Isolation of a specific antigen with alkaline phosphatase activity from soluble extracts of Paracoccidioides brasiliensis

A specific antigen of Paracoccidioides brasiliensis was isolated from a metabolic extract of the fungus. The extraction was made by specific adsorption to and subsequent elution from a column containing a cross linked polymer to which the antibodies of a monospecific rabbit serum had been covalently attached. The purity of the final product was demonstrated by immunodiffusion analysis of the eluate using immune serum produced in a sensitized rabbit. The purified antigen was shown to have cationic electrophoretic mobility and alkaline phosphatase activity.     

Isoenzyme profile of Paracoccidioides brasiliensis

Isoenzyme profiles of 10 strains of Paracoccidioides brasiliensis from different origins (nine strains from patients with different clinical forms of paracoccidioidomycosis and one from the faeces of a penguin) were determined by polyacrylamide gel electrophoresis using 37 different enzymes. Differences in carbonate dehydratase, phosphoglucomutase, phosphoglucoseisomerase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase and beta-esterase were detected among the isolates studied allowing the characterization of nine zymodemes. Two isolates showed identical profiles. There was no correlation between the zymodeme patterns and virulence, clinical forms of the disease nor age of the cultures.     

Cloning and sequence of a 3.835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5-like gene from Candida albicans

We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3'-terminal half of a gene encoding a PEX5-like protein.     

Cloning and nucleotide sequence of amidase gene from Pseudomonas putida

The peptidergic signal substance thyrotropin-releasing hormone (TRH) is inactivated by the TRH-degrading ectoenzyme (TRH-DE), a peptidase that exhibits an extraordinary high degree of substrate specificity and other unusual characteristics. There is no other ectopeptidase known capable of degrading this tripeptideamide, and vice versa, TRH is the only known substrate of this unique enzyme. Thus, studies on this enzyme may reveal new aspects on the function of the TRH signaling system. After succeeding in purifying this enzyme to homogeneity and cloning the cDNA encoding rat TRH-DE, molecular tools became available to study the expression of this enzyme by Northern blot analysis and in situ hybridization histochemistry. The stringent and tissue-specific regulation of the adenohypophyseal TRH-DE by estradiol and thyroid hormones strongly suggests that this enzyme may act as a regulatory element modulating pituitary hormone secretion. In brain, the expression of TRH-DE is not influenced by peripheral hormones but the distinct distribution pattern, and the high activities support the concept that in this tissue TRH-DE may act as a terminator of TRH signals.     

Geographic discrimination of Paracoccidioides brasiliensis strains by randomly amplified polymorphic DNA analysis

The structured clinical interview for diagnosis (axis 1) according to the Diagnostic and Statistical Manual for Mental Disorders (DSM-III-R) was used to assess psychiatric morbidity in 110 infertile patients. They were divided into two groups according to whether referral to the service of psychosomatic medicine was deemed advisable by the physician in charge. Psychiatric disorders were diagnosed in 39 of 56 (69.6%) patients in the referred group and in 13 of 54 (24.1%) in the non-referred group. Psychiatric morbidity was found in 61.1% of females and 21% of males. Adjustment disorders were found in 59.6% (31/52) of all patients, in 59% (24/39) of patients among the referred group and in 61.5% (8/13) of patients among the non-referred group. Fourteen (67%) of 21 women in the referred group with adjustment disorders suffered from anxiety. In addition, 33.3% of patients in the non-referred group showed important psychological dysfunction, although DSM-III-R criteria were not met. Psychiatric morbidity was significantly associated with the number of treatment cycles and female gender in the whole study population, as well as with the type and length of infertility in the non-referred group. Psychological services in an infertility clinic help to identify at an early stage those individuals who are more likely to be vulnerable. This would enable psychological interventions to be targeted towards those in greater need.     

Structural characterization of a new galactofuranose-containing glycolipid antigen of Paracoccidioides brasiliensis

An acidic glycolipid (Band 1), purified from P. brasiliensis by a combination of ion exchange chromatography, HPLC, and HPTLC, was found to be reactive with sera of all patients with paracoccidioidomycosis (PCM). Monosaccharide analysis of Band 1 yielded mannose and galactose in a 2:1 ratio, while mild acid hydrolysis and mild periodate oxidation/NaB3H4 reduction indicated the presence of a terminal galactofuranose. Preliminary analysis of 1H-NMR and MS data suggests that the structure of the glycan is Galf beta 1-->6(Manp alpha 1-->3)Manp beta 1-->2Ins (Ins = myo-inositol). Removal of the galacto-furanose decreased by 60-80% the reactivity of sera from PCM patients with Band 1, suggesting that this residue is immunodominant. With the presumed absence of galactofuranose in mammalian hosts, compounds containing this residue may be useful targets for therapy of several parasitic and fungal diseases.     

Cloning and nucleotide sequence of mouse immunoglobulin epsilon chain cDNA

cDNA corresponding to mouse IgE heavy (epsilon) chain mRNA was cloned from mouse IgE-secreting hybridoma cells. A clone containing the epsilon cDNA insert was identified by hybridization to epsilon mRNA and subsequent translation in vitro to unprocessed epsilon chain reactive with anti-mouse IgE antibodies. This clone was used to select 20 other epsilon cDNA clones by colony hybridization. The clone containing the longest insert was selected and the epsilon cDNA insert was subjected to sequence analysis. The determined sequence is 1,279 nucleotides long and contains the coding regions for part of the constant region (C epsilon) I and all of the C epsilon 2, C epsilon 3, and C epsilon 4 domains and also the entire 3' untranslated region of epsilon mRNA. When the amino acid sequence determined from the nucleotide sequence is compared to that of human epsilon chain, significant homologies between corresponding domains of the two epsilon chains are found, including conservations in cysteine and tryptophan residues and carbohydrate attachment sites.     

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum malic enzyme

The nucleotide sequence of a full-length cDNA encoding NAD(+)-malic enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2269 bases comprises a 5'-leader, a single open reading frame of 1851 bases, and the complete 3'-noncoding region of 340 bases. The first 12 amino acids of the translated sequence are hydrophobic, typical of mitochondrial translocation signals, and do not appear in the purified mature protein. The mature protein contains 605 amino acids and has a molecular mass of 68,478 Da. The amino acid sequences of tryptic peptides from the purified protein and also the N-terminal sequence show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the ascarid protein with the human and rat liver NAD(+)-malic enzymes reveals highly conserved regions interrupted with long stretches of lesser homologous sequences. Structural motifs such as the putative nucleotide binding domains and also the malate binding site are clearly identified by alignment of the three protein sequences.     

Cloning and nucleotide sequence of a complementary DNA encoding Xenopus laevis metallothionein: mRNA accumulation in response to heavy metals

This investigation studied the effect of 50 Hz electric and magnetic fields on the human heart. The electrocardiograms of 27 transmission-line workers and 26 male volunteers were recorded with a Holter recorder both in and outside the fields. The measurements took from half an hour to a few hours. The electric field strength varied from 0.14 to 10.21 kV/m and the magnetic flux density from 1.02 to 15.43 microT. Analysis of the ECG recordings showed that extrasystoles or arrhythmias were as frequent outside the field as in the field. In some cases a small decrease in heart rate was observed after field exposure.     

Cloning and nucleotide sequence of fosfomycin biosynthetic genes of Streptomyces wedmorensis

The biosynthetic pathway for production of the antibiotic fosfomycin by Streptomyces wedmorensis consists of four steps including the formation of a C-P bond and an epoxide. Fosfomycin production genes were cloned from genomic DNA using S. wedmorensis mutants blocked at different steps of the biosynthetic pathway. Four genes corresponding to each of the biosynthetic steps were found to be clustered in a DNA fragment of about 5 kb. Nucleotide sequencing of a large fragment revealed the presence of ten open reading frames, including the four biosynthetic genes and six genes with unknown functions.     

Characterization of rabbit DNA microsatellites extracted from the EMBL nucleotide sequence database

Microsatellite polymorphisms are invaluable for mapping vertebrate genomes. In order to estimate the occurrence of microsatellites in the rabbit genome and to assess their feasibility as markers in rabbit genetics, a survey on the presence of all types of mononucleotide, dinucleotide, trinucleotide and tetranucleotide repeats, with a length of about 20 bp or more, was conducted by searching the published rabbit DNA sequences in the EMBL nucleotide database (version 323). A total of 181 rabbit microsatellites could be extracted from the present database. The estimated frequency of microsatellites in the rabbit genome was one microsatellite for every 2-3 kb of DNA. Dinucleotide repeats constituted the prevailing class of microsatellites, followed by trinucleotide, mononucleotide and tetranucleotide repeats, respectively. The average length of the microsatellites, as found in the database, was 26, 23, 23 and 22 bp for mono-, di-, tri- and tetranucleotide repeats, respectively. The most common repeat motif was AG, followed by A, AC, AGG and CCG. This group comprised about 70% of all extracted rabbit microsatellites. About 61% of the microsatellites were found in non-coding regions of genes, whereas 15% resided in (protein) coding regions. A significant fraction of rabbit microsatellites (about 22%) was found within interspersed repetitive DNA sequences.     

Cloning and nucleotide sequence analysis of a gene encoding an OXA-derived beta-lactamase in Acinetobacter baumannii

A clinical strain of Acinetobacter baumannii (strain Ab41) that was resistant to all beta-lactam antibiotics tested except ceftazidime, ceftriaxone, ceftizoxime, and imipenem produced three beta-lactamases: a presumptive chromosomal cephalosporinase, a TEM-1-like beta-lactamase (pI 5.4), and a novel OXA-derived beta-lactamase named OXA-21 (pI 7.0). The gene encoding OXA-21 was located in an integron. The nucleotide sequence showed three mutations compared with the sequence of OXA-3, with two being silent; the nonsilent mutation generated a substitution of Ile-217 to Met.     

Cloning and nucleotide sequence of cDNA for rhodopsin of the squid Todarodes pacificus

A cDNA for rhodopsin was isolated from a library constructed from poly(A)+RNA of the squid (Todarodes pacificus) retina. One positive clone with the longest insert of cDNA (3.1 kb) was selected by employing a PCR-amplified cDNA fragment as a probe. The nucleotide sequence of the cDNA revealed a single open reading frame of 1,344 bp encoding a polypeptide (M(r)49,833), which covered a complete sequence for the squid opsin. This clone had a very long 3'-non-coding region (1.7 kb) including multiple polyadenylation signals, AATAAA, resembling the clones for Todarodes retinochrome and retinal-binding protein (RALBP). The analysis of hydropathicity demonstrated the presence of seven transmembrane spanning domains, and a possible retinal-binding site, Lys-305, was found in the 7th domain. Todarodes rhodopsin contained characteristic sequences of PPQGY repeated in the C-terminal region, as reported in Loligo and octopus rhodopsins. Structural comparison of those cephalopod rhodopsins is also discussed.     

Generation of chromosome fragment specific bovine DNA sequences by microdissection and DOP-PCR

Trends in causative organisms and sources of infection were studied in a series of 288 episodes of bacteremia in neutropenic cancer patients observed in a single institution from 1986 to 1993. The incidence of bacteremia increased significantly from 20 episodes per 1000 admissions in 1986 to 50 episodes per 1000 admissions in 1993 (p = 0.00001). Over the study period, a continuous increment in gram-positive bacteremia, which reached 81% of episodes in 1993 (p = 0.000001), was observed. Conversely, the incidence of gram-negative bacteremia remained stable. Coagulase-negative staphylococci and viridans group streptococci were the most commonly isolated pathogens. Bacteremia caused by coagulase-negative staphylococci increased from 3 episodes per 1000 admissions to 19 episodes per 1000 admissions (p = 0.0001), and viridans group streptococci bacteremia increased from 0 episodes per 1000 admissions to 19 episodes per 1000 admissions (p = 0.000001). The upward trend in gram-positive bacteremia appeared to be related to a significant increase in both intravascular catheters (p = 0.003) and oral mucositis (p = 0.003) as sources of infection. Specific strategies to prevent chemotherapy-induced mucositis and catheter-related bacteremia merit further investigations.     

Cloning and nucleotide sequence of the gene encoding dinitrogenase reductase (nifH) from the cyanobacterium Nostoc 6720

The nucleotide sequence of the 3' end of the nifU coding sequence, the complete coding sequence of nifH and a substantial part of the 5' end of nifD coding sequence from Nostoc 6720 is presented. The coding sequences are highly conserved with those of Anabaena 7120 and Anabaena sp. L31. However the intergenic region between nifU and nifH contains two segments of short tandemly repetitive repeat sequences (STRRs) that differ from the STRR that is common to both Anabaena7120 and Anabaenasp. L31. Various sequence structures that are common to Nostoc 6720, the Anabaena strains and Plectonema boryanum are discussed.     

Cloning, nucleotide sequence and functions of XPR6, which codes for a dibasic processing endoprotease from the yeast Yarrowia lipolytica

Yarrowia lipolytica DO613, carrying the xpr6-13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys-Arg at the end of the pro-region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys-Arg of an artificial substrate. The XPR6 gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pair SalI-SphI XPR6 fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110,016). The deduced amino acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolated xpr6 alleles were due to leaky mutations. Second, mating type B strains carrying the disrupted XPR6 gene did not mate, but mating type A strains did. Third, the XPR6 disruption strains grew poorly on rich media at pH 5.5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon-like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardless of the pH.     

Cloning and DNA sequence analysis of an aac(3)-Vb gene from Serratia marcescens

The AAC(3)-V resistance mechanism is characterized by high-level resistance to the aminoglycosides gentamicin, netilmicin, 2'-N-ethylnetilmicin, and 6'-N-ethylnetilmicin and moderate resistance levels to tobramycin. Serratia marcescens 82041944 contains an AA(3)-V resistance mechanism as determined from aminoglycoside resistance profiles. This strain, however, does not exhibit hybridization with a probe derived from the previously cloned aac(3)-Va gene, (R. Allmansberger, B. Br?u, and W. Piepersberg, Mol. Gen. Genet. 198:514-520, 1985). High-pressure liquid chromatography analysis of the acetylation products of sisomicin carried out by extracts of S. marcescens 82041944 have demonstrated the presence of an AAC(3) enzyme. We have cloned the gene encoding this acetyltransferase and have designated it aac(3)-Vb. Nucleotide sequence comparisons show that the aac(3)-Va and aac(3)-Vb genes are 72% identical. The predicted AAC(3)-Vb protein is 28,782 Da. Comparisons of the deduced amino acid sequences show 75% identity and 84% similarity between the AAC(3)-Va and AAC(3)-Vb proteins. The use of a DNA fragment internal to the aac(3)-Vb as a hybridization probe demonstrated that the aac(3)-Vb gene is very rare in clinical isolates possessing an AAC(3)-V mechanism.