Evaluation of photo-crosslinked fibrinogen as a rapid and strong tissue adhesive.

Tissue adhesives and sealants are commonly used in surgery either as an adjunct to, or replacement for, sutures. Previously, we have shown that fibrinogen can be crosslinked rapidly to give a high-strength bond in the presence of a ruthenium(II) complex, a persulfate and irradiation with visible light, and that the crosslinked fibrinogen is nontoxic to cells in vitro. This approach addresses limitations to current fibrin sealants that typically have relatively slow curing times and low bond strengths. In the present study, we have evaluated the efficacy and safety of this new biological scaffold sealant in various animal models. When placed as solid implants into rats, the crosslinked fibrinogen persisted for at least 8 weeks but was fully resorbed by 18 weeks with minimal inflammatory responses. When used as a tissue adhesive for repair of skin incisions in rats or as an arterial haemostat in pig, the photo-crosslinked fibrinogen sealed tissue or arrested bleeding within 20 s of application. For the skin incisions, the fibrinogen sealant promoted rapid tissue vascularization and cellular infiltration with no adverse foreign body cell generation. New collagen deposition occurred and with time the matrix had remodelled to acquire large mature collagen fiber bundles which were accompanied by maximum regenerated tensile strength. This biomaterial system may find useful applications in surgical procedures where rapid curing and/or high strength tissue sealing is required.

Introduction and expression of the bacterial glyoxylate cycle genes in transgenic mice.

The glyoxylate cycle, catalysed by two unique enzymes: isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2), is necessary for the net conversion of acetate into glucose. This metabolic pathway operates in microorganisms, higher plants and nematodes. Two bacterial genes, encoding ICL and MS, were modified in order to introduce them into the mouse germ line. The ovine metallothionein-Ia (MT-Ia) promoter-ace B gene-ovine growth hormone (GH) gene (3' GH sequence) construct was fused to the ovine, MT-Ia promoter-ace A gene-ovine GH gene (3' GH sequence). Therefore, in this single DNA sequence, both ace A and ace B are under independent MT-Ia promoter control and can be induced by zinc. Transgenic mice were generated by pronuclear microinjection of the ace B-ace A gene construct. We now report the establishment of four mouse lines carying these two transgenes. Studies on the progeny of these lines indicate that one line (No. 91) is expressing both genes at the mRNA and enzyme levels in the liver and intestine, whereas another line (No. 66) has a much lower expression. Both enzyme activities were detected in the liver and intestine at levels up to 25% of those measured in fully derepressed Escherichia coli cells.

Characterization of Cryptococcus neoformans by random DNA amplification.

Random amplified polymorphic DNA (RAPD) was optimized and used to distinguish between the varieties and serotypes of Cryptococcus neoformans. The RAPD technique distinguished between serotypes A, D or AD within C. neoformans var. neoformans, and revealed further differentiation within each serotype. Four RAPD profiles were clearly recognizable within C. neoformans var. gattii, although its two serotypes, B and C, were only differentiated with one primer combination out of seven.

Concordance of clinical and environmental isolates of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA analysis and PCR fingerprinting.

Sixty-one clinical and forty-nine environmental isolates of Cryptococcus neoformans var. gattii from Australia and the United States were analyzed by random amplification of polymorphic DNA (RAPD), using 12- to 22-mer primers in pairs, and/or PCR fingerprinting with a single primer derived from the microsatellite core sequence of the wild-type phage M13 (5' GAGGGTGGCGGTTCT 3'). Three major genetic profiles were identified by both typing techniques. A single RAPD profile (VGI) predominated among clinical isolates (44 of 48, 92%) and isolates from host eucalypts (45 of 45, 100%) from Australia. Of the 94 Australian isolates, 4 (3 clinical and 1 environmental) were assigned to profile VGII; 2 of these were recovered from patients and one was recovered from plant debris from Western Australia. Only one Australian clinical isolate was assigned to profile VGIII. A different distribution of RAPD profiles (four VGIII, two VGII, and one VGI) was found among four clinical and three environmental isolates from the United States. RAPD profiles of 8 of the 101 isolates studied revealed minor genetic variants, 4 of profile VGI and 4 of profile VGII. Genetic concordance between the majority of clinical and environmental isolates in Australia is consistent with the hypothesis that human disease is acquired from exposure to host eucalypts. Profiles of clinical isolates were independent of body site of infection, and profiles of all isolates were stable over time. Analysis by PCR fingerprinting confirmed the RAPD results. A second RAPD profile (VGII) was associated with infection in southwest Western Australia, where the two host eucalypts do not occur naturally. This raises the possibility of an alternative and as yet unidentified natural habitat of C. neoformans var. gattii. Our results indicate that RAPD analysis is a sensitive and useful method for investigating environmental sources of human infection with this biotype.

Natural environmental sources of Cryptococcus neoformans var. gattii.

We sought evidence for new environmental sources of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA (RAPD) analysis of isolates from 29 animals with a restricted territorial range in five Australian states. Twenty-three of the 29 isolates and 45 of 45 eucalypt isolates tested previously exhibited one RAPD profile, VGI. RAPD profile VGII was identified in 6 of 17 isolates from domesticated species but in none of 12 native species. Four VGII isolates originated from an area of Western Australia with no natural stands of known eucalypt host, indicating the existence of at least one unrecognized natural source of C. neoformans var. gattii.

Identification by random amplification of polymorphic DNA of a common molecular type of Cryptococcus neoformans var. neoformans in patients with AIDS or other immunosuppressive conditions.

Sixty clinical isolates of Cryptococcus neoformans var. neoformans were analyzed by random amplification of polymorphic DNA (RAPD) using 12- to 22-mer primers in pairs. Five major profiles, which clearly distinguished between serotypes A (profiles I-III), AD (profile IV), and D (profile V), were identified. Forty-two of 58 serotype A isolates were assigned to profile I, 13 to profile II, and 3 to profile III. Profile I compromised 5 subtypes (profiles Ia-Ie), with 37 to 42 isolates in profile Ia. Twenty-seven of 28 isolates from patients with AIDS belonged to profile Ia (P<.001), as did 7 of 10 isolates from otherwise immunocompromised patients. Isolates from immunocompetent hosts were broadly distributed (profile I, 8 isolates; profile II, 10 isolates; profile III, 2 isolates). RAPD profiles were independent of body site and geographic origin of isolates. Isolates pairs from 3 patients produced identical profiles. A predominant genetic profile among serotype A strains from AIDS patients has not been reported previously.

Cloning and sequencing of a cDNA encoding an ovine oestrus-associated oviducal protein.

Ovine oestrus-associated oviducal glycoprotein (oEGP) is synthesized and secreted specifically by the ampullary region of the ovine oviduct during the peri-ovulatory stages of the oestrous cycle. A cDNA that encodes oEGP was isolated and sequenced. Isolation of oEGP was achieved using the polymerase chain reaction (PCR) with primers based on a bovine oestrus-associated oviducal glycoprotein cDNA (bOGP) sequence. A 1599-bp cDNA encodes, in part, a deduced 519-amino acid sequence of mature protein which carries two potential N-linked glycosylation sites. The deduced amino acid sequence is more than 95% identical to that of bOGP and more than 74% identical to the first 491 amino acids of human oestrogen-dependent oviducal glycoprotein (hOGP). Northern blot hybridizations of RNA from several sheep tissues detected mRNA (2.4 kb) only in an ampulla oviduct sample.

The 5S ribosomal RNA gene is linked to large and small subunit ribosomal RNA genes in the oomycetes, Phytophthora vignae, P. cinnamomi, P. megasperma f.sp. glycinea and Saprolegnia ferax.

Southern hybridization and polymerase chain reaction data indicate that the 5S ribosomal RNA gene is linked to the ribosomal RNA gene repeat unit in the oomycetes, Phytophthora vignae, P. cinnamomi, P. megasperma f.sp. glycinea and Saprolegnia ferax, and is apparently transcribed in the same direction as the large and small subunit ribosomal RNA genes. The polymerase chain reaction has been used to amplify all components of the entire ribosomal RNA gene repeat unit for each of these oomycetes. The total size of all amplified products is identical to the size of the ribosomal RNA gene repeat unit, as determined by Southern analysis.

Molecular evolution of the fungi: relationship of the Basidiomycetes, Ascomycetes, and Chytridiomycetes.

Establishing the phylogeny of fungi and protists often has proved difficult owing to the simple morphologies and convergent characters in these organisms. We used DNA sequences of nuclear small-subunit ribosomal RNA genes to determine phylogenetic relationships among three major classes of organisms considered to be fungi--Basidiomycetes, Ascomycetes and Chytridiomycetes--and to assess the taxonomic position of Neocallimastix, an economically important anaerobic rumen microorganism whose classification is controversial. The Basidiomycetes and Ascomycetes, two classes of nonflagellated fungi, are the most closely related taxa. Chytridiomycetes, though bearing flagella, group with these higher fungi rather than with the protists. Neocallimastix, a eukaryote lacking mitochondria and variously classified as a protist or as a fungus, shows closest molecular affinities with the Chytridiomycete fungi in the order Spizellomycetales.

Remarkably AT-rich genomic DNA from the anaerobic fungus Neocallimastix.

The genomic DNA of an anaerobic rumen phycomycete of the genus Neocallimastix has been purified and characterized. The non-repetitive fraction of the DNA has a G.C content of only 13%. The ribosomal RNA genes are highly reiterated, making up about 30% of the total DNA, and are evident as a more G.C-rich satellite with a repeating unit of about 9.4 kilobases (Kb). A.T-rich regions of DNA are highly dispersed and possess some sequence complexity. Chemical analysis of the DNA constituents reveals no evidence of modified bases. The genome of this anaerobic fungus has the highest A.T content of any organism so far described.

Cloning of the regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans.

The areA gene, which mediates nitrogen metabolite repression in the fungus Aspergillus nidulans, lies sufficiently close to a telomere that no indispensable gene can be distal to it. We were able therefore to exploit the existence of a near terminal pericentric inversion to devise a method for cloning areA plus the region beyond it towards the telomere. In crosses heterozygous for this inversion a class of duplication-deficient progeny lacking areA and the region centromere-distal to it is obtained. We, therefore, sought clones from an A. nidulans gene library in lambda Charon 4 able to hybridize to total genomic DNA from a wild-type strain but not to that from a duplication-deficiency strain. A clone, containing an 11.6-kb insert, which hybridised weakly to duplication-deficiency DNA, overlapped chromosome breakpoints of three different aberration-associated areA alleles and was able to transform an areA mutant to areA+. Southern blotting and genetic analysis established that the transforming sequence had integrated in the region centromere distal to areA. The cloning method yielded other clones from the region centromere-distal to areA which were used to show that the translocation associated with a mutant areA allele is reciprocal rather than non-reciprocal, a fact which could not be established by classical genetics. Finally, analysis of the cloned portion of the dispensable region centromere-distal to areA indicates that this region contains at least 0.5% of the A. nidulans genome.

Regulation of gene expression by pH of the growth medium in Aspergillus nidulans.

In the fungus Aspergillus nidulans the levels of a number of enzymes whose location is at least in part extracellular (e.g. acid phosphatase, alkaline phosphatase, phosphodiesterase) and of certain permeases (e.g. that for gamma-amino-n-butyrate) are controlled by the pH of the growth medium. For example, at acidic pH, levels of acid phosphatase are high and those of alkaline phosphatase are low whereas at alkaline pH the reverse is true. Mutations in five genes, palA, B, C, E and F, mimic the effects of growth at acid pH whereas mutations in pacC mimic the effects of growth at alkaline pH. palA, B, C, E and F mutations result in an intracellular pH (pHin) which is more alkaline than that of the wild type whereas pacC mutations result in a pHin more acidic than that of the wild type. This indicates that these mutations exert their primary effects on the regulation of gene expression by pH rather than on the pH homeostatic mechanism but that the expression of at least some component(s) of the pH homeostatic mechanism is subject to the pH regulatory system. It is suggested that pacC might be a wide domain regulatory gene whose product acts positively in some cases (e.g. acid phosphatase) and negatively in others (e.g. alkaline phosphatase). The products of palA, B, C, E and F are proposed to be involved in a metabolic pathway leading to synthesis of an effector molecule able to prevent the (positive and negative) action of the pacC product.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitrate uptake in Aspergillus nidulans and involvement of the third gene of the nitrate assimilation gene cluster.

In Aspergillus nidulans, chlorate strongly inhibited net nitrate uptake, a process separate and distinct from, but dependent upon, the nitrate reductase reaction. Uptake was inhibited by uncouplers, indicating that a proton gradient across the plasma membrane is required. Cyanide, azide, and N-ethylmaleimide were also potent inhibitors of uptake, but these compounds also inhibited nitrate reductase. The net uptake kinetics were problematic, presumably due to the presence of more than one uptake system and the dependence on nitrate reduction, but an apparent Km of 200 microM was estimated. In uptake assays, the crnA1 mutation reduced nitrate uptake severalfold in conidiospores and young mycelia but had no effect in older mycelia. Several growth tests also indicate that crnA1 reduces nitrate uptake. crnA expression was subject to control by the positive-acting regulatory gene areA, mediating nitrogen metabolite repression, but was not under the control of the positive-acting regulatory gene nirA, mediating nitrate induction.

Nitrogen metabolite repression in Aspergillus nidulans: A farewell to tamA?

Previous work has established that nitrogen metabolite repression in Aspergillus nidulans is mediated by the positive acting regulatory gene areA. Pateman and Kinghorn (1977) proposed that the gene tamA plays an equally important regulatory role in nitrogen metabolite repression as the result of work with "tamA(r)-50," an "allele" leading to inability to utilise nitrogen sources other than ammonium, and "tamA(d)-1," an "allele" leading to nitrogen metabolite derepression. Both "tamA(r)-50" and "tamA(d)-1" were subsequently lost. We have therefore attempted to reconstruct Pateman and Kinghorn's work with tamA. We propose that "tamA(r)-50" was in fact a pyroB(-) tamA(-) double mutation. pyroB(-) mutations lead to a block in vitamin B6 biosynthesis which can be supplemented by extremely high concentrations of ammonium. tamA(-) mutations, possibly as the result of a membrane alteration, reduce the concentration of ammonium required to supplement the pyroB(-) auxotrophy. There is, however, no evidence that pyroB(-) or tamA- mutations, alone or in combination, affect the regulation of the levels of a number of enzymes subject to nitrogen metabolite repression. Reversion of pyroB(-) strains constitutes a powerful positive selection technique for obtaining a wide variety of mutations in glnA, the probable structural gene for glutamine synthetase. We suggest that the nitrogen metabolite derepressed phenotype attributed to "tamA(d)-1" might have resulted from an extremely leaky glnA(-) mutation.

Purification and characterization of two high-affinity (adenosine 3',5'-monophosphate)-binding proteins from yeast. Identification as multiple forms of glyceraldehyde-3-phosphate dehydrogenase.

Two high-affinity cAMP-binding proteins (I and II) have been purified to homogeneity from baker's yeast by a procedure avoiding proteolytic damage. These proteins have been identified as multiple forms of glyceraldehyde-3-phosphate dehydrogenase. The two cAMP-binding proteins are similar in affinities for cAMP, have identical elution volumes on gel filtration, and contain one type of subunit (Mr 37 500). The form II of glyceraldehyde-3-phosphate dehydrogenase is free of NAD+ and has a Kd of 1.3 X 10(-6) M with respect to cAMP. A marked concentration-dependent self-association of the subunits of the form-II protein was revealed by Yphantis sedimentation equilibrium studies. Significant monomer concentrations are present at total concentrations less than 0.02 mg/ml. Conventional sedimentation equilibrium analyses indicated a tetramer Mr of 170 000. The high-affinity binding of cAMP to glyceraldehyde-3-phosphate dehydrogenase may significantly reduce intracellular cAMP levels and is also discussed in relation to the nature of eukaryote cAMP-binding proteins with similar native or subunit Mr values which are at present functionally undefined.

The ligand specificity of the (adenosine 3',5'-monophosphate)-binding site of yeast glyceraldehyde-3-phosphate dehydrogenase. Interaction with adenosine derivatives and pharmacologically-active compounds.

The high-affinity cAMP-binding site of form-II yeast glyceraldehyde-3-phosphate dehydrogenase has a marked specificity for adenosine derivatives, such ligands including N6-substituted adenosine derivatives active as cytokinins n plant systems and adenine nucleotides. Of a wide range of nucleotides and nucleosides examined only adenosine derivatives bind to the cAMP binding site. A variety of antimitotic compounds (including colchicine, colcemid and phenylcarbamate derivatives), adrenergic receptor antagonists (alprenolol and propranolol) and non-steroidal anti-inflammatory agents (notably indomethacin and flufenamic acid) displace cAMP from glyceraldehyde-3-phosphate dehydrogenase. Colchicine, colcemid, N6-furfuryladenosine, indomethacin, flufenamic acid and propranolol inhibit cAMP binding to the enzyme in an apparently competitive fashion. Given the evolutionary conservatism and abundance of glyceraldehyde-3-phosphate dehydrogenase, the affinity of the cAMP-binding site of this enzyme for a variety of structurally-disparate pharmacologically-active compounds compromises simple one-site interpretations of physiological responses to these agents.

Enzymology and genetic regulation of a cyclic nucleotide-binding phosphodiesterase-phosphomonoesterase from Aspergillus nidulans.

A cyclic nucleotide-binding phosphohydrolase that possesses both a phosphomonoesterase and a phosphodiesterase catalytic function has been partially purified from Aspergillus nidulans. The enzyme hydrolyzes both p-nitrophenylphosphate and bis-(p-nitrophenyl)-phosphate. o'-Nucleoside monophosphates are the best physiological phosphomonesterase substrates but 5'- and 2'-nucleoside monophosphates are also hydrolyzed. The enzyme catalyzes the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and 2',3'- and 3'5'-cyclic nucleotides, but not of ribonucleic acid, deoxyribonucleic acid, or nicotinamide adenine dinucleotide. The enzyme has acid pH optima and is not activated by divalent cations. Nucleosides and nucleotides inhibit the enzyme. Cyclic nucleotides are competitive inhibitors of the phosphodiesterase-phosphomonoesterase. The enzyme can occur extracellularly. The phosphodiesterase-phosphomonoesterase is present at high levels in nitrogen-starved mycelium, and it is strongly repressed during growth in media containing ammonium or glutamine and weakly repressed during growth in glutamate-containing medium. Experiments with various area mutants show that this regulatory gene is involved in the control of the enzyme. No evidence for regulation of the enzyme by carbon or phosphorus starvation has been found.