The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

Interventional radiological treatment of non-colorectal liver metastases

Transcatheter treatment was performed in 81 patients with unresectable non-colorectal liver metastases. Effectiveness increased in the following order: hepatic artery infusion--arterial chemoembolization--combined, arterial and portal vein oily chemoembolization. The mean survival rates for these methods were 8.2 +/- 5.3, vs 11.7 +/- 12.9 vs 13.6 +/- 6.8 mo, and 1-year survival rates 29% vs 46% vs 65%, respectively. Chemoembolization with doxorubicin-in-oil and gelatin sponge was the most effective technique. Interventional radiological procedures were effective in neuroendocrine liver metastases. The mean survival, 1- and 3-year survival rates were as high as 34 mo, 100% and 80%, respectively, for hepatic metastases from resected malignant carcinoid tumors. Also good results were achieved after chemoembolization of metastatic ovarian carcinoma and arterial infusion for gastric carcinoma metastatic to the liver. Transcatheter treatment was ineffective in liver metastases from pancreatic carcinoma, gallbladder cancer, and unknown (and non-resected) tumors. The initial results of the use of interventional radiological procedures in non-colorectal liver metastases are promising, so following clinical trails are needed.     

Three-dimensional structure of an unusual Kunitz (STI) type trypsin inhibitor from Copaifera langsdorffii

The crystallographic structure of a novel trypsin inhibitor (CTI) from Copaifera langsdorffii is reported. The structure was solved by MIRAS procedure and refined to a crystallographic residual of 17.3% (R(free) = 20.3%) at 1.8 A resolution. Two isomorphous derivatives were obtained by quick cryo-soaking approach. CTI is the first structure of a member of Kunitz (STI) family formed by two noncovalently bound polypeptide chains and only one disulfide bridge. A standard Kunitz-type inhibitor has a single polypeptide chain and two disulfide bridges. Structural features granting CTI high inhibitory activity are discussed.     

Average protein density is a molecular-weight-dependent function

The mass density of proteins is a relevant basic biophysical quantity. It is also a useful input parameter, for example, for three-dimensional structure determination by protein crystallography and studies of protein oligomers in solution by analytic ultracentrifugation. We have performed a critical analysis of published, theoretical, and experimental investigations about this issue and concluded that the average density of proteins is not a constant as often assumed. For proteins with a molecular weight below 20 kDa, the average density exhibits a positive deviation that increases for decreasing molecular weight. A simple molecular-weight-depending function is proposed that provides a more accurate estimate of the average protein density.     

Crystal structure of the Kunitz (STI)-type inhibitor from Delonix regia seeds

The three-dimensional structure of a novel Kunitz (STI) family member, an inhibitor purified from Delonix regia seeds (DrTI), was solved by molecular replacement method and refined, respectively, to R(factor) and R(free) values of 21.5% and 25.3% at 1.75A resolution. The structure has a classical beta-trefoil fold, however, differently from canonical Kunitz type (STI) inhibitors, its reactive site loop has an insertion of one residue, Glu68, between the residues P1 and P2. Surprisingly, DrTI is an effective inhibitor of trypsin and human plasma kallikrein, but not of chymotrypsin and tissue kallikrein. Putative structural grounds of such specificity are discussed.     

Divergence in macromolecular assembly: X-ray crystallographic structure analysis of lumazine synthase from Brucella abortus

We have determined the three-dimensional structure of 6, 7-dimethyl-8-ribityllumazine synthase (lumazine synthase) from Brucella abortus, the infectious organism of the disease brucellosis in animals. This enzyme catalyses the formation of 6, 7-dimethyl-8-ribityllumazine, the penultimate product in the synthesis of riboflavin. The three-dimensional X-ray crystal structure of the enzyme from B. abortus has been solved and refined at 2.7 A resolution to a final R-value of 0.18 (R(free)=0.23). The macromolecular assembly of the enzyme differs from that of the enzyme from Bacillus subtilis, the only other lumazine synthase structure known. While the protein from B. subtilis assembles into a 60 subunit icosahedral capsid built from 12 pentameric units, the enzyme from B. abortus is pentameric in its crystalline form. Nonetheless, the active sites of the two enzymes are virtually identical indicating inhibitors to theses enzymes could be effective pharmaceuticals across a broad species range. Furthermore, we compare the structures of the enzyme from B. subtilis and B. abortus and describe the C teminus structure which accounts for the differences in quaternary structure.     

Amino acid sequence of piratoxin-II, a myotoxic lys49 phospholipase A(2) homologue from Bothrops pirajai venom

The complete amino acid sequence of the 121 amino acid residues of piratoxin II, a phospholipase A(2) like myotoxin from Bothrops pirajai venom, is reported. PrTX-II is a basic protein with a molecular mass of 13740 Da, a calculated pI of 9.03, but an experimental pI of 8.4 +/- 0.2, showing sequence similarity with other bothropic (90-99%) or non-bothropic ( approximately 80%) Lys49 PLA(2)-like myotoxins. This similarity falls to approximately 70% when this sequence is aligned with that of Asp49 PLA(2). Due to the substitution of Asp49 by Lys49 and alterations in the calcium binding loop structure, as the replacement of Gly32 by Leu32, piratoxin-II shows no PLA(2) activity when assayed on egg yolk. Piratoxin-II showed the same primary structure as piratoxin-I, except that it has Lys116 for Leu116. Despite this slightly higher basicity at the C-terminal region, piratoxin-II was shown to be less myotoxic than piratoxin-I. The change Leu --> Lys induced an alteration of the molecule surface shape and probably of the environment charge high enough to slightly decrease the myotoxic activity. When aligned with B. jararacussu bothropstoxin-I and with B. asper Basp-II, piratoxin-II revealed a single (position 132) and a quintuple (positions 17, 90, 111, 120 and 132) amino acid substitution, respectively, suggesting a common evolutionary origin for these three myotoxins.     

啤酒酵母菌的细胞融合

啤酒多倍体酵母菌原生质体已成功地与单倍体原生质体进行融合。经细胞壁再生后,稳定的融合重组体被分离出来。这些融合体的基因分析表明,融合体中含有双亲的基因型。孢子形成良好,且每个子囊中含有四个孢子,每个孢子确实是二倍体。这样原生质体融合就提供了一个对啤酒酿造酵母进行遗传分析的方法。但是如果没有一个方便的杂交技术,这个方法将是很困难的。