Wood adhesives prepared from lucerne fiber fermentation residues of Ruminococcus albus and Clostridium thermocellum.

Fermentation residues (consisting of incompletely fermented fiber, adherent bacterial cells, and a glycocalyx material that enhanced bacterial adherence) were obtained by growing the anaerobic cellulolytic bacteria Ruminococcus albus 7 or Clostridium thermocellum ATCC 27405 on a fibrous fraction derived from lucerne (Medicago sativa L.). The dried residue was able to serve as an effective co-adhesive for phenol-formaldehyde (PF) bonding of aspen veneer sheets to one another. Testing of the resulting plywood panels revealed that the adhesive, formulated to contain 30% of its total dry weight as fermentation residue, displayed shear strength and wood failure values under both wet and dry conditions that were comparable with those of industry standards for PF that contained much smaller amounts of fillers or extenders. By contrast, PF adhesives prepared with 30% of dry weight as either unfermented lucerne fiber or conventional fillers or extenders rather than as fermentation residues, displayed poor performance, particularly under wet conditions.

The effect of fungal ribosome inactivating proteins upon feeding choice in C. freemani, and indications of a mutualistic relationship with A. restrictus. Environmental mycology.

Carpophilus freemani beetles' feeding on the fungus Aspergillus nidulans was substantially inhibited when A. nidulans was transformed and induced to secrete the ribosome inactivating protein, restrictocin (genetic source: Aspergillus restrictus). No inhibition of feeding was observed when A. nidulans was transformed and induced to produce an inactive form of restrictocin with a single amino-acid substitution in the active site. Similarly, there was no inhibition of feeding upon transgenic strains when the production of restrictocin was not induced. Feeding inhibition of C. freemani by restrictocin requires that the ribonuclease be active and is not due to other characteristics of the protein or the transgenic host fungus.

Expression of human recombinant beta 2-microglobulin by Aspergillus nidulans and its activity.

The light chain of HLA class I protein (beta 2m) has been expressed in Aspergillus nidulans. The cDNA of beta 2m was modified using the polymerase chain reaction to include overlapping extensions for its subsequent fusion into an Aspergillus vector. This fusion resulted in beta 2m cDNA being flanked by the Aspergillus awamori glucoamylase promoter and the Aspergillus niger glucoamylase terminator. Expression of beta 2m was induced by the addition of starch to the culture medium. In preliminary mass culture trials, 177 micrograms/liter of f beta 2m were obtained in 60-liter fermentations. N-terminal sequencing of purified human beta 2m produced in fungi (f beta 2m) revealed that 28% of the purified protein was of proper sequence and 61% of the protein had an additional serine and lysine residue derived from the C-terminus of the fungal leader. Purified f beta 2m from culture supernatants appeared biochemically similar to beta 2m obtained from human urine (u beta 2m) as seen in immunoblot analysis. Functionally, f beta 2m effectively interacted as a subunit of class I MHC molecules. This was seen both in a sandwich ELISA for detecting properly folded HLA class I heavy chain and in assays showing cell-surface beta 2m exchange into the mouse class I MHC H-2Kd. In these experiments the biological activity of f beta 2m was indistinguishable from u beta 2m. The successful expression of biologically active beta 2m in A. nidulans suggests that fungal systems might be useful for the production of other active components of the HLA class I MHC complex.

Increased xylose reductase activity in the xylose-fermenting yeast Pichia stipitis by overexpression of XYL1.

The Pichia stipitis xylose reductase gene (XYL1) was inserted into an autonomous plasmid that P. stipitis maintains in multicopy. The plasmid pXOR with the XYL1 insert or a control plasmid pJM6 without XYL1 was introduced into P. stipitis. When grown on xylose under aerobic conditions, the strain with pXOR had up to 1.8-fold higher xylose reductase (XOR) activity than the control strain. Oxygen limitation led to higher XOR activity in both experimental and control strains grown on xylose. However, the XOR activities of the two strains grown on xylose were similar under oxygen limitation. When grown on glucose under aerobic or oxygen-limited conditions, the experimental strain had XOR activity up to 10 times higher than that of the control strain. Ethanol production was not improved, but rather it decreased with the introduction of pXOR compared to the control, and this was attributed to nonspecific effects of the plasmid.

Production of caproic acid by cocultures of ruminal cellulolytic bacteria and Clostridium kluyveri grown on cellulose and ethanol.

Ruminal cellulolytic bacteria (Fibrobacter succinogenes S85 or Ruminococcus flavefaciens FD-1) were combined with the non-ruminal bacterium Clostridium kluyveri and grown together on cellulose and ethanol. Succinate and acetate produced by the cellulolytic organisms were converted to butyrate and caproate only when the culture medium was supplemented with ethanol. Ethanol (244 mM) and butyrate (30 mM at pH 6.8) did not inhibit cellulose digestion or product formation by S85 or FD-1; however caproate (30 mM at pH 6.8) was moderately inhibitory to FD-1. Succinate consumption and caproate production were sensitive to culture pH, with more caproic acid being produced when the culture was controlled at a pH near neutrality. In a representative experiment under conditions of controlled pH (at 6.8) 6.0 g cellulose l-1 and 4.4 g ethanol l-1 were converted to 2.6 g butyrate l-1 and 4.6 g caproate l-1. The results suggest that bacteria that efficiently produce low levels of ethanol and acetate or succinate from cellulose should be useful in cocultures for the production of caproic acid, a potentially useful industrial chemical and bio-fuel precursor.

Lactose fed-batch overexpression of recombinant metalloproteins in Escherichia coli BL21 (DE3): process control yielding high levels of metal-incorporated, soluble protein.

A method for producing recombinant proteins in pilot scale fermentation equipment using a glucose fed-batch initial growth, followed by a midlog phase feeding of a glucose and lactose mixture is described. Using the host strain Escherichia coli BL21(DE3), the diiron protein stearoyl-acyl carrier protein delta 9 desaturase has been overexpressed at a biomass level of up to 12 g x liter-1 dry cell weight, representing a 12-fold increase in volumetric productivity relative to that obtained from batch fermentations. Under these conditions, a maximum of 36% of the total cellular protein accumulates as the desaturase polypeptide. A correlation between the slowed growth rate of the fed-batch culture, a continued, albeit slower, exponential growth under inducing conditions, and a favorable partitioning between formation of the soluble holoprotein and inclusion bodies is reported. This correlation suggests that fed-batch techniques can be used to beneficially influence rate-limiting processes in the maturation of overexpressed proteins, such as metal uptake and incorporation proposed here. By using cells produced from the fed-batch method, the iron-containing, soluble desaturase can be purified in a yield of up to 66 mg x g-1 dry cell weight (approximately 500 mg x liter-1 culture), representing a three to fivefold increase in the yield relative to that obtained from batch fermentations. In addition, these methods are suitable for the production of the Anabena 7120 vegetative [2Fe 2S] ferredoxin in E. coli BL21(DE3) pLysS, a host strain used for the overexpression of toxic proteins.

Effects of leader sequences upon the heterologous expression of restrictocin in Aspergillus nidulans and Aspergillus niger.

The effects of altered leader sequences on the secretion and localization of restrictiocin expression in Aspergillus nidulans and Aspergillus niger were investigated. The region encoding the leader sequence of the Aspergillus restrictus restrictocin (res) gene was altered and variants were expressed under the glucoamylase (glaA) promoter in A. nidulans and A. niger. The entire restrictocin leader sequence was replaced by the glaA leader sequence in one variant. In another, the signal sequence of restrictocin was replaced with the glaA signal, leaving a hybrid with the putative restrictocin pro region in place of the glaA pro region. The putative pro region was deleted from the restrictocin leader of a third variant. Toxic effects, such as reduced transcript levels and cellular lysis, were minimal when restrictocin was expressed with the native leader sequence, but became more pronounced as the leader sequence was varied. These toxic effects were inversely proportional to the level of restrictocin secreted. In all transformed strains, restrictocin secretion appeared at the periphery of colonies and was observed to occur at the tips of hyphae. Localization of restrictocin to differentiated structures (conidiophores), as occurs in A. restrictus, was observed only in transformed strains containing the complete restrictocin leader sequence.

Purification and characterization of the oxygen-sensitive 4-hydroxybutanoate dehydrogenase from Clostridium kluyveri.

Cell extracts of Clostridium kluyveri grown on ethanol plus succinate contained a NAD(H) dependent 4-hydroxybutanoate dehydrogenase (EC 1.1.1.61) at 66 U/mg. This enzyme was purified 42-fold under anaerobic conditions to homogeneity. Heat treatment, ion exchange chromatography on DEAE-cellulose, nondenaturing polyacrylamide gel electrophoresis, hydrophobic interaction chromatography on phenyl agarose, and gel filtration on Sephadex G-100 were used in the purification. The molecular mass of the enzyme was estimated to be 41.6 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 86 kDa by gel filtration which indicates the active form of the enzyme is dimeric. The protein contains two atoms of Cu and one atom of Fe per monomeric unit. The enzyme exhibits maximum activity at pH 6.1 for the reduction of succinic semialdehyde and at pH 9.4 for the oxidization of 4-hydroxybutanoate. The Km values for NADH and succinic semialdehyde were 150 +/- 20 microM and 560 +/- 80 microM, respectively. In the reverse direction, the Km values were 670 +/- 80 microM and 55 +/- 16 mM for NAD and 4-hydroxybutanoate, respectively. The enzyme is inactivated by oxygen. The inactivation occurs with a t1/2 = 4.5 min at pH 8.2 and 30 degrees C.

Heterologous expression of the cytotoxin restriction in Aspergillus nidulans and Aspergillus niger.

The cDNA clone of restrictocin was placed under the control of the glucoamylase promoter from Aspergillus awamori and was transformed into Aspergillus nidulans and Aspergillus niger. Site-specific changes were introduced into cDNA constructs and these were transformed into A. nidulans. The secretion signal sequence was deleted from one form of the gene and three mutations introduced single amino acid substitutions into the protein. Culture conditions were optimized for maximum expression levels of restrictocin. The activities of the expressed proteins were characterized with an in vitro rabbit reticulocyte assay. Protein synthesis in this assay was inhibited 50% by 2.5 ng/ml wild-type restrictocin, 3.5 ng/ml E95G, 30 ng/ml E95C, and 600 ng/ml H136L. Toxic effects of restrictocin were observed in the A. nidulans expression system with reduced levels of cellular protein and messenger RNA upon induction of restrictocin expression as well as the formation of the alpha-fragment product of ribosomal RNA cleavage. Toxic effects were most highly pronounced in strains expressing restrictocin without the signal sequence, less so in strains expressing native restrictocin, and absent in strains expressing H136L restrictocin.

Aspergillus ribotoxins react with IgE and IgG antibodies of patients with allergic bronchopulmonary aspergillosis.

Asp f I has been identified as a major allergen of Aspergillus fumigatus and shows amino acid sequence homology with the mitogillin family of cytotoxins. In this study we compared the various cytotoxins from Aspergillus species for their reactivity with immunoglobulin E (IgE) and immunoglobulin G (IgG) antibodies in the serum samples of patients with allergic bronchopulmonary aspergillosis. Elevated levels of IgE and IgG antibodies to all A. fumigatus antigens and ribotoxins were demonstrated in a majority of ABPA patients as compared with allergic patients with asthma and normal subjects. In approximately 75% of patients with allergic aspergillosis, IgE antibody to ribotoxins showed marked elevation, whereas in the remaining patients it was below the baseline, although crude A. fumigatus antigen showed higher specific serum IgE. These results suggest that cytotoxins play a major role in the pathogenesis of allergic bronchopulmonary aspergillosis.

Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.

Glucose and carbon dioxide metabolism by Succinivibrio dextrinosolvens.

Growth rates and culture conditions affect the molar yields of catabolic end products and cells of Succinivibrio dextrinosolvens growing on glucose. When growth in chemostats occurred, a trend toward decreased succinate and acetate formation, increased lactate formation, and a higher yield of cells correlated with an increase in the growth rate. End product and cellular yields on defined medium indicate a high maintenance requirement for S. dextrinosolvens and are consistent with energy conservation steps during the formation of acetate and succinate. Simultaneous carbon dioxide consumption and production were determined from batch studies with NaH14CO3, and the amounts were used to calculate a fermentation balance. These data also indicated that CO2 consumption lags behind CO2 production early in the growth phase, becoming equivalent to it toward stationary phase. Significantly more CO2 was fixed by S. dextrinosolvens when the organism was cultured in chemostats sparged with CO2. Formate is in part derived from free CO2 in the medium, as shown by 13C nuclear magnetic resonance studies, and may be sensitive to CO2 availability. Nuclear magnetic resonance data are consistent with the carboxylation of a C3 intermediate of the Embden-Meyerhof-Parnas pathway of glycolysis to a C4 compound to eventually form succinate.

Studies on the role of the V3 loop in human immunodeficiency virus type 1 envelope glycoprotein function.

Mutations within the principal neutralizing determinant (the V3 loop) of the HIV-1 surface envelope glycoprotein gp120 block or greatly reduce the ability of the HIV-1 envelope glycoprotein to induce cell fusion in CD4+ HeLa T4 cells while keeping its CD4 binding ability. However, when either cysteine or both cysteines forming the V3 disulfide bridge were mutated, the resultant glycoprotein could not mediate cell fusion, undergo proteolytic processing, or bind CD4. To investigate the role that the V3 loop plays in gp160 processing and CD4 binding, we deleted the entire V3 loop region of the HIV-1 env gene. The resultant glycoprotein could not mediate cell fusion in the HeLa T4 cell line and no proteolytic processing of gp160 or CD4 binding could be detected. To test whether any domain of the V3 loop is involved in attaining the proper envelope glycoprotein conformation required for proteolytic processing and CD4 binding, we introduced a series of deletions into the coding region of the V3 loop. Most of the residues within the V3 loop could be removed while retaining gp160 processing and CD4 binding. Our results indicate that the cysteines that form the V3 loop or the disulfide bond itself are important for proper envelope glycoprotein folding and processing. Because many of the mutants constructed in this study do not contain the type-specific neutralizing determinant of HIV-1, they may be potential reagents to bind group-specific neutralizing antibodies or to elicit a group-specific neutralizing response against HIV-1.

Effects of amino-terminal extensions and specific mutations on the activity of restrictocin.

The cytotoxic activities of restrictocin with aminoterminal extensions and specific mutations were investigated using in vivo and in vitro systems. Genes were constructed from the cDNA clone of restrictocin which encode: the native form of restrictocin (including the leader sequence); Met-prorestrictocin, in which a codon for methionine was placed before a putative pro region; Met-mature restrictocin, with a methionine codon prior to the mature form of restrictocin; and three mutated forms of Met-mature restrictocin, E95G, E115G/H136L, and H136L. These constructions were placed under the control of the GAL1 promoter and were transformed into Saccharomyces cerevisiae. Transformants were killed, and a new RNA band formed when any of these genes except those containing the H136L mutation were expressed. Restrictocin protein was detected by immunoblot only in cells expressing the native form of restrictocin and the forms containing the H136L mutation. Native restrictocin, Met-prorestrictocin, and Met-mature restrictocin mRNA were translated in an in vitro system resulting in proteins of the expected molecular weight and inactivation of the translation system. Restrictocin was not inactivated by the presence of the leader sequence and the putative prosequence. Amino acid His136 is putatively in the active site of restrictocin by analogy to ribonuclease U2 and the elimination of toxic effects in the S. cerevisiae expression and in vitro translation systems.

Production and localization of restrictocin in Aspergillus restrictus.

The production and secretion of restrictocin (a cytotoxin that cleaves ribosomal RNA) by cultures of the fungus Aspergillus restrictus was investigated. Previous studies have indicated that restrictocin production in liquid culture coincides with the appearance of differentiated cell structures. A study of the correlation between the appearance of differentiated structures and restrictocin production was conducted with A. restrictus grown on agar medium. Restrictocin was found to be associated with the cell mass of the agar-grown culture (in contrast to liquid cultures), and was first observed when aerial hyphae emerged. Restrictocin levels increased until the time of conidiation, after which they fell off sharply. No restrictocin could be found in the agar medium. The presence of restrictocin upon and within various cell structures was determined by immunofluorescent laser microscopy. This study showed that restrictocin became localized to the conidiophores and phialides during the process of conidiation. Prior to this, restrictocin was found within the hyphae in localized concentrations that may correspond to secretory vesicles.

Regulation of restrictocin production in Aspergillus restrictus.

The production of restrictocin (a cytotoxin that specifically cleaves ribosomal RNA) by cultures of Aspergillus restrictus grown in liquid medium was investigated. The function of restrictocin, the method of its accumulation and the mode of resistance to restrictocin in A. restrictus are unknown. Previous studies have indicated that restrictocin accumulates in the medium with culture age. These observations have been extended in this study by cloning the cDNA of the res gene and using this cDNA clone to probe the onset of messenger RNA synthesis in the cells. The results of the Northern analysis were compared to the production and accumulation of restrictocin and morphological differentiation of the cells in culture. Restrictocin was found in the medium at the same time that mRNA was detected in the cells. This suggests that the leader sequence encoded by the cDNA provides an efficient secretion system for the protein. Both the protein and the mRNA were detected coincident with the formation of differentiated cell structures. These structures develop into conidiophores with one layer of sterigmata and conidia forming from the sterigmata. These results suggest that restrictocin is either involved in the process of conidiation or is coordinately regulated with differentiation leading to conidiation.

Antibodies from human immunodeficiency virus-infected individuals bind to a short amino acid sequence that elicits neutralizing antibodies in animals.

A total of 18 overlapping peptides were synthesized that covered envelope amino acid sequences (amino acids 288-472 of the III-B isolate) of the human immunodeficiency virus type 1 (HIV-1). Antibodies from human immunodeficiency virus-1-infected individuals bound to three of the peptides tested. Guinea pigs were immunized with each of the overlapping peptides and the resultant sera analyzed for biologic activity. One of the peptides elicited antibodies that had both neutralizing and fusion blocking activities that were type specific. This peptide, designated 1-69, was also one of the peptides reactive with the human immunodeficiency virus type 1-positive human sera.

Antibodies reactive with human immunodeficiency virus gag-coded antigens (gag reactive only) are a major cause of enzyme-linked immunosorbent assay reactivity in a blood donor population.

Normal blood donors were examined for human immunodeficiency virus (HIV)-reactive antibodies with both virus- and Escherichia coli-expressed env- and gag-coded antigens. The frequency of samples from normal (low-risk) donors that were repeatedly reactive with an HIV enzyme-linked immunosorbent assay blood screening test (Du Pont Co.) was 0.6%. Two classes of HIV serological reactivity were identified: a minor env-reactive class (0.03 to 0.06% of donors) and the predominant env-nonreactive gag-reactive class (gag reactive only [GRO]) (0.4 to 0.5% of donors). Assignment of env reactivity was made by a synthetic (recombinant) env enzyme-linked immunosorbent assay and virus immunoblot. Most GRO sera reacted with p15/p17 bands on HIV immunoblot. Antibody specificity in GRO sera was confirmed by competition-binding studies with viral gag and E. coli-expressed p55gag. This study provides independent verification that gag-specific antibodies are present in many env-nonreactive sera. More serological and virological studies of individuals with this antibody pattern should be pursued to determine the origin of these gag-reactive antibodies.

Stable Carbon Isotope Fractionation by Methanosarcina barkeri during Methanogenesis from Acetate, Methanol, or Carbon Dioxide-Hydrogen.

Methanosarcina barkeri was cultured on methanol, H(2)-CO(2), and acetate, and the C/C ratios of the substrates and the methane produced from them were determined. The discrimination against C in methane relative to substrate decreased in the order methanol > CO(2) > acetate. The isotopic fractionation for methane derived from acetate was only one-third of that observed with methanol as the substrate. The data presented indicate that the last enzyme of methanogenesis, methylreductase, is not the primary site of isotopic discrimination during methanogenesis from methanol or CO(2). These results also support biogeochemical interpretations that gas produced in environments in which acetate is the primary methane precursor will have higher C/C ratios than those from environments where other substrates predominate.

Analysis of human serum antibodies to human immunodeficiency virus (HIV) using recombinant ENV and GAG antigens.

Recombinant proteins representing gag and env amino acid sequences of the Human Immunodeficiency Virus (HIV) (HTLV-IIIb) were produced in Escherichia coli and used to analyze sera for the presence of antibodies to HIV. ENV-9 is a protein representing the carboxy terminus of gp120 and part of gp41 which is highly immunoreactive. GAG-1 represents 83% and GAG-55 100% of the amino acids of the gag open reading frame. The purified proteins allow sensitive detection by enzyme linked immunosorbent assay (ELISA) of antibodies directed against either env or gag of HIV. We have determined the reactivity of sera from several HIV exposed individuals, either form high risk populations or with clinically defined conditions, in the ENV-9, GAG-55, and GAG-1 assays and found that two major seropositive groups are observed. The quantitative analysis of sera with env and gag antigens by ELISA showed AIDS patients had very low gag reactivity while retaining high env reactivity. Results obtained with authentic p24 viral protein in both ELISA and radioimmunoassay correlated to those from the GAG-55 ELISA. This correlation and the analysis of sera with both the ENV and GAG ELISAs indicate that the antibodies reactive to gag are specifically affected relative to env reactivity and that different levels of antibodies to separate viral components in these sera may correlate with disease state.