Partial purification and properties of Galleria mellonella larvae proteolytic inhibitors acting on Metarhizium anisopliae toxic protease

Gel permeation, preparative isoelectric focusing, and affinity chromatography were used to purify three inhibitors of proteolytic activity from perchloric acid extracts of last instar Galleria mellonella larvae. Electrofocusing experiments revealed three isoinhibitors with different isoelectric points: inhibitor I-1 with p1 of pH 5.6, inhibitor I-2, pH 7.7, and inhibitor I-3 (of small inhibitory activity), pH 8.6. By affinity chromatography on trypsin-Sepharose 4B the I-1 was purified 9.7 ×, but 71.1% of inhibitory activity was lost. Molecular mass of the inhibitory complex was 12,600 Da. I-1 and I-2 are relatively stable to heat at several pHs with minor stability at pH 10. I-1 and I-2 inhibit serine proteases about 2.5 times as much as sulfhydryl proteases. In the same ratio protease P-1 and protease P-2 from Metarhizium anisopliae are inhibited.     

Survival and metabolism of the harpacticoid copepod Thompsonula hyaenae (Thompson) fed on different diatoms

Survival times and oxygen consumption rates have been determined for a benthic harpacticoid copepod, Thompsonula hyaenae (Thompson), when fed different algal diets. Nauplii and adults lived slightly longer on a diet of Navicula sp. than did those fed N. pelliculosa or a mixture of both naviculoid species. Mean numbers of harpacticoids hatched were significantly higher in cultures of N. pelliculosa. Metabolic rates of non-gravid females fed for one week on N. sp. were significantly lower than those fed N. pelliculosa or the mixture. There was no significant difference in oxygen uptake between animals fed N. pelliculosa and those fed the mixed culture. The smaller size and lower food energy content of N. pelliculosa are reflected in the higher respiratory rates of animals led this diatom species.Based on a thesis submitted in partial fulfillment of the M.S. degree in Marine Science at the University of South Carolina. Belle W. Baruch Institute for Marine Biology and Coastal Research Contribution No. 114. Research supported by the Oceanography Section, National Science Foundation, NSF Grant DES72-01573 A01.     

Evidence that the C-terminus of OprM is involved in the assembly of the VceAB-OprM efflux pump

Although the architecture of tripartite multiple drug resistance (MDR) efflux pumps of Gram-negative bacteria has been well characterized, the means by which the components recognize each other and assemble into a functional pump remains obscure. In this study we present evidence that the C-terminal domain of the Pseudomonas aeruginosa OprM and the α-helical hairpin domain of Vibrio cholerae VceA play an important role in the recognition/specificity/recruitment step in the assembly of a functional, VceAB-OprM chimeric efflux pump. To our knowledge, this is the first evidence directly linking the C-terminal domain of an outer membrane efflux protein to its recruitment during the assembly of a tripartite efflux pump.     

Photoinactivation of Photosystem II in leaves

Photoinactivation of Photosystem II (PS II), the light-induced loss of ability to evolve oxygen, inevitably occurs under any light environment in nature, counteracted by repair. Under certain conditions, the extent of photoinactivation of PS II depends on the photon exposure (light dosage, x), rather than the irradiance or duration of illumination per se, thus obeying the law of reciprocity of irradiance and duration of illumination, namely, that equal photon exposure produces an equal effect. If the probability of photoinactivation (p) of PS II is directly proportional to an increment in photon exposure (p = kΔx, where k is the probability per unit photon exposure), it can be deduced that the number of active PS II complexes decreases exponentially as a function of photon exposure: N = N_oexp(−kx). Further, since a photon exposure is usually achieved by varying the illumination time (t) at constant irradiance (I), N = N_oexp(−kI t), i.e., N decreases exponentially with time, with a rate coefficient of photoinactivation kI, where the product kI is obviously directly proportional to I. Given that N = N_oexp(−kx), the quantum yield of photoinactivation of PS II can be defined as −dN/dx = kN, which varies with the number of active PS II complexes remaining. Typically, the quantum yield of photoinactivation of PS II is ca. 0.1μmol PS II per mol photons at low photon exposure when repair is inhibited. That is, when about 10⁷ photons have been received by leaf tissue, one PS II complex is inactivated. Some species such as grapevine have a much lower quantum yield of photoinactivation of PS II, even at a chilling temperature. Examination of the longer-term time course of photoinactivation of PS II in capsicum leaves reveals that the decrease in N deviates from a single-exponential decay when the majority of the PS II complexes are inactivated in the absence of repair. This can be attributed to the formation of strong quenchers in severely-photoinactivated PS II complexes, able to dissipate excitation energy efficiently and to protect the remaining active neighbours against damage by light.     

Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60–85%) and the γ-subunit of nerve growth factor (by 55–93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the γ-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), IV), and (VI)], papain [(I), (II), IV), and (VI)], subtilisin [(V) and (VI)], α-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 × 10^?2 M, while, at the concentration of 1 × 10^?3 M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.     

2种嵩草属植物形态特征对生境干旱化的响应

该研究于西藏自治区东南部的东达山高山草甸沿生境干旱化梯度设置10个样地,采用线性回归方法分析优势种高山嵩草(Kobresia pygmaea)和矮生嵩草(K.humilis)叶片数量与其它构件数值(分株数量、茎基直径、根系数量和根系长度)之间的关系,并采用线性回归斜率测度分株功效、叶片萌生能力、根系分生功效和根系伸长功效,以探讨嵩草的分株能力、茎基生长和根系生长对生境干旱化过程的响应机制。结果显示:(1)2种嵩草植物的叶片数量与4种构件数值均为显著线性正相关关系。(2)随着生境干旱化程度增加,高山嵩草分株数量增加,矮生嵩草分株受干旱抑制程度高,分株数量呈下降趋势;2种嵩草分株功效下降,即单株叶片数量因干旱化程度增加而减少。(3)2种嵩草的茎基直径、叶片萌生能力随着生境干旱化程度增加而下降;高山嵩草叶片萌生能力的变化与生境干旱化梯度一致,具有连续性;矮生嵩草叶片萌生能力对生境干旱化的适应性弱,干旱到一定程度发生骤降。(4)随着生境干旱化,高山嵩草的根系数量和长度均增加,矮生嵩草根系长度增加,但数量却无规律变化;2种嵩草的根系分生功效和根系伸长功效均下降。研究表明:嵩草属植物分布的最适宜生境为表面稍有积水的沼生生境。为适应生境干旱化,高山嵩草降低叶片萌生率以减少蒸腾作用,增加分株数量以增强对空间的占有能力,并增加根系数量和长度来提高对土壤水分的吸收能力,因此对干旱有较强适应性且分布范围广;矮生嵩草只通过降低叶片萌生率,增加根系长度响应干旱化生境,其分布范围较窄。     

Primeval cells: Possible energy-generating and cell-division mechanisms

Summary It is proposed that the first entity capable of adaptive Darwinian evolution consisted of a liposome vesicle formed of (1) abiotically produced phospholipidlike molecules; (2) a very few informational macromolecules; and (3) some abiogenic, lipid-soluble, organic molecule serving as a symporter for phosphate and protons and as a means of high-energy-bond generation. The genetic material had functions that led to the production of phospholipidlike materials (leading to growth and division of the primitive cells) and of the carrier needed for energy transduction. It is suggested that the most primitive exploitable energy source was the donation of 2H⁺+2e^– at the external face of the primitive cell. The electrons were transferred (by metal impurities) to internal sinks of organic material, thus creating, via a deficit, a protonmotive force that could drive both the active transport of phosphate and high-energy-bond formation.This model implies that proton translocation in a closed-membrane system preceded photochemical or electron transport mechanisms and that chemically transferable metabolic energy was needed at a much earlier stage in the development of life than has usually been assumed. It provides a plausible mechanism whereby cell division of the earliest protocells could have been a spontaneous process powered by the internal development of phospholipids. The stimulus for developing this evolutionary sequence was the realization that cellular life was essential if Darwinian survival of the fittest was to direct evolution toward adaptation to the external environment.     

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e₂ = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H₂O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II.

2. The initial phase of H⁺ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H⁺ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.

3. Dibromothymoquinone reduction, observed as O₂ evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O₂ evolution agreeing well with the steady-state rate observed under continuous illumination.

4. Such measurements of the initial rates of O₂ evolution and H⁺ uptake yield an H⁺/e⁻ ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H⁺/e⁻ ratio of 1.7 at pH 7.6.)

5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H⁺ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P_i and hexokinase, when added individually, have no effect on proton uptake in this system.

6. The involvement of a proton uptake reaction with an H⁺/e⁻ ratio of 0.5 in the Photosystem II partial reaction H₂O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e₂) and proton uptake (H⁺/e⁻) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.     

Stimulation of mucosal uptake of selenium from selenite by some thiols at various sites of rat intestine

The influence of several thiols (conc. 1 mmol/L) on mucosal uptake of⁷⁵Se from⁷⁵Se-labeled selenite (conc. 10 μmol/L) across the brush border of rat jejunum and cecum was investigated in vitro using a short-term uptake technique.l-Cysteine (l-Cys) stimulated⁷⁵Se uptake in the mid- and distal jejunum and cecum, but not in the proximal jejunum. The effect was maximal in the distal jejunum.d-Cys was less effective in the jejunum and similarly effective in the cecum.l-Leucine (l-Leu) andl-glutamic acid significantly reduced the stimulatory effect ofl-Cys on Se uptake in the distal jejunum, whereas the respective effect ofd-Cys was not diminished byl-Leu. Cysteamine stimulated mucosal⁷⁵Se uptake at all intestinal sites tested, whereas the effect of mercaptopyruvate was restricted to the distal jejunum. Thioglycolate also enhanced⁷⁵Se uptake in the distal jejunum. The stimulatory effects ofl-Cys, mercaptopyruvate, and thiologlycolate were Na⁺-dependent, whereas the effect of cysteamine also occurred in the absence of Na⁺. Mercaptosuccinate,d-penicillamine, ergothioneine, and thiosulfate did not enhance mucosa⁷⁵Se uptake. It is concluded from these findings that the reaction of some thiols with selenite results in Se compounds that are rapidly absorbed by the intestinal epithelium through various Na⁺-dependent and Na⁺-independent, mechanisms. The high bioavailability of Se from selenite found by others might thus be the result of the presence of thiols in the gastrointestinal tract.     

Mass production,infectivity, and field application studies with the entomogenous fungus Paecilomyces farinosus

Hyphal bodies (HB) of the entomogenous fungus Paecilomyces farinosus mass produced in shaken liquid cultures are infective per os and topically to Spodoptera exigua larvae. Pathogenicity of HB was greatest at pH 5.5. It was reduced by exposure for more than one-half hour to temperatures above 30°C. P. farinosus infected, killed, and produced mycelia on the surface of first-instar S. exigua larvae at a temperature—relative humidity combination as low at 15°C and 70% RH. Using two types of microdroplet applicators (droplet in the size range 1–30 and 30–90 μm, respectively) under field conditions, HB were delivered to distances of 76 m from the site of spraying. The number of HB deposited on the leaf surfaces of treated crops caused mortality of 70 to 90% in bioassayed larvae.     

Mechanisms of metal carcinogenesis

Experimental observations that pertain to mechanisms of metal carcinogenesis are summarized, with emphasis upon (a) interactions of metals with nucleic acids in vitro; (b) impairment by metals of the fidelity of DNA replication by DNA polymerase in vitro; (c) mutagenicity of metals in microorganisms; (d) cytogenetic aberrations induced by metals in tissue culture cells; (e) induction by metals of neoplastic transformation of tissue culture cells; and (f) nuclear uptake of metals in vivo and concomitant inhibitory effects of metals on synthesis of nucleic acids. Considered in toto, the experimental data support the somatic mutation hypothesis of chemical carcinogenesis. Sufficient experimental evidence is available regarding four carcinogenic metals (As, Be, Cr, and Ni) to permit speculations about the molecular reactions whereby these metals may induce somatic mutations. This article is an updated outgrowth of a review presented at the A. O. Beckman Conference on the Biochemistry of Cancer that was held in San Antonio, Texas, on September 6–8, 1978. The earlier draft of this article is being published in the proceedings volume.     

Effects influencing boar semen

The aim of this study was to analyze the main influences on the quality and quantity of boar semen. A total of 230,705 records of semen collections were utilised to estimate statistics of semen traits of 2712 boars belonging to the following breeds: Czech Meat Pig, Duroc, Hampshire, Landrace, Large White, Czech Large White and Pietrain, and various crosses of these breeds. The evaluation was based on semen volume (VO), concentration of spermatozoa (CO), progressive motion of spermatozoa (MO), abnormal spermatozoa (AB), total number of spermatozoa (NO_T) and corrected number of spermatozoa (NO_C). The breeds differed significantly for all examined traits. The maximal differences between the breeds were 95 ml for VO, 109 × 10³ mm⁻³ for CO, 9% for MO, 1.6% for AB, 24 × 10⁹ for NO_T and 19 × 10⁹ for NO_C. The maximal heterosis effect reached 12% for VO, 17% for CO, 4% for MO, −14% for AB, and 8% for NO_T and NO_C. The results demonstrate that the year-season effect has a clear effect on semen quality. The lowest values of semen traits were observed in summer while the highest values were found in autumn and winter. Age of boar was found to have a strong impact on sperm output. Sperm output tended to increase up to a boars’ age of 3.5 years. An acceptable level of semen volume occurred after a sexual pause of 3 days and the pool of spermatozoa was restoring after 5–7 days and fully after 10–11 days.     

Amino acid sequences of ovomucoid third domain from 25 additional species of birds

Ovomucoids were isolated from 25 avian species other than the 101 studied in Laskowskiet al. (1987,Biochemistry 26, 202–221). These were subjected to limited proteolysis with an appropriate enzyme, and connecting peptide extended ovomucoid third domains were isolated and sequenced to the end in a protein sequencer. Of the 25 new sequences, 13 duplicate ones were already known, and 12 are unique. Probably the most striking findings are a Pro¹⁴ Ser¹⁴ replacement in weka, an Ala¹⁴Thr¹⁵ replacement in Bulwer's pheasant, the discovery of two additional amino acid residues Ile¹⁸ and Gly¹⁸ at the P₁ reactive site position in Kalij pheasant and tawny frogmouth, respectively, and the first finding of a negative (Glu³⁴) rather than positive (Lys³⁴ or Arg³⁴) amino acid residue at the NH₂ terminus of the helix in caracara ovomucoid third domain. These results complete the determination of all the sequences of ovomucoid third domains in the four species genusGallus, in the five species genusSyrmaticus, and in the two species generaAix andPavo.     

Biochemical and Stability Properties of Recombinant Human MnSOD

The light absorption spectral properties of recornbinant human MnSOD. which contains an N-terminal additional methionyl residue, were investigated as a function of pH in the range 4.5–10.5. Whereas the extinction coefficient, ?_M at the UV maximum (282 nm) was essentially independent of pH, the ?_M values of the visible spectrum maximum (482 nm) displayed a bell-shaped dependence with a plateau between pH 6.5 and B. Those spectral changes were reversible and the enzymatic activity was not affected by exposure to buffered solutions at 25°C in the pH range 5–10.5. The stability of MnSOD was determined between 25 and 60°C at two different pH: 6.5 and 8.2. The enzyme was found to be considerably more stable at pH 6.5 than at pH 8.2, both toward aggregation and degradation. The gel permeation properties of MnSOD were investigated: the enzyme is a tetramer, with a subunit of 22.2 kD; however. it elutes from a Superose 12 column (Pharmacia) with an apparent molecular weight of ～60kD. Under dissociative conditions (such as guanidine-HCI). molecular weights corresponding to the dimer and monomer could also be demonstrated. It thus appears that the tetramer adopts a non-globular shape. which causes the deviation from the Stokes radius corresponding to its molecular weight.     

Circular dichroism analysis of the secondary structures of bovine blood coagulation factor IX, factor X, and prothrombin

Analysis of the far-ultraviolet circular dichroism spectrum of bovine blood coagulation factor IX reveals the presence of approximately 14% helical structures 26% -sheets, 20% -turns, and 40% coils. These values are essentially the same for the activation products of this zymogen, factor IXa and factor IXa. Similar analysis for bovine factor X permits calculation of these secondary structural as approximately 11% helices, 31% -structures, 22% -turns, and 36% random structures. Bovine prothrombin contains approximately 12% helical structures, 35% -structures, 24% -turns, and 29% coils. None of these values is substantially altered as a result of increase of thepH from 7.4 to 10.5, or upon addition of Ca²⁺ to a concentration of at least 20 mM. Analysis of the near-ultraviolet spectra of factor IX and prothrombin suggests that several aromatic amino acid residues and the disulfide bond present in their -carboxyglutamic acid-containing regions are exposed to solvent and are perturbed by the abovepH adjustment and Ca²⁺ addition. Similar effects are observed in the case of factor X; in addition, the Trp residue at the amino terminus of the heavy chain appears to be influenced by the abovepH alteration. The results reported in this paper show that these vitamin K-dependent blood coagulation proteins are similar in their ordered secondary structures, which are dominated by -sheets and -turns. Their overall secondary structures are not influenced by Ca²⁺ binding and are stable to alkalinepH changes. However, these same environmental alterations appear to be effective probes of aromatic residues in the -carboxyglutamic acid regions.     

The Method of Unweighted Means in Unbalanced Analysis of Variance

For factorial experiments with unbalanced data the method of unweighted means is an alternate method of analysis which is computationally simpler than likelihood ratio based F-tests. The quality of the null approximation to the F-distribution of the statistic for the method of unweighted means and the power of this test relative to the likelihood ratio test are discussed. Recommendations are made of when in the course of data analysis the superior operating characteristics of the likelihood ratio test may not outweigh the computational simplicity of the method of unweighted means test.     

Gelation of high methoxy pectin in the presence of pectin methylesterases and calcium

High methoxy pectin was submitted to various amounts of a fungal pectin methylesterase (PME) from Aspergillus aculeatus and of a plant PME from orange in the presence of calcium. The systems were characterized by rheological means during the gelation process. By the way of in situ demethoxylation with low amount of orange PME, it was possible to gel pectin from the beginning of the reaction although its high degree of methylation around 70. To understand this unusual properties, the behaviour of the two enzymes was investigated in pectic gels and in solution through the analysis of content and distribution of the remaining methyl esters. In the gel, the degree of methylation decreased slowly with orange PME and rapidly with Aspergillus PME. The degree of methylation and degree of blockiness after treatment with each PME in solution or in gels were slightly different. Possible explanations for this are evolving visco-elastic properties, including gel formation or influence of calcium on the enzyme–substrate complex.     

Conservation of genetic resources for five traditional crops from Jazan,SW Saudi Arabia,at the KACST Gene-Bank

The conservation of five traditional crops is an important aspect of achieving national food security. In the present study, we aimed to collect and conserve germplasm of five local crops from the Jazan region of southwestern Saudi Arabia: Sorghum: Sorghum bicolor (L.) Moench); Barley (Hordeum vulgare L.) Millet (Pennisetum glaucum (L.) R. Br.); Sesame (Sesamum indicum L.) and Guar (Cyamopsis tetragonoloba (L.) Taub). Forty-one seed accessions of these five crops were collected and tested to determine seed moisture content (MC%) and quality as indicators of their potential to survive during long-term dry storage at ?18 °C (i.e. ex-situ conservation of genetic resources). Seed viability was assessed using germination tests, the tetrazolium chloride (TZ) test and X-ray imaging. Seeds of the five crops had very low MC% and high viability (fully developed embryos and germination >91%), indicating that they were of good quality and had high potential for long-term survival in gene banks. The genetic resources of these crops (seeds) have now been preserved at the gene-bank of King Abdulaziz City for Science and Technology (KACST-BGB), Riyadh, Saudi Arabia.     

Identification of highly related references about gene-disease association

Background

Curation of gene-disease associations published in literature should be based on careful and frequent survey of the references that are highly related to specific gene-disease associations. Retrieval of the references is thus essential for timely and complete curation.

Results

We present a technique CRFref (Conclusive, Rich, and Focused References) that, given a gene-disease pair < g, d>, ranks high those biomedical references that are likely to provide conclusive, rich, and focused results about g and d. Such references are expected to be highly related to the association between g and d. CRFref ranks candidate references based on their scores. To estimate the score of a reference r, CRFref estimates and integrates three measures: degree of conclusiveness, degree of richness, and degree of focus of r with respect to < g, d>. To evaluate CRFref, experiments are conducted on over one hundred thousand references for over one thousand gene-disease pairs. Experimental results show that CRFref performs significantly better than several typical types of baselines in ranking high those references that expert curators select to develop the summaries for specific gene-disease associations.

Conclusion

CRFref is a good technique to rank high those references that are highly related to specific gene-disease associations. It can be incorporated into existing search engines to prioritize biomedical references for curators and researchers, as well as those text mining systems that aim at the study of gene-disease associations.