Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 60 S ribosomal subunit proteins L4, L5, L7, L9, L11, L12, L13, L21, L22, L23, L26, L27, L30, L33, L35', L37, and L39.

The proteins of the large subunit of rat liver ribosomes were separated into seven groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Seventeen proteins (L4, L5, L7, L9, L11, L12, L13, L21, L22, L23, L26, L27, L30, L33, L35', L37, and L39) were isolated from three of the groups (B60, D60, G60) by ion exchange chromatography on carboxymethylcellulose and by filtration through Sephadex. The amount of protein obtained varied from 0.5 to 15 mg. Eight of the proteins (L9, L11, L13, L21, L22, L35', L37 and L39) had no detectable contamination; the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.     

Purification of Drosophila ribosomal proteins. Isolation of proteins S8, S13, S14, S16, S19, S20/L24, S22/L26, S24, S25/S27, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 7/8, 9, and 11

The proteins of Drosophila melanogaster embryonic ribosomes were separated into seven groups (A80 through G80) by stepwise elution from carboxymethylcellulose with lithium chloride at pH 6.5 by procedures previously described [Chooi, W. Y., Sabatini, L. M., MacKlin, M. D., & Fraser, W. (1980) Biochemistry 19, 1425-1433]. Three relatively acidic proteins, S14, S25/S27, and 7/8, have now been isolated from group A80 by ion-exchange chromatog raphy on carboxymethylcellulose eluted with a linear gradient of lithium chloride at pH 4.2. Fractions containing the relatively basic proteins (groups B80 through G80) were furher combined into a total of 24 "pools". The criterion for combination was the migration patterns in one-dimensional polyacrylamide gels containing sodium dodecyl sulfate (NaDodS04) of every fifth fraction from the carboxymethylcellulose column. Each pool contained between 1 and 12 major proteins. Proteins S8, S13, S16, S19, S20/L24, S22/L26, S24, S26, S29, L4, L10/L11, L12, L13, L16, L18, L19, L27, 1, 9, and 11 have now been isolated from selected pools by gel filtration through Sephadix G-100. The amount of each protein recovered from a starting amount of 1.8 g of total 80S proteins varied form 0.2 to 10.8 mg. Five proteins had no detectable contamination, and in each of the others the impurities were no greater than 9%. The amino acid composition of the individual purified proteins was determined. The molecular weights of the proteins were estimated by polyacrylamide gel electrophoresis in NaDodSO4.     

Relative photorefractoriness, prolactin, and reproductive regression in a flexibly breeding sonoran desert passerine, the rufous-winged sparrow, Aimophila carpalis

We tested the hypothesis that adult male rufous-winged sparrows, Aimophila carpalis, exhibit relative photorefractoriness. This condition results in partial loss of sensitivity to photoperiod as a reproductive stimulus after prolonged exposure to long photoperiods and is similar to the mammalian condition called photoperiodic memory. Captive birds were exposed either to 8 h of light/16 h of dark per day (8L) or to 16L for 11 weeks and were then exposed either to 8L, 13L, 14L, or 16L. Testicular diameter, plasma luteinizing hormone (LH), and plasma prolactin (PRL) were measured to assess reproductive system activity in response to photostimulation. In free-living birds, testicular diameter, plasma LH, and PRL were compared in birds caught in September in a year when birds were breeding and in a year when birds were not breeding to further evaluate the role of PRL in the termination of seasonal breeding. Testes completely developed after transfer from 8L to 14L or to 16L and partially developed after transfer from 8L to 13L. However, after 11 weeks of 16L exposure, transfer to 14L caused partial regression and transfer to 13L caused complete regression of the testes. Plasma LH increased in all birds that were transferred from 8L to a longer photoperiod. PRL showed a weak response to longer photoperiod treatment and was elevated in birds after chronic 16L exposure in comparison to birds exposed to chronic 8L. These data indicate that male rufous-winged sparrows lose sensitivity to photoperiod after long photoperiod exposure consistent with the relative photorefractoriness and photoperiodic memory models. Lower PRL in birds that developed testes on 13L and 14L compared to birds that regressed testes on 13L and 14L are consistent with the hypothesis that PRL regulates relative photorefractoriness. However, PRL does not appear to regulate interannual differences in the timing of testicular regression.     

Solid-state conformation of copolymers of β-benzyl-L-aspartate with L-alanine,L-leucine,L-valine, γ-benzyl-L-glutamate,or ϵ-carbobenzoxy-L-lysine

The solid-state conformation of copolymers of β-benzyl-L -aspartate [L -Asp(OBzl)] with L -leucine (L -Leu), L -alanine (L -Ala), L -valine (L -Val), γ-benzyl-L -glutamate [L -Glu(OBzl)], or ?-carbobenzoxy-L -lysine (Cbz-L -Lys) has been studied by ir spectroscopy and circular dichroism (CD). The ir spectra in the region of the amide I and II bands and in the region of 700–250 cm^?1 have been determined. The results from the ir studies are in good agreement with data obtained by CD experiments. Incorporation of the amino acid residues mentioned above into poly[L -Asp(OBzl)] induces a change from the left-handed into the right-handed α-helix. This conformational change for the poly[L -Asp(OBzl)] copolymers was observed in the following composition ranges: L -Leu, 0–15 mol %; L -Ala, 0–32 mol %; L -Val, 0–8 mol %; L -Glu(OBzl), 3–10 mol %; and Cbz-L -Lys, 0–9 mol %.     

European yellow lupine, Lupinus luteus, and narrow-leaf lupine, Lupinus angustifolius, as hosts for the pea aphid, Acyrthosiphon pisum

The pea aphid, Acyrthosiphon pisum Harris (Homoptera: Aphididae), fed, developed, and reproduced on yellow lupine, Lupinus luteus L. (Fabaceae: Genisteae). No clear preferences for any variety within L. luteus were found. Acyrthosiphon pisum showed negative values of relative growth rate and no aphid completed development on any variety of narrow-leaf lupine Lupinus angustifolius L. Aphids did not ingest phloem sap while probing on L. angustifolius and the probes were very short. All varieties of L. angustifolius were rejected by aphids during an early stage of probing in peripheral tissues, that is, epidermis or mesophyll. There were qualitative and quantitative differences in alkaloid and soluble sugar content between the two lupine species. Within species, the relative content of individual compounds differed among the varieties. Lupinus angustifolius contained four quinolizidine alkaloids (13-hydroxylupanine, dehydrolupanine, lupanine, and angustifoline), while L. luteus contained two (lupanine and sparteine). Lupanine occurred in all varieties of both lupine species. The total content of soluble carbohydrates was similar in L. luteus and L. angustifolius . The following cyclitols were found in both lupine species: myo -inositol, D-ononitol, and D-pinitol. Lupinus angustifolius also contained D- chiro -inositol. The study of aphid probing behaviour, development, and reproduction demonstrated that L. luteus is a suitable host plant for A. pisum while L. angustifolius is not. It is likely that the rejection of L. angustifolius by A. pisum was caused by chemical factors detected by aphids at the epidermis and mesophyll level.     

Antioxidant properties of essential oils from lemon, grapefruit, coriander, clove, and their mixtures

Antioxidant properties of individual essential oils from lemon (Citrus limon L.), pink grapefruit (Citrus paradise L.), coriander (Coriandrum sativum L.), and clove (Caryophyllus aromaticus L.) buds and their mixtures were studied by capillary gas-liquid chromatography. Antioxidant activity was assessed by oxidation of the aliphatic aldehyde hexanal to the carboxylic acid. The lowest and highest antioxidant activities were exhibited by grapefruit and clove bud essential oils, respectively. Mixtures containing clove bud essential oil also strongly inhibited oxidation of hexanal. Changes in the composition of essential oils and their mixtures in the course of long-term storage in the light were studied. The stability of components of lemon and coriander essential oils in mixtures increased compared to individual essential oils.     

Separation of Leydigia louisi Jenkin, 1934 from L. leydigi (Schoedler, 1863) (Chydoridae, Anomopoda, Cladocera)

The aim of the present article was to contribute to the systematics of the leydigi-like species of Leydigia consisting of a few (probably 3) formal species with: (1) a large basal spine on the postabdominal claw (as long as claw thickness at the base); (2) a short setulation at anterior margin of labral keel; (3) no longitudinal striation on the female valves; (4) at least three lateral setae in each fascicle on the postabdomen; (5) three large lateral setae on exopodite III. The morphology of L. leydigi (Schoedler, 1863) and L. louisi Jenkin, 1934 are redescribed, and type material of L. macrodonta Sars, 1916 is studied. In contrast to previous suggestions (Jenkin, 1934; Smirnov, 1971), I found that: (1) L. louisi is a valid species, not a subspecies of L. macrodonta; (2) L. macrodonta is not a member of the L. leydigi-group. The third member of leydigi-group, Leydigia macrodonta longiseta Chen Shou-zhong, 1992, was described from China. It is not a subspecies of L. macrodonta, but a relative of L. leydigi; most probably, it is a valid species, but this opinion must to be confirmed by examination of original Asian material. In this article, the presence of L. leydigi in Palearctic only, and that of L. louisi in only Africa was confirmed. A new subspecies of L. louisi, found in Mexico, will be reported separately.     

Recombinant and truncated tetanus neurotoxin light chain: cloning, expression, purification, and proteolytic activity

Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L). The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus. Here we describe the cloning of the L chain of TeNT from Clostridium tetani strain Y-IV-3 (WS 15) and its expression in Escherichia coli as a glutathione S-transferase fusion protein. The full-length recombinant L chain, corresponding to residues 1-457, was obtained as a mixture of proteins of slightly different mass with identical N-terminal ends. To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1-427) was cloned, expressed, and purified with high yield. This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysis of VAMP. Preliminary experiments of crystallization of the truncated recombinant L chain gave encouraging results.     

Quinolizidine alkaloid profiles of Lupinus varius orientalis, L. albus albus, L. hartwegii, and L. densiflorus

Alkaloid profiles of two Lupinus species growing naturally in Egypt (L. albus albus [synonym L. termis], L. varius orientalis) in addition to two New World species (L. hartwegii, L. densiflorus) which were cultivated in Egypt were studied by capillary GLC and GLC-mass spectrometry with respect to quinolizidine alkaloids. Altogether 44 quinolizidine, bipiperidyl and proto-indole alkaloids were identified; 29 in L. albus, 13 in L. varius orientalis, 15 in L. hartwegii, 6 in L. densiflorus. Some of these alkaloids were identified for the first time in these plants. The alkaloidal patterns of various plant organs (leaves, flowers, stems, roots, pods and seeds) are documented. Screening for antimicrobial activity of these plant extracts demonstrated substantial activity against Candida albicans, Aspergillus flavus and Bacillus subtilis.     

Structure and evolution of the L11, L1, L10, and L12 equivalent ribosomal proteins in eubacteria, archaebacteria, and eucaryotes

The genes corresponding to the L11, L1, L10, and L12 equivalent ribosomal proteins (L11e, L1e, L10e, and L12e) of Escherichia coli have been cloned and sequenced from two widely divergent species of archaebacteria, Halobacterium cutirubrum and Sulfolobus solfataricus, and the L10 and four different L12 genes have been cloned and sequenced from the eucaryote Saccharomyces cerevisiae. Alignments between the deduced amino acid sequences of these proteins and to other available homologous proteins of eubacteria and eucaryotes have been made. The data suggest that the archaebacteria are a distinct coherent phylogenetic group. Alignment of the proline-rich L11e proteins reveals that the N-terminal region, believed to be responsible for interaction with release factor 1, is the most highly conserved region and that there is specific conservation of most of the proline residues, which may be important in maintaining the highly elongated structure of the molecule. Although L11 is the most highly methylated protein in the E. coli ribosome, the sites of methylation are not conserved in the archaebacterial L11e proteins. The L1e proteins of eubacteria and archaebacteria show two regions of very high similarity near the center and the carboxy termini of the proteins. The L10e proteins of all kingdoms are colinear and contain approximately three fourths of an L12e protein fused to their carboxy terminus, although much of this fusion has been lost in the truncated eubacterial protein. The archaebacterial and eucaryotic L12e proteins are colinear, whereas the eubacterial protein has suffered a rearrangement through what appear to be gene fusion events. Within the L12e derived region of the L10e proteins there exists a repeated module of 26 amino acids, present in two copies in eucaryotes, three in archaebacteria, and one in eubacteria. This modular sequence is apparently also present in the L12e proteins of all kingdoms and may play a role in L12e dimerization, L10e-L12e complex formation, and the function of the L10e-L12e complex in translation.     

Functionalized benzophenone, thiophene, pyridine, and fluorene thiosemicarbazone derivatives as inhibitors of cathepsin L

A series of thiosemicarbazone analogs based on the benzophenone, thiophene, pyridine, and fluorene molecular frameworks has been prepared by chemical synthesis and evaluated as small-molecule inhibitors of the cysteine proteases cathepsin L and cathepsin B. The two most potent inhibitors of cathepsin L in this series (IC(50)<135 nM) are brominated-benzophenone thiosemicarbazone analogs that are further functionalized with a phenolic moiety (2 and 6). In addition, a bromo-benzophenone thiosemicarbazone acetyl derivative (3) is also strongly inhibitory against cathepsin L (IC(50)=150.8 nM). Bromine substitution in the thiophene series results in compounds that demonstrate only moderate inhibition of cathepsin L. The two most active analogs in the benzophenone thiosemicarbazone series are highly selective for their inhibition of cathepsin L versus cathepsin B.     

The complete primary structure of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus

The amino acid sequences of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus have been completely determined. This has been achieved by sequence analyses of peptides derived from enzymatic digestions of the proteins with trypsin, chymotrypsin, pepsin, Staphylococcus aureus protease, and Armillaria mellea protease as well as by chemical cleavage with hydroxylamine and cyanogen bromide. Based on the primary structures of the six proteins, their secondary structures were predicted using four different computer prediction programs. A comparison of the amino acid sequences of the studied proteins from B. stearothermophilus with the homologous proteins from Escherichia coli revealed that in four proteins (L1, L15, L24 and L29) between 40-50% of the residue in the sequences are identical, whereas this value is significantly higher (69%) for L14 and lower (28%) for L23. The distribution of those amino acid residues which are identical in the corresponding proteins from the two bacteria is not random along the protein chain: some regions are highly conserved whereas others are not. This finding indicates that the regions which are conserved during evolution are important for the spatial structure and/or function of the protein.     

Bax activation by engagement with, then release from, the BH3 binding site of Bcl-xL

Bcl-2 homologues (such as Bcl-x(L)) promote survival in part through sequestration of "activator" BH3-only proteins (such as Puma), preventing them from directly activating Bax. It is thus assumed that inhibition of interactions between activators and Bcl-x(L) is a prerequisite for small molecules to antagonize Bcl-x(L) and induce cell death. The biological properties, described here of a terphenyl-based alpha-helical peptidomimetic inhibitor of Bcl-x(L) attest that displacement of Bax from Bcl-x(L) is also critical. Terphenyl 14 triggers Bax-dependent but Puma-independent cell death, disrupting Bax/Bcl-x(L) interactions without affecting Puma/Bcl-x(L) interactions. In cell-free assays, binding of inactive Bax to Bcl-x(L), followed by its displacement from Bcl-x(L) by terphenyl 14, produces mitochondrially permeabilizing Bax molecules. Moreover, the peptidomimetic kills yeast cells that express Bax and Bcl-x(L), and it uses Bax-binding Bcl-x(L) to induce mammalian cell death. Likewise, ectopic expression of Bax in yeast and mammalian cells enhances sensitivity to another Bcl-x(L) inhibitor, ABT-737, when Bcl-x(L) is present. Thus, the interaction of Bcl-x(L) with Bax paradoxically primes Bax at the same time it keeps Bax activity in check, and displacement of Bax from Bcl-x(L) triggers an apoptotic signal by itself. This mechanism might contribute to the clinical efficiency of Bcl-x(L) inhibitors.     

Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 60 S ribosomal subunit proteins La, Lb, Lf, P1, P2, L13', L14, L18', L20, and L38

The proteins of the large subunit of rat liver ribosomes were separated into seven groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Ten proteins (La, Lb, Lf, P1, P2, L13', L14, L18', L20, and L38) were isolated from three groups (A60, B60, and D60) by ion exchange chromatography on carboxymethylcellulose and DEAE-cellulose, and by filtration through Sephadex. The amount of protein obtained varied from 0.3 to 3.8 mg. Two of the proteins (La and L18') had no detectable contamination; the impurities in the others were not greater than 8%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined. Several additional acidic proteins were identified: P1a and P1b are phosphorylated derivatives of P1; P2a, P2b, and P2c are phosphorylated derivatives of P2. P1 and P2 are distinct proteins but both have large amounts of alanine (20.4 and 17.5 mol %).     

Plasma homocysteine levels in cycling, pregnant, and spayed bitches

The aim of this study was to evaluate the physiological range of homocysteine (Hcy) exhibited by bitches during the follicular (pro-oestrus), luteal (dioestrus) and anoestrus phases of cycling animals, and in pregnant and spayed bitches. The lowest concentrations of Hcy were observed during dioestrus (3.2+/-1.27mumol/L) and in pregnant bitches (3.9+/-1.72mumol/L), and the highest concentrations during anoestrus (7.8+/-0.6mumol/L) and in spayed bitches (12.1+/-5.16mumol/L).     

Expression,purification and characterization of the interferon-inducible,antiviral and tumour-suppressor protein,human RNase L

The interferon (IFN)-inducible, 2′,5′-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) plays key role in antiviral defense of mammalian cells. Induction by IFN and activation by double-stranded RNA lead to 2-5A cofactor synthesis, which activates RNase L by causing its dimerization. Active RNase L degrades single-stranded viral as well as cellular RNAs causing apoptosis of virus-infected cells. Earlier, we had reported that expression of recombinant human RNase L caused RNA-degradation and cell-growth inhibition in E. coli without the need for exogenous 2-5A. Expression of human RNase L in E. coli usually leads to problems of leaky expression, low yield and degradation of the recombinant protein, which demands number of chromatographic steps for its subsequent purification thereby, compromising its biochemical activity. Here, we report a convenient protocol for expression of full-length, soluble and biochemically active recombinant human RNase L as GST-RNase L fusion protein from E. coli utilizing a single-step affinity purification with an appreciable yield of the highly purified protein. Recombinant RNase L was characterized by SDS-PAGE, immunoblotting and MALDI-TOF analysis. A semi-quantitative agarose-gel-based ribonuclease assay was developed for measuring its 2-5A-dependent RNase L activity against cellular large rRNAs as substrates. The optimized expression conditions minimized degradation of the protein, making it a convenient method for purification of RNase L, which can be utilized to study effects of various agents on the RNase L activity and its protein–protein interactions.     

Reliability of unfamiliar, multijoint, uni- and bilateral strength tests: effects of load and laterality

To better understand the reliability of unfamiliar multijoint strength tests, 16 resistance-trained men performed maximum velocity uni- (1 leg [1L]) and bilateral (2 legs [2L]) lifts on an unfamiliar semiprone leg squat machine with loads equivalent to 40 and 70% of maximum isometric force on 2 separate occasions. Peak force was highly reproducible between testing occasions at the heavy load under both uni- and bilateral conditions (intraclass correlation coefficient [ICC](1L70%) = 0.91, ICC(2L70%) = 0.92), was slightly reduced in the light load bilateral condition (ICC(2L40%) = 0.85), and was significantly (p < 0.05) reduced in the light load unilateral condition (ICC(1L40%) = 0.57). Test reliability was not related to total load lifted (2L 70% > 1L 70% > 2L 40% > 1L 40%) or to the peak force developed during the tests (2L 70% > 1L 70% = 2L 40% > 1L 40%), but it was somewhat related to the time taken to attain peak force (2L 70% = 1L 70% > 2L 40% > 1L 40%). To obtain reliable strength data from athletes, more familiarization seems to be needed when they perform modified versions of common multijoint strength tests, or unfamiliar strength tests, under light load, unilateral conditions. The marked differences in reliability resulting from variation in loading conditions suggests that the reliability of a test needs to be reestablished when it is modified, before it is used to assess athlete/subject strength performance.     

植物乳杆菌ZJ316生产细菌素

[目的]研究植物乳杆菌ZJ316生长和产细菌素的最佳培养基成分和发酵条件,以提高该菌产plantaricin ZJ316的能力.[方法]改变培养基成份和发酵条件,考察不同氮源、碳源等培养基成分和不同的发酵温度等条件对ZJ316生长和产细菌素的影响.[结果]最佳培养基为MRS培养基;优化后的培养基配方为葡萄糖10 g/L,麦芽糖10 g/L,酵母提取物10 g/L,蛋白胨10 g/L,柠檬酸三铵2 g/L,吐温80为1 Ml/L,K2HPO4·3H2O 6 g/L,乙酸钠5 g/L,硫酸镁0.2 g/L,硫酸锰0.05 g/L.培养基初始Ph6.5,30℃静置培养24 h.[结论]通过培养基成分和发酵条件的优化,细菌素产量提高了2.3倍,为进一步研究和规模化生产奠定基础.