Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition

Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization. We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis. First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer. The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely unaltered by detergent extraction. Residual desmosomes, as can be seen in thin sections, were also well- preserved. The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation. The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders. This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts. Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold. Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders. The 52,000-mol-wt nuclear matrix protein was confined to the chromatin- depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions. The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold.     

Bidirectional effect of met-enkephalin on macrophage effector functions

Summary Met-enkephalin (ME) exerts a bimodal effect on functional activities of rat peritoneal macrophages (PM); in a range of low concentration (10^-9-10^-7 M) antibody dependent cellular cytotoxicity (ADCC)was markedly stimulated with a simultaneous decrease of Fc receptor (FcR) mediated phagocytosis while the opposite was observed at 10^-6-10^-5 M concentrations.Studying the possible underlying mechanism(s) the followings were recorded: (1) ME in all applied concentrations induced an early Na⁺ influx which was followed by a Ca²⁺ efflux in the range of low concentrations. In the range of high concentrations Na⁺ influx was accompanied by a Ca²⁺ influx. (2) ME at 10^-8 M concentration induced a rise in cGMP level with a plateau in the 60–120th min of incubation. This effect was prevented by 10^-5 M of naloxone. At 10^-6 M concentration a transient rise of cAMP level was recorded which was not affected by naloxone. (3) Verapamil in 10^-6 M abolished both the Ca²⁺ influx and the rise in cAMP level induced by 10^-6-10^-5 M ME but not the rise in cGMP level induced by lower ME concentrations. (4) cAMP elevation by high ME concentrations was abolished by enkephalinase inhibitory puromycin. (5) PM-enkephalinase as assessed by the cleavage of fluorogenic substrate L-alanine beta naphthylamide (ABNA), was inhibited by 10^-6-10^-5 M of ME. This inhibition was abolished by verapamil, but not affected by naloxone. In the range of low concentrations ME appears to act on specific delta opioid receptors and its action is positively coupled to guanylate cyclase. In relatively higher concentrations ME-action is not mediated by specific delta opioid receptors and it appears to involve Ca²⁺ influx, adenylate cyclase activation as well as the processing of hormone by PM-enkephalinase.     

Long-acting contraceptive agents: Norethisterone esters of arylcarboxylic acids

The synthesis of esters of norethisterone (17α-ethynyl-17β-hydroxy-estr-4-en-3-one) with acids containing a benzene ring is described, two methods of esterification being compared in terms of yield and convenience. The activities of these esters as long-acting contraceptive agents have been evaluated.     

Aliesterase activity and reproduction in Musca domestica: Effects of DDT and metepa

The chemosterilant metepa, administered orally and topically to adult M. domestica, was found to reduce aliesterase activity, to reduce the hatch of eggs, and when administered orally it also prolonged the pre-ovipositional period. Orally administered DDT had little effect on aliesterase but did reduce egg viability. Selection of a strain of M. domestica with diazinon, in an attempt to increase its resistance to diazinon, had the unexpected result of the flies becoming more susceptible but concomitantly their level of aliesterase was significantly increased. The aliesterase level of the immature stages of M. domestica is reported.

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Zusammenfassung Es wurde festgestellt, daß das Chemosterilisans Metepa bei oraler oder topischer Applikation bei erwachsenen Stubenfliegen die Aliesterase-Aktivität herabsetzt, die Schlüpfrate der Eier vermindert und bei oraler Anwendung auch die Präovipositionszeit verlängert. Oral verabreichtes DDT hat wenig Einfluß auf die Aliesterase, setzt aber die Lebensfähigkeit der Eier herab. Die Selektion einer Linie von Musca domestica durch Diazinon mit dem Ziel, ihre Resistenz gegen Diazinon zu erhöhen, hatte das unerwartete Ergebnis, daß die Fliegen empfindlicher wurden, zugleich aber ihr Aliesterase-Spiegel signifikant anstieg. Der Aliesterase-Spiegel der nicht erwachsenen Stubenfliegen-Stadien wird angegeben.

     

Mapping of B-cell epitopes of the human hepatitis B virus X protein.

The immune response to the X protein of human hepatitis B virus (HBV) was studied by epitope mapping by using a set of MS2-HBx fusion proteins and synthetic peptides. Antibodies in sera of patients with acute and chronic HBV infection showed a multispecific immune response. Each serum contained antibodies to a different set of epitopes, which taken together cover most of the HBx sequence. Some of the epitopes were detectable only by immunoblotting with fusion proteins; others were detectable only by an enzyme-linked immunosorbent assay (ELISA) with synthetic peptides. The carboxy-terminal half of the HBx protein was preferentially recognized by antibodies from patients with chronic hepatitis and contained a short immunodominant antigenic region with at least two major nonoverlapping epitopes. Anti-HBx antibody titers as revealed by peptide ELISAs were highest and most frequent in patients with chronic hepatitis and usually low in acutely infected patients and asymptomatic carriers. The data demonstrate a remarkable qualitative and quantitative heterogeneity of the humoral HBx immune response which can be monitored by HBx-specific peptide ELISAs. Such tests may become useful diagnostic tools.     

Two new loci for hybrid sterility in cultivated rice (Oryza sativa L.)

Female gamete abortion in Indica-Japonica crosses of rice was earlier identified to be due to an allelic interaction at the S-5 locus on chromosome 6. Recently, in other crosses of rice, similar allelic interactions were found at loci designated as S-7 and S-8, located on chromosomes 7 and 6 respectively. All of them are independent of each other. At the S-5 locus, Indica and Japonica rice have S-5 ⁱ and S-5 ^j alleles respectively and Javanicas, such as Ketan Nangka, have a neutral allele S-5 ⁿ .The S-5 ⁱ /S-5 ^j genotype is semi-sterile due to partial abortion of female gametes carrying S-5 ^j , but both the S-5 ⁿ /S-5 ⁱ and S-5 ⁿ /S-5 ^j genotypes are fertile. The S-5 ⁿ allele is thus a wide-compatibility gene (WCG), and parents homozygous for this allele are called wide-compatible varieties (WCV). Such parents when crossed with Indica or Japonica varieties do not show F₁ hybrid sterility. Wide-compatible parents have been used to overcome sterility barriers in crosses between Indica and Japonica rice. However, a Javanica variety, Ketan Nangka (WCV), showed typical hybrid sterility when crossed to the Indian varieties N22 and Jaya. Further, Dular, another WCV from India, showed typical hybrid sterility when crossed to an IRRI line, IR2061-628-1-6-4-3(IR2061-628). By genetic analyses using isozyme markers, a new locus causing hybrid sterility in crosses between Ketan Nangka and the Indicas was located near isozyme loci Est-1 and Mal-1 on chromosome 4, and was designated as S-9. Another new locus for hybrid sterility in the crosses between Dular and the IR2061-628 was identified and was found linked to four isozyme loci, Sdh-1, Pox-2, Acp-1 and Acp-2, on chromosome 12. It was designated as S-15. On the basis of allelic interactions causing female-gamete abortion, two alleles were found at S-9, S-9 ^kn in Ketan Nangka and S-9 ⁱ in N22 and Jaya. In the heterozygote, S-9 ^kn /S-9 ⁱ , which was semisterile, female gametes carrying S-9 ^kn were aborted. The hybrid of Dular and IR2061-628, with a genetic constitution of S-15 ^Du /S-15 ⁱ , was semi-sterile and the female gametes carrying S-15 ^Du were aborted. A Japonica tester variety, Akihikari, and an Indica variety, IR36, were found to have neutral alleles, S-9 nand S-15 n, at these loci, in addition to S-7 nand at S-7. The accumulation of three neutral alleles into a breeding line should help solve the hybrid sterility problem in wide crosses of rice.     

棉铃虫致病菌BT—931菌株的应用研究

１９９２－１９９３年,作者从菏泽、聊城棉田采集的自然罹病死亡棉铃虫幼虫体内,分离到２个较高毒效的Ｂｔ菌株,编号为ＢＴ—９３１和ＢＴ—０２１,经室内毒力测定和田间药效试验表明,防效及保蕾效果接近或超过化学农药,菌药混剂３—５天平均防治效果达８７．０－９１．６％,增效作用显著。经１９９４年大田防治示范表明,利用ＢＴ—９３１菌剂。配合化学农药防治棉铃虫,具有成本低、保护天敌、持效期长、增产效益高等优点。本研究对菌剂的田间应用技术进行了试验示范。     

磁水对蕃茄酯酶同工酶的影响

蕃茄（ＬｙｃｏｐｅｒｓｉｃｏｍｅｓｃｕｌｅｎｔｕｍＭｉｌｌ）在各生长发育期酯酶同工酶谱带无明显变化,但经磁水处理后各生长发育阶段功能叶中酯酶同工酶酶谱及磷素含量与对照组相比存在显著差异,且具有酶谱负极端带数增加,正极端带数减少的趋势。总磷含量测定结果表明处理组均比对照组显著提高。这些变化在磁水处理著前后代中不存在,故认为磁水只是致使非编码顺序局部复制引起ＤＮＡ分子高级结构改变或是通过活化调节基因来调控酯酶同工酶,活化磷代谢,达到促进生长和增产的效果,而并非导致可遗传变异的结果。