein molecule. The conformation is stable even at pH 10.5 or 3.0. The spectrum in the near-UV region consists of fine structures that are disturbed in acidic or alkaline solution. The environments around Trp2 and Trp82 of this protein were studied by intrinsic fluorescence and solute quenching. They give an emission peak at 345 nm, and about 87% of them are accessible to quenching by acrylamide. Correlating the quenching effect of CsCl and Kl on the protein fluorescence to the charged groups along the polypeptide chain suggests the difference in the "local charge" around the two tryptophan residues. The presence of ZnCl2 in the protein solution effects no change in the circular dichroism but perturbs the fluorescence due to Trp82. Analysis of the fluorescence data suggests a Zn(2+)-binding site on the protein, which cannot coordinate with both Ca2+ and Mg2+. The association constant for the complex formation is 1.35 x 10(5) +/- 0.04 x 10(5) M-1 at pH 7.4.     

Quantitative contribution of the acid production to the intracellular acidification in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

A chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP), induced an acidification of cytosol by about 0.05 pH units in 30 sec followed by an alkalinization in human neutrophils. The quantitative contribution of acid production to the acidification was studied. The superoxide (O₂ ^–) production stimulated by fMLP was not involved in the acidification because the production of acids in neutrophils from patients with chronic granulomatous disease who do not produce O₂ ^–, was the same as that in normal neutrophils. The intracellular acidification was completely inhibited by deoxyglucose, suggesting that energy metabolism enhanced upon stimulation by fMLP might be the main source of the acidification. Although enhancement of the lactate formation by fMLP was 0.8 nmol/10⁶ cells, which could lower intracellular pH by 0.08 pH units, the lactate production could not explain the initial acidification because the production of lactate started at 1 min after the stimulation while the intracellular acidification began immediately after the stimulation. Mitochondrial respiratory inhibitors such as KCN and rotenone had no effects on the fMLP-induced intracellular acidification. The fMLP-induced production of CO₂ in 30 sec through the hexose monophosphate shunt was only 2.6 pmol/10⁶ cells, which was calculated to decrease intracellular pH by only 0.0014. Thus, changes of energy metabolism induced by fMLP does not explain the acidification.Abbreviations fMLP N-formyl-methionyl-leucyl-phenylalanine - BCECF-AM 2,7-bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester - PMA phorbol 12-myristate 13-acetate - CGD chronic granulomatous disease - HMP hexose monophosphate - pHi intracellular pH     

Vinculin Proteolysis Unmasks an ActA Homolog for Actin-based Shigella Motility

In polarized Madin-Darby canine kidney (MDCK) cells, the transferrin receptor (TR) is selectively delivered to the basolateral surface, where it internalizes transferrin via clathrin-coated pits and recycles back to the basolateral border. Mutant tailless receptors are sorted randomly in both the biosynthetic and endocytic pathways, indicating that the basolateral sorting of TR is dependent upon a signal located within the 61–amino acid cytoplasmic domain. To identify the basolateral sorting signal of TR, we have analyzed a series of mutant human TR expressed in MDCK cells. We find that residues 19–41 are sufficient for basolateral sorting from both the biosynthetic and endocytic pathways and that this is the only region of the TR cytoplasmic tail containing basolateral sorting information. The basolateral sorting signal is distinct from the YTRF internalization signal contained within this region and is not tyrosine based. Detailed functional analyses of the mutant TR indicate that residues 29–35 are the most important for basolateral sorting from the biosynthetic pathway. The structural requirements for basolateral sorting of internalized receptors from the endocytic pathway are not identical. The most striking difference is that alteration of G₃₁DNS₃₄ to YTRF impairs basolateral sorting of newly synthesized receptors from the biosynthetic pathway but not internalized receptors from the endocytic pathway. Also, mutations have been identified that selectively impair basolateral sorting of internalized TRs from the endocytic pathway without affecting basolateral sorting of newly synthesized receptors. These results imply that there are subtle differences in the recognition of the TR basolateral sorting signal by separate sorting machinery located within the biosynthetic and endocytic pathways.     

Infectivity enhancement by human immunodeficiency virus type 1 Nef is independent of its association with a cellular serine/threonine kinase.

Nef proteins from human immunodeficiency virus type 1 isolate SF2 (HIV-1SF2) and simian immunodeficiency virus isolate mac239 (SIVmac239) have been found to associate with a cellular serine/threonine kinase designated NAK. We have recently shown that the association of Nef with NAK is isolate dependent. To identify the structural basis for Nef-kinase association, several chimeric molecules were constructed between SF2 Nef (binding NAK) and 233 Nef (a primary isolate not binding NAK) and stably expressed in HuT-78 human T cells via retrovirus-mediated gene transfer. The Nef 233/SF2/SF2 chimera in which the N-terminal 37 amino acids of SF2 Nef were replaced by those of 233 Nef showed the same ability as SF2 Nef to bind NAK. The Nef 233/SF2/233 chimera in which the N-terminal 37 amino acids and the C-terminal 72 amino acids of SF2 Nef were replaced by corresponding sequences from 233 Nef completely lost the ability to associate with the kinase activity. Furthermore, replacement of the C-terminal 72 amino acids of 233 Nef with the equivalent SF2 sequence (chimera 233/233/SF2) fully restored kinase association to 233 Nef. These results suggest that (i) the core of Nef is not sufficient for NAK binding, (ii) the C terminus of SF2 Nef contains structural determinants important for association with NAK, and (iii) the failure of 233 Nef to bind NAK is due to a defect in its C terminus. Taking advantage of the C terminus of 233 Nef being nonfunctional and using an infectious clone of HIV-1SF2, we show that association with NAK is not required for Nef-mediated infectivity enhancement. While the strong and reproducible association of some Nef isolates with NAK has been clearly established, the role of NAK in Nef function remains to be fully elucidated.     

Interaction of an adenovirus 14.7-kilodalton protein inhibitor of tumor necrosis factor alpha cytolysis with a new member of the GTPase superfamily of signal transducers.

The adenovirus (Ad) 14.7-kDa E3 protein (E3-14.7K), which can inhibit tumor necrosis factor alpha (TNF-alpha) cytolysis, was used to screen HeLa cell cDNA libraries for interacting proteins in the yeast two-hybrid system. A new member of the low-molecular-weight (LMW) GTP-binding protein family with Ras and ADP-ribosylation factor homology was discovered by this selection and has been named FIP-1 (14.7K-interacting protein). FIP-1 colocalized with Ad E3-14.7K in the cytoplasm especially near the nuclear membrane and in discrete foci on or near the plasma membrane. Its interaction with E3-14.7K was dependent on the FIP-1 GTP-binding domain. The stable expression of FIP-1 antisense message partially protected the cells from TNF-alpha cytolysis. FIP-1 was associated transiently with several unknown phosphorylated cellular proteins within 15 min after treatment with TNF-alpha. FIP-1 mRNA was expressed ubiquitously but at higher levels in human skeletal muscle, heart, and brain. In addition to homology to other LMW GTP-binding proteins, FIP-1 has regions of homology to two prokaryotic metalloproteases. However, there was no homology between FIP-1 and any of the recently isolated death proteins in the TNF-alpha or Fas/APO1 cytolytic pathway and no interaction with several members of the Bcl-2 family of inhibitors of apoptosis. These data suggest that FIP-1, as a cellular target for Ad E3-14.7K, is either a new intermediate on a previously described pathway or part of a novel TNF-alpha-induced cell death pathway. FIP-1 has two consensus sequences for myristoylation which would be expected to facilitate membrane association and also has sequences for Ser/Thr as well as Tyr phosphorylation that could affect its function.