A putative two-component regulatory system involved in secondary metabolism in Streptomyces spp. A DNA fragment stimulating actinorhodin, undecylprodigiosin, and A-factor production in Streptomyces lividans 66 was cloned from Streptomyces coelicolor A3(2). Nucleotide sequencing revealed the presence of an open reading frame of 225 codons, named afsQ1, that showed great similarity in amino acid sequence to the response regulators of typical prokaryotic two-component regulatory systems responsible for adaptive responses. The termination codon, TGA, of afsQ1 overlapped the initiation codon, GTG, of a second open reading frame, afsQ2, of 535 codons. The afsQ2 gene product showed homology with the sensory histidine protein kinases of two-component systems. In agreement with the assumption that the AfsQ1 and AfsQ2 proteins comprise an aspartate-histidine phosphotransfer system, an amino acid replacement from Asp to Glu at residue 52 of AfsQ1, generated by site-directed mutagenesis, resulted in loss of the protein's ability to stimulate antibiotic production in S. lividans. Primer extension experiments indicated that transcription of the afsQ1 and afsQ2 genes initiates at the translational start codon (GTG) of the afsQ1 gene. The afsQ1 and afsQ2 genes were physically mapped at a chromosomal position near the actinorhodin biosynthetic gene cluster (act) by hybridization to Southern blots of restriction fragments separated by pulsed-field gel electrophoresis. Disruption of either afsQ1 or afsQ2 on the S. coelicolor chromosome by use of phage phi C31KC515 led to no detectable change in secondary metabolite formation or morphogenesis. The afsQ1 gene on pIJ922 suppressed the S. coelicolor absA mutation and caused actinorhodin production but did not suppress the absB mutation. Southern blot hybridization showed that sequences homologous to afsQ1 and afsQ2 are present in almost all of the actinomycetes examined.


Analysis of operons encoding 23S rRNA of Clostridium botulinum type A. Southern hybridization analysis of Clostridium botulinum type A chromosomal DNA indicated the presence of six copies of the 23S rRNA gene. Fragments of DNA encoding 23S rRNA were amplified by polymerase chain reaction and cloned in Escherichia coli. Three clones examined by restriction enzyme and sequence analysis were found to be derived from different operons. Sequence determination of the entire insert of two clones revealed nine nucleotide changes in the genes coding for 23S rRNA (99.7% sequence identity) between operons encoded on the same chromosome, showing microheterogeneity in the rRNA operons of this organism.


Cloning of a promoter-like soybean DNA sequence responding to IAA induction in Escherichia coli K12. We have constructed a soybean genomic DNA library in Escherichia coli K12 strain KC13 using plasmid pPV33, which consists of a promoter-less tetracycline resistance (Tcr) gene. A recombinant clone, KC13(pAU-SB1)+, was obtained by selecting for resistance to tetracycline in the presence of indole-3-acetic acid (IAA). Restriction enzyme cleavage and Southern hybridization analysis revealed that the pAU-SB1 plasmid has a 250 bp soybean DNA insert fused with the Tcr gene. In the presence of a selected group of auxins, induction of the Tcr phenotype and mRNA synthesis of the Tcr gene are observed only in KC13(pAU-SB1)+ cultures. On the other hand, induction of the Tcr phenotype and mRNA synthesis of the Tcr gene are absent in cells harboring the cloning vector pPV33 or a recombinant plasmid containing the 250 bp insert in the reverse orientation, pAU-SB1ro. This demonstrated a need for the insertion of the 250 bp soybean DNA and the specificity of its orientation in response to IAA induction. The start point of mRNA transcription in response to IAA, IBA, IPA, 2,4,5-T, and a-NAP is at base pair -96 or -95 upstream of the translational start site of the Tcr gene and base pair -98 with 2,4-D.


Molecular cloning of the bovine CD9 antigen from ocular ciliary epithelial cells. The ciliary epithelium is a bilayer of epithelial cells responsible for the formation and secretion of aqueous humor in the mammalian eye. We have isolated a cDNA clone from a lambda gt11 cDNA library of bovine ocular ciliary epithelial cells encoding the CD9 antigen, a member of a new family of transmembrane proteins. The bovine CD9 clone contains an open reading frame of 226 amino acids (M(r) 24,860). The deduced amino acid sequence from the bovine CD9 cDNA clone shows 83.5% identity with the human counterpart isolated from megakaryocytes, and a lower degree of identity with a group of related antigens (TAPA-1, C0-029, CD53, MRC OX-44, ME491, CD63, CD37, and Sm23) involved in growth regulation. Analysis of bovine ocular tissues reveals that the CD9 gene encodes a 1.4 kb mRNA which is detectable predominantly in cornea and at low levels in ciliary epithelium, retina, iris, and lens. Normal and transformed cell lines established from the ocular ciliary epithelium exhibited significant levels of CD9 transcripts. These results raise questions regarding possible roles of CD9 in the anterior segment of the eye.


Gene structure and multiple mRNA species of Drosophila melanogaster aldolase generating three isozymes with different enzymatic properties. Genomic clones encoding the Drosophila aldolase gene were isolated and the organization of the gene was determined. The protein-coding region spanning nearly 3.5 kb consists of five coding exons (exon 2, 3, 4 alpha, 4 beta, and 4 gamma). The insect exon 2 corresponds to exons 2 to 7 of vertebrate aldolase genes and thus appears to have been formed by the fusion of these 6 exons into a single exon during evolution. The Drosophila aldolase gene is predicted to generate mRNAs for three isozymes (alpha-, beta-, and gamma-types) from the primary transcripts by alternative usage of the final three exons. The reverse transcriptase-PCR assay revealed the occurrence of mRNAs for the three isozymic forms at different developmental stages, and tissue-specific expression was also found to occur in adult flies. In addition to the usual type mRNA species for the alpha-, beta-, and gamma-isozymes, two novel forms of mRNAs, alpha beta- and beta gamma-type mRNAs, were detected tissue-specifically in adult flies, although their functions are unpredictable. The alpha beta-mRNA is an alpha-type mRNA in which exon 4 beta remains unspliced, while the beta gamma-mRNA is a beta-type mRNA with the exon 4 gamma remaining unspliced. Recombinant enzymes expressed in Escherichia coli were all active and exhibited different enzymatic properties.


Differential expression of cell surface heparan sulfate proteoglycans in human mammary epithelial cells and lung fibroblasts. Treating the liposome-intercalatable heparan sulfate proteoglycans from human lung fibroblasts and mammary epithelial cells with heparitinase and chondroitinase ABC revealed different core protein patterns in the two cell types. Lung fibroblasts expressed heparan sulfate proteoglycans with core proteins of approximately 35, 48/90 (fibroglycan), 64 (glypican), and 125 kDa and traces of a hybrid proteoglycan which carried both heparan sulfate and chondroitin sulfate chains. The mammary epithelial cells, in contrast, expressed large amounts of a hybrid proteoglycan and heparan sulfate proteoglycans with core proteins of approximately 35 and 64 kDa, but the fibroglycan and 125-kDa cores were not detectable in these cells. Phosphatidylinositol-specific phospholipase C and monoclonal antibody (mAb) S1 identified the 64-kDa core proteins as glypican, whereas mAb 2E9, which also reacted with proteoglycan from mouse mammary epithelial cells, tentatively identified the hybrid proteoglycans as syndecan. The expression of syndecan in lung fibroblasts was confirmed by amplifying syndecan cDNA sequences from fibroblastic mRNA extracts and demonstrating the cross-reactivity of the encoded recombinant core protein with mAb 2E9. Northern blots failed to detect a message for fibroglycan in the mammary epithelial cells and in several other epithelial cell lines tested, while confirming the expression of both glypican and syndecan in these cells. Confluent fibroblasts expressed higher levels of syndecan mRNA than exponentially growing fibroblasts, but these levels remained lower than observed in epithelial cells. These data formally identify one of the cell surface proteoglycans of human lung fibroblasts as syndecan and indicate that the expression of the cell surface proteoglycans varies in different cell types and under different culture conditions.


Major histocompatibility complex (MHC)-encoded HAM2 is necessary for antigenic peptide loading onto class I MHC molecules. The mutant murine lymphoma cell line RMA-S is unable to present endogenous antigens due to its inability to efficiently assemble class I major histocompatibility complex molecules and antigenic peptides. Therefore, it has been suggested that RMA-S cells are defective either in peptide generation or in peptide transport into the endoplasmic reticulum, where class I major histocompatibility complex molecule assembly is believed to occur. As proteasomes and the putative peptide transporters HAM1 and HAM2 have been implicated in class I antigen processing, we have investigated their expression in RMA-S and its wild-type counterpart RMA. Both proteasomes and HAM1 proteins are expressed at similar levels and show identical subcellular distributions in the two cell lines. However, only one copy of the HAM2 gene is present in RMA-S cells, and it contains a point mutation that leads to a premature stop codon. Thus, the HAM2 protein is absent from RMA-S cells. These data demonstrate that HAM2 is essential for peptide loading onto class I molecules.


Characterization of the mmsAB operon of Pseudomonas aeruginosa PAO encoding methylmalonate-semialdehyde dehydrogenase and 3-hydroxyisobutyrate dehydrogenase. A 5417-base pair (bp) region of Pseudomonas aeruginosa PAO chromosomal DNA containing the mmsAB operon and an upstream regulatory gene (mmsR) has been cloned and characterized. The operon contains two structural genes involved in valine metabolism: mmsA, which encodes methylmalonate-semialdehyde dehydrogenase; and mmsB, which encodes 3-hydroxyisobutyrate dehydrogenase. mmsA and mmsB share the same orientation and are separated by a 16-bp noncoding region. The transcriptional start site for the operon has been pinpointed to a cytidine residue located 77 bp from the translational start site of the operon. mmsR is located on the opposite strand and begins 134 bp from the translational start site of mmsA. MmsR has been identified as a member of the XylS/AraC family of transcriptional regulators and appears to act as a positive regulator of the mmsAB operon. Sequence comparison of MmsA to other proteins in the data bases revealed that MmsA belongs to the aldehyde dehydrogenase (NAD+) superfamily. MmsB shares a 44% amino acid identity with 3-hydroxyisobutyrate dehydrogenase from rat liver. Mutants with insertionally inactivated mmsR, mmsA, and mmsB grow slowly on valine/isoleucine medium and exhibit reduced enzyme activity in cell-free extracts compared to P. aeruginosa PAO.


Regulation of gene expression of rat skeletal muscle/liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Isolation and characterization of a glucocorticoid response element in the first intron of the gene. At least two genes encode isoenzymes of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Alternative splicing of one of these genes generates a skeletal muscle-specific transcript from an upstream promoter and a liver-specific transcript from a downstream promoter. A potent glucocorticoid response element was identified in the first intron of the gene, i.e. between liver exon I and exon II. The element is approximately 3.5 kilobase pairs (kb) downstream of the liver isoenzyme transcription start site and 13 kb upstream of exon II of the gene and confers dexamethasone-sensitive expression of chloramphenicol acetyltransferase (CAT) activity from a heterologous thymidine kinase promoter and from both homologous 5'-flanking regions of the gene. This glucocorticoid response element also exhibits androgen- but not estrogen-sensitive expression of CAT activity in HeLa cells cotransfected with the appropriate receptor expression vector. DNase footprint and sequence analysis revealed that the element is comprised minimally of two adjacent 15-mer glucocorticoid receptor dimer binding sites situated in opposite orientations. Glucocortcoid regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in liver and skeletal muscle is mediated by a single complex glucocorticoid response element located in the first intron of the skeletal muscle/liver gene.


4-Oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer. The xylH gene encoding 4-oxalocrotonate tautomerase (4-OT) has been located on a subclone of the Pseudomonas putida mt-2 TOL plasmid pWW0 and inserted into an Escherichia coli expression vector. Several of the genes of the metafission pathway encoded by pWW0 have been cloned in E. coli, but the overexpression of their gene products has met with limited success. By utilizing the E. coli alkaline phosphatase promoter (phoA) coupled with the proper positioning of a ribosome-binding region, we are able to express functional 4-OT in yields of at least 10 mg of pure enzyme/liter of culture. 4-OT has been previously characterized and shown to be an extremely efficient catalyst (Whitman, C. P., Aird, B. A., Gillespie, W. R., and Stolowich, N. J. (1991) J. Am. Chem. Soc. 113, 3154-3162). Kinetic and physical characterization of the E. coli-expressed protein show that it is identical with that of the 4-OT isolated from P. putida. The functional unit is apparently a pentamer of identical subunits, each consisting of only 62 amino acid residues. This is the smallest enzyme subunit reported to date. The amino acid sequence, determined in part from automated Edman degradation and also deduced from the primary sequence of xylH, did not show homology with any of the sequences in the current data bases nor with any of the sequences of enzymes that catalyze similar reactions. We propose that the active site of 4-OT may be established by an overlap of subunits and comprised of amino acid residues belonging to several, if not all, of the subunits.


Primary structure and characteristics of the melibiose carrier of Klebsiella pneumoniae. The melB gene coding for the melibiose carrier of Klebsiella pneumoniae was cloned and sequenced. There were two potential translation initiation sites. It was predicted that the melibiose carrier consists of 471 (or 467) amino acid residues. Seventy-eight percent of the 471 amino acids were identical to the Escherichia coli melibiose carrier. Sugar transport characteristics were studied using an E. coli mel- mutant expressing cloned K. pneumoniae melB gene. Accumulation of melibiose via the K. pneumoniae melibiose carrier was not stimulated by adding NaCl or LiCl which stimulates melibiose accumulation via the E. coli melibiose carrier. Lactose was accumulated only in the presence of LiCl. TMG (methyl-1-thio-beta-D-galactopyranoside) was accumulated in the absence of added NaCl or LiCl. The accumulation was stimulated by LiCl but not by NaCl. Rapid H+ uptake was observed when melibiose or TMG was added to cell suspensions. These results suggest that the preferred cation couplings via K. pneumoniae melibiose carrier are H(+)-melibiose, Li(+)-lactose, and H+/Li(+)-TMG. This coupling spectrum is quite different from that of the E. coli melibiose carrier. It is of special interest that the K. pneumoniae melibiose carrier seems to be lacking the ability to recognize Na+ which is a preferred coupling cation of the E. coli melibiose carrier for all known sugar substrates. Further investigation of these two carriers may give us insight into the Na+ recognition site.


Localized mutagenesis and evidence for post-transcriptional regulation of MAK3. A putative N-acetyltransferase required for double-stranded RNA virus propagation in Saccharomyces cerevisiae. The MAK3 gene of Saccharomyces cerevisiae is necessary for the propagation of the L-A double-stranded RNA virus and its satellites, such as M1 that encodes a killer toxin. We cloned the MAK3 gene based on its genetic map position using physically mapped lambda-clones covering nearly all of the yeast genome. The minimal sequence necessary to complement the mak3-1 mutation contained 3 open reading frames (ORFs). Only one (ORF3) was necessary to complement mak3-1. A deletion insertion mutant of ORF3 grew slowly on nonfermentable carbon sources, an effect not due simply to its loss of L-A. Although ORF3 alone is sufficient for MAK3 activity when expressed from an expression vector, in its native context an additional 669 base pairs 3' to the ORF and complementary to the gene for a non-histone protein are necessary for expression, but not for normal steady state transcript levels. This suggests a post-transcriptional control of MAK3 expression by the 3' region. The MAK3 protein has substantial homology with several N-acetyltransferases with consensus patterns h..h.h. . . Y..[HK]GI[AG][KR].Lh. . .h and h.h[DE]. . . .N..A. . .Y . . .GF. . . .. . . .Y . . [DE]G, (h = hydrophobic). Mutation of any of the underlined conserved residues (94GI----AA, 123N----A, 130Y----A, 134GF----SL, 144Y----A, and 149G----A) inactivated the gene, supporting the hypothesis that MAK3 encodes an N-acetyltransferase.


The identification of a cis-acting element involved in cyclic 3',5'-adenosine monophosphate regulation of bovine vasopressin gene expression. Cyclic adenosine 3',5'-monophosphate (cAMP) has been implicated as an intracellular messenger mediating osmotic regulation of expression of the gene encoding the neuropeptide vasopressin (VP) in the hypothalamus. We have used a heterologous transient transfection system to demonstrate that cAMP regulates the bovine VP gene promoter following transfection into CV1 cells. Mutational analysis identified a bovine VP cAMP-responsive element (BVP-CRE) 120-112 base-pairs upstream of the start of transcription. DNase I footprint analysis using nuclear protein extract from CV1 cells showed protection at the site of the BVP-CRE. Protection of the BVP-CRE was also observed using purified AP1 protein, while there was a weak interaction with the BVP-CRE using purified rat CREB protein. Nuclear proteins purified from the rat supraoptic nucleus bind to the BVP-CRE. As transgenic mouse studies have shown that the bovine VP gene is subject to appropriate physiological regulation in the mouse hypothalamus (Ang, H. L., Funkhouser, J., Carter, D. A., Ho, M. Y., and Murphy, D. (1991) Soc. Neurosci. Abstr. 513, 12), these data indicate a role for the BVP-CRE element in mediating VP gene expression in vivo. These data demonstrate that cAMP regulates bovine VP gene expression in vitro via a cis-acting element within the VP promoter, and this activation may be mediated by members of the AP1/ATF/CREB family of transcription factors.


Amplification and molecular cloning of the ornithine decarboxylase gene of Leishmania donovani. A strain of Leishmania donovani has been described that is resistant to DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (OD-Case) activity, and contains 15-fold greater amounts of ODCase activity and protein than the wild type strain from which it was derived (Coons, T., Hanson, S., Bitonti, A.J., McCann, P.P., and Ullman, B. (1990) Mol. Biochem. Pharmacol. 39, 77-90). From this mutant strain, another ODCase overproducing L. donovani strain, DFMO16, was generated by virtue of its ability to proliferate under even higher concentrations of DFMO. To investigate the mechanism by which DFMO-resistant cells overexpress ODCase, the leishmanial ODCase gene was isolated by hybridization to a fragment of the L. donovani ODCase gene that was generated by the polymerase chain reaction. The nucleotide sequence of a 4.5-kilobase DNA fragment encompassed an open reading frame encoding 707 amino acids (Mr = 77,350). The leishmanial protein contained an extra approximately 200 amino acid NH2-terminal extension and lacked the COOH terminus of the mammalian ODCase. Northern blot analysis revealed two leishmanial OD-Case transcripts of 4.8 and 6.5 kilobases, both of which were amplified 10-20-fold in the DFMO16 cells. Genomic Southern blot analysis established that the augmented amount of ODCase activity and ODCase mRNA in the DFMO16 strain could be attributed to a approximately 10-20-fold amplification of the ODCase gene copy number. DFMO16 cells exhibited an unstable phenotype in that the amplification of the ODCase gene, the increased amount of ODCase transcript, the overproduction of ODCase activity, and the DFMO-resistance growth phenotype all reverted synchronously in the absence of selective pressure.


The human type VI collagen gene. mRNA and protein variants of the alpha 3 chain generated by alternative splicing of an additional 5-end exon. The amino- and carboxyl-terminal globular domains of type VI collagen are composed of several homologous modules similar to the type A collagen-binding modules present in von Willebrand factor. The human alpha 3(VI) chain that contributes most of the amino-terminal globule appears heterogeneous in size as a result of alternative splicing of two exons (Stokes D. G., Saitta, B., Timpl, R., and Chu, M.-L. (1991) J. Biol. Chem. 266, 8626-8633). In the present study, we report a further characterization of the 5'-end of the gene of the human alpha 3(VI) chain and show that transcription initiates at multiple sites. Southern blotting and DNA sequencing indicate that there is an additional type A exon (A9/N10) at about 1.8 kilobase pairs downstream of the exon coding for the signal peptide. The open reading frame of this additional exon reveals 1 cysteine and three potential N-glycosylation sites. Polymerase chain reaction, Northern blotting, and RNase protection assays demonstrate that exon A9/N10 is subject to alternative splicing in normal and tumor cell lines and that this generates more protein variants of the alpha 3(VI) chain than expected before. A comparison with the corresponding amino-terminal globule of the chicken alpha 3(VI) chain shows the presence of 1 additional cysteine in this portion of the molecule and suggests that human type VI collagen has more possibilities for structural and functional variations compared to chicken type VI collagen.


A member of the aldoketo reductase family confers methotrexate resistance in Leishmania. Methotrexate (MTX)-resistant mutants of the parasitic protozoan Leishmania have been used as models for the mechanism and genetic basis of drug resistance in trypanosomatids and other cells. Three resistance mechanisms to MTX, a dihydrofolate reductase inhibitor, have been described in Leishmania: decreased uptake and accumulation of MTX via the folate/MTX transporter, amplification and overexpression of the dihydrofolate reductase-thymidylate synthase gene, and extrachromosomal amplification of H region DNA. We have now identified hmtxr as the H region gene conferring MTX resistance using a transfection-based approach. Data base searches show that the predicted HMTXr protein is related to members of the polyol dehydrogenase/carbonyl reductase family of aldoketo reductases, whose substrates include polyols, quinones, steroids, prostaglandins, fatty acids, and pterins. We therefore propose that HMTXr is also an oxidoreductase and suggest several biochemical mechanisms of resistance in Leishmania that could be exploited in the design of parasite-specific inhibitors.


Primary structure deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase. Evidence that the enzyme is a homodimer of identical subunits homologous to rat liver-specific alloantigen F. 4-Hydroxyphenylpyruvic acid dioxygenase is an important enzyme in tyrosine catabolism in most organisms. From porcine and human liver cDNA libraries we isolated complementary DNA inserts for the enzyme. Protein sequence analysis of the porcine enzyme revealed a block of the amino terminus of the mature enzyme. Comparison of the amino acid sequence determined by Edman degradation of peptides derived from porcine liver 4-hydroxyphenylpyruvic acid dioxygenase with the nucleotide sequences revealed the primary structure of the porcine and human enzymes. The mature human and porcine enzymes have an 89% amino acid sequence identity in amino acid residues and are composed of 392 amino acid residues. A computer-assisted homology search revealed that the enzyme is 88% identical in amino acid sequence to rat liver-specific alloantigen F. A monoclonal antibody (mob 51), which can immunoprecipitate both the human and porcine enzymes, was developed. Cultured BMT-10 cells transfected with the cDNA insert of the human enzyme, using the expression vector pCAGGSneodE, produced a polypeptide with an M(r) of 43,000, which was immunoprecipitated with mob 51. Enzymic activity of the enzyme was detected in the transfected cells but not in the mock transfected cells. These findings suggest that the human 4-hydroxyphenylpyruvic acid dioxygenase is a homodimer of two identical subunits with an M(r) of 43,000. Liver-specific alloantigen F seems to be closely related to the enzyme or possibly to the subunit of the enzyme itself. Elucidation of the complete amino acid sequence of the enzyme is expected to reveal structure-function relationships of this metabolically important enzyme and to shed light on inherited disorders related to tyrosine metabolism, especially tyrosinemia types 1 and 3.


Molecular cloning of a fourth member of a human alpha (1,3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x, and difucosyl sialyl Lewis x epitopes. We and others have previously described the isolation of three human alpha (1,3)fucosyltransferase genes which form the basis of a nascent glycosyltransferase gene family. We now report the molecular cloning and expression of a fourth homologous human alpha (1,3)fucosyltransferase gene. When transfected into mammalian cells, this fucosyltransferase gene is capable of directing expression of the Lewis x (Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc), sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4 [Fuc alpha 1-->3]GlcNAc), and difucosyl sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3 Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc) epitopes. The enzyme shares 85% amino acid sequence identity with Fuc-TIII and 89% identity with Fuc-TV but differs substantially in its acceptor substrate requirements. Polymerase chain reaction analyses demonstrate that the gene is syntenic to Fuc-TIII and Fuc-TV on chromosome 19. Southern blot analyses of human genomic DNA demonstrate that these four alpha (1,3)fucosyltransferase genes account for all DNA sequences that cross-hybridize at low stringency with the Fuc-TIII catalytic domain. Using similar methods, a catalytic domain probe from Fuc-TIV identifies a new class of DNA fragments which do not cross-hybridize with the chromosome 19 fucosyltransferase probes. These results extend the molecular definition of a family of human alpha (1,3)fucosyltransferase genes and provide tools for examining fucosyltransferase gene expression.


Molecular cloning and functional expression of cDNA encoding a mammalian inorganic pyrophosphatase. Extracts of soluble proteins from bovine retina contain multiple species of inorganic pyrophosphatase (PPase) that can be resolved by hydroxylapatite or ion exchange chromatography. We have purified one of these isoforms by a combination of chromatography and electrophoresis under denaturing conditions and have partially sequenced four peptides generated from it by CNBr digestion. This sequence information was used to clone PPase cDNA from a retinal cDNA library. Of five cDNA inserts, three were 1.3 kilobase pairs in length and two of these contained a complete open reading frame that was 867 base pairs long and encoded a 289-amino acid protein of 33 kDa. The deduced amino acid sequence is 49.5% identical to that of PPase from Saccharomyces cerevisiae, and contains identical amino acid residues at all of the positions previously identified as essential for catalytic activity in that enzyme. When the bovine PPase cDNA was expressed in Escherichia coli, catalytically active PPase was produced that comigrated with bovine retinal PPase in a nondenaturing gel and was clearly distinguishable from the host PPase. Northern analysis of poly(A)+ RNA from human, canine, and bovine retinas revealed that each contained a single major band of 1.4 kilobases that hybridized strongly with a pyrophosphatase cDNA probe. Southern analysis of bovine genomic DNA was consistent with the existence of one PPase gene. Thus, the multiple forms separated by chromatography may be derived from a common precursor or from mRNAs of very similar size.


Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity. Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function. And its nucleotide sequence revealed that it contains an open reading frame encoding a 525-amino acid protein. Homology comparison indicated that pre-pro-RPI consists of three domains: (1) an NH2-terminal prepro domain not found in the mature form of RPI, (2) a protease domain homologous to the trypsin family of serine proteases, and (3) a COOH-terminal domain homologous to the COOH-terminal part of Oerskovia xanthineolytica beta-1,3-glucanase and the NH2-terminal part of the ricin B chain, a lectin isolated from the part of the ricin B chain, a lectin isolated from the castor bean. The RPI gene and its mutant were subsequently expressed in Escherichia coli under its beta-galactosidase promoter to investigate the function of the COOH-terminal domain. The mutant RPI, whose COOH-terminal domain was truncated by site-directed mutagenesis, lost both its mannose-binding and yeast-lytic activity, although the protease activity was not affected. These findings suggest that the COOH-terminal domain actually participates in the mannose-binding activity and is required for yeast-lytic activity.


Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity. A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.


Visible light-inducible photolyase gene from the goldfish Carassius auratus. By introducing and expressing a cDNA library constructed from mRNA of the cultured goldfish Carassius auratus cells in Escherichia coli, a gene encoding photolyase of the vertebrate was isolated, the first example from metazoa. The amino acid sequence deduced from the nucleotide sequence differs significantly from those of microorganisms. Five out of 6 tryptophan residues strictly conserved in photolyases from microorganisms and thought to play important roles in DNA and chromophore binding of the enzyme are substituted by other residues of different characteristics. By Northern analysis the expression of the photolyase gene was found to be induced more than 10 times by exposure of the cells to visible light. These results indicate a unique evolution of the photolyase gene and a novel mechanism of gene regulation, in which visible light triggers the production of the light-dependent enzyme for repair of DNA damages induced by harmful ultraviolet part of sunlight.


A novel complex locus UGT1 encodes human bilirubin, phenol, and other UDP-glucuronosyltransferase isozymes with identical carboxyl termini. Two human liver UDP-glucuronosyltransferase (transferase) cDNAs, HUG-Br1 and HUG-Br2, were previously isolated (Ritter, J. K., Crawford, J. M., and Owens, I. S. (1991) J. Biol. Chem. 266, 1043-1047), and each was shown to encode a bilirubin transferase isozyme which catalyzes the formation of all physiological conjugates of bilirubin IX alpha following expression in COS-1 cells. Sequence data showed that the cDNAs contained identical 3' ends (1469 base pairs in length) to each other and to that of the human phenol transferase cDNA, HLUG P1 (Harding, D., Fournel-Gigleux, S., Jackson, M. R., and Burchell, B. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8381-8385). Here we report that the two corresponding bilirubin transferases and the phenol transferase are encoded by a novel locus, UGT1, which is also predicted to encode three other bilirubin transferase-like isozymes all having identical carboxyl termini. The transcriptional arrangement utilizes six nested promoter elements, each of which is positioned upstream of a unique exon 1. Each exon 1 encodes the NH2-terminal domain (286 amino acids) and confers the substrate specificity of the isoform. The 3' end of the locus contains 4 common exons which encode the identical carboxyl termini (246 amino acids). It is predicted that six nested primary transcripts are synthesized and that each exon 1 is differentially spliced to the 4 common exons to produce six unique, mature mRNAs. Although the gene organization is present as a single copy, it provides the flexibility of independent regulation of each isoform which is known to occur in the case of bilirubin and phenol transferase activities. With an understanding of the gene structure, lethal, as well as the nonlethal defects, associated with bilirubin transferase activity can now be determined.


Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase. The TIS10 cDNA was cloned as a primary response gene transcript whose mRNA rapidly accumulates in 3T3 cells treated with serum, polypeptide growth factors, or phorbol esters. The sequence of the TIS10 cDNA suggested that the gene encodes a protein with strong similarities to prostaglandin G/H synthase/cyclooxygenase (EC 1.14.99.1). Transient transfection into COS-1 cells of an expression vector driving the TIS10 cDNA leads to production and secretion of prostaglandin E2. Microsomes prepared from COS-1 cells transfected with this construct demonstrate both hydroperoxidase and cyclooxygenase activities similar to that demonstrated by cells transfected with a vector encoding the ovine prostaglandin G/H synthase. These data demonstrate that the TIS10 gene encodes a functional prostaglandin synthase/cyclooxygenase distinct from the prostaglandin synthase/cyclooxygenase whose cDNAs and/or genes have previously been cloned from sheep, mouse, and man. The structure of the TIS10 gene, determined by a combination of sequencing of genomic clones and polymerase chain reactions from genomic clones, demonstrates remarkable exon-intron conservation with the human prostaglandin synthase/cyclooxygenase gene. A 1-kilobase sequence located immediately proximal to the start site of transcription of the TIS10 gene can confer phorbol ester and serum inducibility to a luciferase reporter gene following transient transfection into NIH 3T3 cells, suggesting that this region of the gene is responsible for transcriptional regulation of the TIS10 gene by mitogens in fibroblasts.


Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho- forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis. The mechanism by which cAMP-dependent protein kinase-catalyzed phosphorylation modulates the activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was examined after site-specific mutation of the cAMP-dependent phosphorylation site (Ser32) to aspartic acid or alanine. The mutant and wild-type enzymes were overexpressed in Escherichia coli in a rich medium to levels as high as 30 mg/liter and were then purified to homogeneity. The kinetic properties of the Ser32-Ala mutant were identical with the dephosphorylated wild-type bifunctional enzyme. Mutation of Ser32 to aspartic acid mimicked several effects of cAMP-dependent phosphorylation: there was an increase in the Km for fructose 6-phosphate for 6-phosphofructo-2-kinase and an increase in the maximal velocity of fructose-2,6-bisphosphatase. Fructose-2,6-bisphosphatase activity of the Ser32-Asp mutant was 75% that of the phosphorylated wild-type enzyme, the mutant's kinase reaction had an identical dependence on fructose 6-phosphate, while its maximum velocity was only 60% that of the phosphorylated wild-type enzyme over a wide pH range. Furthermore, catalytic subunit-catalyzed in vitro phosphorylation of the Ser32-Ala mutant on Ser33 increased the Km for fructose 6-phosphate by 4-fold for the 6-phosphofructo-2-kinase. The results support the hypothesis that Ser32 is an important residue in the regulation of the activities of the bifunctional enzyme and that phosphorylation of Ser32 can be functionally substituted by aspartic acid. The results suggest a role for negative charge in the effect of phosphorylation.


High expression of beta-adrenergic receptor kinase in human peripheral blood leukocytes. Isoproterenol and platelet activating factor can induce kinase translocation. Receptor phosphorylation is a key step in the process of desensitization of the beta-adrenergic and other related receptors. A selective kinase (called beta-adrenergic receptor kinase, beta ARK) has been identified which phosphorylates the agonist-occupied form of the receptor. Recently the bovine beta ARK cDNA has been cloned and the highest levels of specific mRNA were found in highly innervated tissues. It was proposed that beta ARK may be primarily active on synaptic receptors. In the present study, the cDNA of human beta ARK was cloned and sequenced. The sequence was very similar to that of the bovine beta ARK (the overall amino acid homology was 98%). Very high levels of beta ARK mRNA and kinase activity were found in peripheral blood leukocytes and in several myeloid and lymphoid leukemia cell lines. Since agonist-induced beta ARK translocation is considered the first step involved in beta ARK-mediated homologous desensitization, we screened a number of G-protein-coupled receptor agonists for their ability to induce beta ARK translocation. In human mononuclear leukocytes, beta-AR agonist isoproterenol and platelet-activating factor were able to induce translocation of beta ARK from cytosol to membrane. After 20 min of exposure to isoproterenol (10 microM), the cytosolic beta ARK activity decreased to 61% of control, while membrane-associated beta ARK activity increased to 170%. 20-min exposure to platelet-activating factor (1 microM) reduced the cytosolic beta ARK activity to 42% of control with concomitant increase in membrane beta ARK activity to 214% of control. The high levels of beta ARK expression in human peripheral blood leukocytes together with the ability of isoproterenol and platelet-activating factor to induce beta ARK translocation, suggest a role for beta ARK in modulating some receptor-mediated immune functions.


Cloning and transcriptional regulation of a novel adipocyte-specific gene, FSP27. CAAT-enhancer-binding protein (C/EBP) and C/EBP-like proteins interact with sequences required for differentiation-dependent expression. We have reported previously the cloning of several cDNAs whose mRNAs are induced during differentiation of the adipogenic cell line TA1. Here we characterize an adipocyte-specific gene, which we refer to as FSP27 (formerly clone 47), that encodes a protein of 27 kDa, the sequence of which is unrelated to any in the current data banks. The FSP27 promoter confers adipocyte-specific expression to a heterologous reporter gene in transfected adipogenic cell lines, e.g. TA1 and 3T3-L1. Analysis of regulatory elements in the FSP27 promoter region indicates the presence of (a) a proximal palindromic sequence that is necessary for adipocyte-specific expression; and (b) a distal differentiation-independent enhancer-like element. The palindromic sequence TTCGAAA is protected from digestion by DNase I using nuclear extracts from TA1 preadipocytes and adipocytes. Heated rat liver nuclear extract, a very abundant source of the transcription factor CAAT-enhancer-binding protein (C/EBP) and related proteins, generates an equivalent footprint over the palindrome. However, C/EBP can account for only a portion of the protein-DNA complexes in TA1 cells because preadipocytes as well as adipocytes contain proteins distinct from C/EBP which interact with the same sequence. We suggest that C/EBP and other C/EBP-like proteins play a critical role in regulating the transcription of the fat-specific gene FSP27.


Defective folding and stable association with protein disulfide isomerase/prolyl hydroxylase of type I procollagen with a deletion in the pro alpha 2(I) chain that preserves the Gly-X-Y repeat pattern. We have studied the folding, processing, and association with two endoplasmic reticulum (ER) resident proteins of the abnormal type I procollagen molecules produced by a strain of fibroblasts harboring a 4.5 kilobase deletion in an allele of COL1A2 (Willing, M. C., Cohn, D.H., Starman, B. Holbrook, K.A., Greenberg, C.R., and Byers, P.H. (1988) J. Biol. Chem. 263, 8398-8404). By sequencing cDNA, we found that the mutant allele encodes pro alpha 2(I) chains that are shortened by 180 amino acids but retain the Gly-X-Y repeat pattern crucial for collagen triple helix formation. The type I procollagen molecules that incorporated the shortened chain were retained intracellularly and were stable. The triple helical domain in these molecules did not attain a normal conformation and remained accessible to posttranslational modifying enzymes amino-terminal to the deletion site for a prolonged period. The abnormal molecules folded into a triple helical conformation more slowly than the normal molecules, and the amino-terminal ends of the pro alpha 1(I) chains failed to become protease-resistant. While the abnormal procollagen molecules were not bound by the ER-resident protein BiP, they stably associated with protein disulfide isomerase, the beta-subunit of prolyl-4-hydroxylase. These results indicate that some mutations in type I collagen genes both transiently delay folding and permanently disrupt the structure of the triple helix and suggest that binding to prolyl-4-hydroxylase helps to retain certain abnormal procollagen molecules within the ER.


Cloning and expression in Escherichia coli of the gene encoding Aspergillus flavus urate oxidase. Amino acid sequencing of peptides obtained after proteolytic hydrolysis of Aspergillus flavus urate oxidase (uricase) permitted the design of oligodeoxynucleotide probes that were used to obtain 1.2- and 5-kilobase pair DNA fragments from A. flavus cDNA and genomic libraries, respectively. The cDNA fragment contained the entire coding region for uricase, and comparison with the genomic fragment revealed the presence of two short introns in the coding region of the gene. A. flavus uricase has around 40% overall identity with uricases from higher organisms but with many conserved amino acids. Hitherto highly conserved consensus patterns found in other uricases were found to be modified in the A. flavus enzyme, notably the sequence Val-Leu-Lys-Thr-Thr-Gln-Ser near position 150, which in the filamentous fungus is uniquely modified to Val-Leu-Lys-Ser-Thr-Asn-Ser. Silent mutations were introduced by cassette mutagenesis near the 5'-extremity of the coding sequence in order to conform with Escherichia coli codon usage, and the uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form.


Cloning and characterization of two human skeletal muscle alpha-actinin genes located on chromosomes 1 and 11. Conserved sequences of dystrophin, beta-spectrin, and alpha-actinin were used to plan a set of degenerate oligonucleotide primers with which we amplified a portion of a human alpha-actinin gene transcript. Using this short clone as a probe, we isolated and characterized full-length cDNA clones for two human alpha-actinin genes (ACTN2 and ACTN3). These genes encode proteins that are structurally similar to known alpha-actinins with approximately 80% amino acid identity to each other and to the previously characterized human nonmuscle gene. ACTN2 is the human homolog of a previously characterized chicken gene while ACTN3 represents a novel gene product. Northern blot analysis demonstrated that ACTN2 is expressed in both skeletal and cardiac muscle, but ACTN3 expression is limited to skeletal muscle. As with other muscle-specific isoforms, the EF-hand domains in ACTN2 and ACTN3 are predicted to be incapable of binding calcium, suggesting that actin binding is not calcium sensitive. ACTN2 was mapped to human chromosome 1q42-q43 and ACTN3 to 11q13-q14 by somatic cell hybrid panels and fluorescent in situ hybridization. These results demonstrate that some of the isoform diversity of alpha-actinins is the result of transcription from different genetic loci.


Identification of two enhancer elements in the gene encoding the type 1 glucose transporter from the mouse which are responsive to serum, growth factor, and oncogenes. The type 1 glucose transporter (GLUT1) gene encodes an integral membrane glycoprotein responsible for facilitating transfer of glucose across plasma membrane and is rapidly activated by serum, growth factors, and by oncogenic transformation. To elucidate the molecular mechanisms of regulation of GLUT1 gene expression, we isolated and characterized the mouse GLUT1 gene. DNA elements regulating transcription of the gene were analyzed in transient expression assays after transfection of NIH/3T3 cells with a low background chloramphenicol acetyltransferase (CAT) vector system pSVOOCAT. We identified two enhancer elements; the first one is located 2.7 kilobases upstream of the cap site of the gene which contains the homologous sequences with two 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs), a serum response element, a cyclic AMP-responsive element (CRE) and three GC boxes, and the second one is located in the second intron of the gene which contains the homologous sequences with two TREs and one CRE. With the promoter alone the transcription of the gene is activated by src, only slightly activated by ras and is not activated by serum and platelet-derived growth factor. When the gene is accompanied by one of these enhancers, the transcription is activated by all these stimuli.


Identification of a novel annexin in Hydra vulgaris. Characterization, cDNA cloning, and protein kinase C phosphorylation of annexin XII. As a first step toward the elucidation of a simple animal model in which to investigate annexin function, we identified, isolated, and characterized a novel annexin from Hydra vulgaris, annexin XII. A hydra cDNA library was screened using a probe generated by polymerase chain reaction from primers based on the partial amino acid sequence of annexin XII. Annexin XII cDNA was cloned and the functional protein was expressed in high yields in Escherichia coli. The annexin XII cDNA sequence predicted a 316-amino acid protein that had between 44 and 54% sequence identity with the Ca2+-binding core domains of previously characterized vertebrate and Drosophila annexins. The amino-terminal domain of annexin XII did not have sequence similarity with other known annexins except at and around a site that resembled known protein kinase C (PKC) phosphorylation sites in other annexins. As anticipated from its sequence, annexin XII was a high affinity substrate for purified rat brain PKC; half-maximal phosphorylation occurred below 0.1 microM annexin XII, and incorporation of up to 0.8 mol of phosphate/mol of annexin XII was observed. A PKC-like activity in hydra extracts also phosphorylated annexin XII. In summary, hydra promises to be a valuable model system for investigating the biological function of annexins and for determining how this function is modulated by PKC phosphorylation.


The Drosophila l(1)zw10 gene product, required for accurate mitotic chromosome segregation, is redistributed at anaphase onset. Mutations in the gene l(1)zw10 disrupt the accuracy of chromosome segregation in a variety of cell types during the course of Drosophila development. Cytological analysis of mutant larval brain neuroblasts shows very high levels of aneuploid cells. Many anaphase figures are aberrant, the most frequent abnormality being the presence of lagging chromosomes that remain in the vicinity of the metaphase plate when the other chromosomes have migrated toward the spindle poles. Finally, the centromeric connection between sister chromatids in mutant neuroblasts treated with colchicine often appears to be broken, in contrast with similarly treated control neuroblasts. The 85-kD protein encoded by the l(1)zw10 locus displays a dynamic pattern of localization in the course of the embryonic cell cycle. It is excluded from the nuclei during interphase, but migrates into the nuclear zone during prometaphase. At metaphase, the zw10 antigen is found in a novel filamentous structure that may be specifically associated with kinetochore microtubules. Upon anaphase onset, there is an extremely rapid redistribution of the zw10 protein to a location at or near the kinetochores of the separating chromosomes.


Adipogenesis in a myeloid supporting bone marrow stromal cell line. The bone marrow stroma contains pre-adipocyte cells which are part of the hemopoietic microenvironment. Cloned stromal cell lines differ both in their ability to support myeloid and lymphoid development and in their ability to undergo adipocyte differentiation in vitro. These processes have been examined in the +/+2.4 murine stromal cell line and compared to other stromal and pre-adipocyte cell lines. In long-term cultures, the +/+2.4 stromal cells support myeloid cell growth, consistent with their expression of macrophage-colony stimulating factor mRNA. However, despite the presence of mRNA for the lymphoid supportive cytokines interleukins 6 and 7, +/+2.4 cells failed to support stromal cell dependent B lineage lymphoid cells in vitro, suggesting that these stromal cells exhibit only a myelopoietic support function. The +/+2.4 cells differentiate into adipocytes spontaneously when cultured in 10% fetal bovine serum. The process of adipogenesis can be accelerated by a number of agonists based on morphologic and gene marker criteria. Following induction with hydrocortisone, methylisobutylxanthine, indomethacin, and insulin in combination, a time dependent increase in the steady state mRNA and enzyme activity levels of the following adipocyte specific genes was observed: adipocyte P2, adipsin, CAAT/enhancer binding protein, and lipoprotein lipase. In contrast, adipogenesis was accompanied by a slight decrease in the signal intensity of the macrophage-colony stimulating factor mRNA level, similar to that which has been reported in other bone marrow stromal cell lines. These data demonstrate that although the lympho-hematopoietic support function of pre-adipocyte bone marrow stromal cell lines is heterogeneous, they share a common mechanism of adipogenesis.


Identification of sequence types among the M-nontypeable group A streptococci. Streptococcal diseases, namely, acute glomerulonephritis and acute rheumatic fever, are common features in the aboriginal population of the Northern Territory of Australia. We examined the group A streptococcal M types identified during various surveys conducted since 1987. Streptococci were predominantly isolated from skin infections. A high proportion of the isolates could not be serotyped by conventional means and were designated M nontypeable (MNT). M-specific DNA sequences from the MNT isolates were examined, and sequence types were proposed for the classification of MNTs. This allowed a more precise estimate of the M types present in a population study.


Antibody-resistant mutants of Borrelia burgdorferi: in vitro selection and characterization. We used polyclonal antisera and monoclonal antibodies (mAbs) to inhibit the growth of clonal populations of two strains of Borrelia burgdorferi, the Lyme disease agent, and thereby select for antibody-resistant mutants. mAbs were directed at the outer membrane proteins, OspA or OspB. Mutants resistant to the growth-inhibiting properties of the antibodies were present in the populations at frequencies ranging from 10(-5) to 10(-2). The several escape variants that were examined were of four classes. Class I mutants were resistant to all mAbs; they lacked OspA and OspB and the linear plasmid that encodes them. Two other proteins were expressed in larger amounts in class I mutants; mAbs to these proteins inhibited the mutant but not the wild-type cells. Class II mutants were resistant to some but not all mAbs; they had truncated OspA and/or OspB proteins. Class III mutants were resistant only to the selecting mAb; they had full-length Osp proteins that were not bound by the selecting antibody in Western blots. In two class III mutants resistant to different anti-OspA mAbs, missense mutations were demonstrated in the ospA genes. Class IV mutants were likewise resistant only to selecting antibody, but in this case the selecting antibody still bound in Western blots.


Nucleotide sequence of the rhaR-sodA interval specifying rhaT in Escherichia coli. The precise location of the rhaT gene, encoding rhamnose permease, has been established between sodA and rhaC at 3605-3607 kb of Kohara's physical map, which corresponds to 88.4 min on the Escherichia coli chromosomal map. The dependence of the activity of the rhaT product on the function of rhaC, the rhamnose operon regulatory gene, was established by measuring rhamnose transport in wild-type and rhaC-deficient strains. The sequence of the sodA-rhaC interval displayed a single ORF corresponding to rhaT, which is transcribed counterclockwise on the E. coli chromosome. The ORF was shown to be preceded by a ribosome binding consensus sequence and a catabolite repression protein consensus sequence. The derived amino acid sequence displayed very low homology with any other permease and was clearly dissimilar to the homologous group formed by the xylose, arabinose, galactose and several glucose transporters. Analysis of the rhaT primary sequence identified potential membrane-spanning regions, possibly defining a protein structure model different from the one corresponding to the above-mentioned homologous group.


Molecular analysis of an esterase-encoding gene from a lipolytic psychrotrophic pseudomonad. An esterase gene (estA) from a lipolytic psychotroph (Pseudomonas sp. LS107d2), was cloned in Escherichia coli and its nucleotide sequence was determined, revealing an ORF encoding a polypeptide of 389 amino acid residues, with a molecular mass of 42276 Da. Labelling of plasmid-encoded proteins with [35S]methionine, using the maxicell procedure, gave a single polypeptide of molecular mass 42 kDa, consistent with that calculated from the ORF. Colonies of E. coli cells containing estA produced a clear halo when grown on solid media containing tributyrin; no clearance was produced when cells were grown on media containing triolein. Extracts of cells containing estA also hydrolysed water-soluble nitrophenol esters, but were unable to cleave water-insoluble substrates. The preference for water-soluble substrates indicates that the gene product is an esterase.


Changes in specific cleavability of the Sendai virus fusion protein: implications for pathogenicity in mice. Sendai virus mutants, KDe-21 and KDe-62, which had undergone multiple cycles of replication in Madin Darby canine kidney (MDCK) cells in the absence of exogenous proteases were isolated. The fusion (F) protein of the mutants regained proteolytic cleavability in MDCK cells and chick embryos, but the F protein remained non-cleavable in other cell lines. Unlike the F protein of wild-type (wt) virus, the mutant F was resistant to trypsin but was sensitive to elastase and, to a lesser extent, to chymotrypsin. Sequence analyses of the F gene and the F protein revealed an amino acid substitution at the cleavage site, Arg(116) to Ile, which conferred trypsin resistance and enhanced cleavability at Ile(116) by elastase and host proteases present in MDCK cells and in chicken embryos. In contrast to the pneumopathogenicity in mice of wt Sendai virus, the KDe mutants were non-pathogenic; cleavage activation of the F protein did not occur in the lungs and thereby infection was terminated after an initial cycle of replication.


Envelope protein sequences of dengue virus isolates TH-36 and TH-Sman, and identification of a type-specific genetic marker for dengue and tick-borne flaviviruses. Complementary DNAs were synthesized from the envelope protein genes of two isolates of dengue virus (TH-36 and TH-Sman, previously suggested as possible dengue virus type 5 and dengue virus type 6 respectively) and amplified by the polymerase chain reaction using sense and antisense primers designed from conserved dengue virus gene sequences. The amplified cDNA clones were sequenced in both directions by double-stranded dideoxynucleotide sequencing. Alignment with published dengue virus sequences enabled us to assign these viruses accurately to classified serotypes, confirming that TH-36 and TH-Sman are strains of dengue virus type 2 and dengue virus type 1 respectively. Amino acid changes between the proteins encoded by these two isolates and strains of their respective serotypes may account for the significant antigenic differences observed during previous serological typing of these viruses. Moreover, sequence alignment of flavivirus envelope proteins revealed a hypervariable region, within which members of the dengue and tick-borne virus antigenic complexes show unique peptide sequences. This type-specific hypervariable domain may be useful as a genetic marker for typing dengue and tick-borne flaviviruses.


Nucleotide sequence of carnation ringspot dianthovirus RNA-2. RNA-2 of carnation ringspot virus (CRSV), the type member of the dianthovirus group, has been cDNA cloned and sequenced. CRSV RNA-2 is 1394 nucleotides in length and contains a single open reading frame encoding a 304 amino acid polypeptide of 33.8K. Amino acid sequence alignment of this polypeptide with the cell-to-cell movement proteins encoded by RNA-2 of red clover necrotic mosaic virus (RCNMV) Australian (Aus) and Czechoslovakian (TpM-34) isolates indicates 59.6% and 55.7% sequence identity, respectively. The N-terminal 230 amino acids are more highly conserved, with 64.3% and 62.6% sequence identity, respectively. The cell-to-cell movement proteins of the two RCNMV isolates are themselves 82.5% and 91.7% identical when the amino-terminal 230 amino acids are compared. Structural prediction comparison of the RCNMV-Aus, RCNMV-TpM-34 and tobacco mosaic virus cell-to-cell movement proteins to the putative CRSV RNA-2-encoded movement protein suggests that even though no primary amino acid sequence similarity exists, the movement protein polypeptides are possibly similar in structure and function.


Nucleotide sequence of shallot virus X RNA reveals a 5'-proximal cistron closely related to those of potexviruses and a unique arrangement of the 3'-proximal cistrons. The 8890 nucleotide RNA sequence of shallot virus X (ShVX), a new virus isolated from shallot, has been determined. The sequence contains six open reading frames (ORFs) which encode putative proteins (in the 5' to 3' direction) of M(r) 194528 (ORF1), 26333 (ORF2), 11245 (ORF3), 42209 (ORF4), 28486 (ORF5) and 14741 (ORF6). The ORF1 protein was found to be highly homologous to the putative potexvirus RNA replicases; ORF2, -3, -5 and -6 proteins also have analogues among the potex- and/or carlavirus-encoded proteins. ORF3 is followed by an AUG-lacking frame coding for an amino acid sequence homologous to that of the 7K to 8K proteins of the triple gene block of the above-mentioned viruses. The putative ORF4 protein has no reliable homology with proteins in the database. The results obtained testify that, except for the unique 42K protein gene, the ShVX genome combines a number of elements typical of both carla- and potexviruses.


The nucleotide sequence and genome organization of strawberry mild yellow edge-associated potexvirus. The nucleotide sequence (5966 nucleotides) of cDNA clones of strawberry mild yellow edge-associated potexvirus was determined. The genome contains six open reading frames (ORFs) encoding putative proteins with Mrs of 149,423, 25,344, 11,576, 8079, 25,714 and 11,216. In the first three putative proteins and the coat protein considerable similarity was found to comparable polypeptides of the potexviruses potato virus X, clover yellow mosaic virus, narcissus mosaic virus, papaya mosaic virus, white clover mosaic virus and lily virus X.


Prediction of homologous binding sites on RB and p107 common for viral oncoproteins and cellular ligands. Hydropathic anticomplementarity of amino acids specifies that peptides translated from complementary DNA strands may acquire amphiphilic conformations and bind to each other. This concept has been coined 'Molecular Recognition Theory' (MRT) or 'complementary peptide theory'. Inactivation of retinoblastoma protein (RB), a tumor suppressor gene product, has been shown to be involved in the pathogenesis of many tumors and to be due to either mutation of the RB gene, hyperphosphorylation or complex formation with viral oncoproteins. The viral oncoproteins share a common RB binding motif with cellular ligands. The exact site on RB associating with this common RB binding motif of viral oncoproteins and cellular ligands has not been identified yet. This study is the first to predict putative binding sites on RB and p107, a cellular protein with RB sequence homology, respectively, by using the hydropathic complementarity approach. These sites are residues 649-654 of RB and 657-662 of p107. Moreover, this paper proposes a structure for a potential antineoplastic agent based on the amino acid sequence of the predicted RB binding site. The data presented herein should have important implications both for the understanding of cancer pathophysiology and for the drug design of antineoplastic compounds.


Proposed interaction between insulin and retinoblastoma protein. Retinoblastoma protein (RB) is a tumor suppressor gene product involved in embryogenesis and cell cycle progression. One of the major mechanisms leading to RB dysfunction is complex formation with viral oncoproteins using the common RB binding motif Leu X Cys X Glu (LXCXE) which has also been identified in cellular ligands, e.g., RBP-1 and RBP-2. p107, a cellular protein with RB sequence homology, has been shown to bind to the same viral oncoproteins associating with RB and is therefore thought to contribute to cell cycle regulation. It has recently been suggested that insulin stimulates gene transcription through direct association with an, as yet, unidentified intracellular transcription factor. Due to the central roles of RB and p107 in coupling external growth signals with the progression of the cell cycle clock, we have hypothesized that these two proteins might be candidates for mediating the effects of insulin on DNA. We report here the identification of a region in the B-chain of human insulin that has the sequence LXCXE. Based on this finding we predict that the insulin B-chain may interact with RB and/or p107. Since we have also identified sequences hydropathically related to LXCXE in insulin-like growth factor I (IGF-I) and II (IGF-II), but not in relaxin, nerve growth factor, epidermal growth factor, glucagon or beta-endorphin, we further propose that both IGF-I and -II may assemble with RB and/or p107, too. Moreover, binding sites on RB and p107 identical with those suggested for viral oncoproteins and cellular ligands are predicted for insulin/IGF-I/IGF-II by using the hydropathic complementarity approach.(ABSTRACT TRUNCATED AT 250 WORDS)


Supramolecular selective transport of Ca2+/Na+ by dicarboxylic ligands across bulk liquid membranes. Bulk liquid membrane transport of Ca2+ and Na+ ions was performed with dicarboxylic type ligands containing (S)-phenylalanine (Phe-2-O) and (S)-benzyltyrosine (Bz-Tyr-2-O) in pH gradients. With Bz-Tyr-2-O, calcium was generally favoured at every pH, the transport rate strongly depending upon the ligand concentration. At pH 8, a reversal of selectivity (Na+/Ca2+) was observed for the first time at low ligand concentration, thus suggesting that a supramolecular-type of transport occurs as a consequence of the different assemblages formed by the ligand according to pH and concentration. This is the first case of M2+/M+ selectivity induced by the carrier concentration.


Recombinant human secretory phospholipase A2: purification and characterization of the enzyme for active site studies. A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.


Drug-DNA sequence-dependent interactions analysed by electric linear dichroism. The interactions between 20 drugs and a variety of synthetic DNA polymers and natural DNAs were studied by electric linear dichroism (ELD). All compounds tested, including several clinically used antitumour agents, are thought to exert their biological activities mainly by virtue of their abilities to bind to DNA. The selected drugs include intercalating agents with fused and unfused aromatic structures and several groove binders. To examine the role of base composition and base sequence in the binding of these drugs to DNA, ELD experiments were carried out with natural DNAs of widely differing base composition as well as with polynucleotides containing defined alternating and non-alternating repeating sequences, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT),poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). Among intercalating agents, actinomycin D was found to be by far the most GC-selective. GC selectivity was also observed with an amsacrine-4-carboxamide derivative and to a lesser extent with methylene blue. In contrast, the binding of amsacrine and 9-aminoacridine was practically unaffected by varying the GC content of the DNAs. Ethidium bromide, proflavine, mitoxantrone, daunomycin and an ellipticine derivative were found to bind best to alternating purine-pyrimidine sequences regardless of their nature. ELD measurements provided evidence for non-specific intercalation of amiloride. A significant AT selectivity was observed with hycanthone and lucanthone. The triphenyl methane dye methyl green was found to exhibit positive and negative dichroism signals at AT and GC sites, respectively, showing that the mode of binding of a drug can change markedly with the DNA base composition. Among minor groove binders, the N-methylpyrrole carboxamide-containing antibiotics netropsin and distamycin bound to DNA with very pronounced AT specificity, as expected. More interestingly the dye Hoechst 33258, berenil and a thiazole-containing lexitropsin elicited negative reduced dichroism in the presence of GC-rich DNA which is totally inconsistent with a groove binding process. We postulate that these three drugs share with the trypanocide 4',6-diamidino-2-phenylindole (DAPI) the property of intercalating at GC-rich sites and binding to the minor groove of DNA at other sites. Replacement of guanines by inosines (i.e., removal of the protruding exocyclic C-2 amino group of guanine) restored minor groove binding of DAPI, Hoechst 33258 and berenil. Thus there are several cases where the mode of binding to DNA is directly dependent on the base composition of the polymer. Consequently the ELD technique appears uniquely valuable as a means of investigating the possibility of sequence-dependent recognition of DNA by drugs.


Spectrofluorimetric study of the intermolecular complexation of monoclonal antibodies with the high potency sweetener N-(p-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid. Molecular complexation between a set of five monoclonal antibodies (MAbs) and a N,N',N"-trisubstituted guanidinium sweetener (TGS) was studied by monitoring the intrinsic fluorescence of the MAbs. Changes in the emission spectral properties of the MAbs were found to be related to the location of tryptophan residues in the antibody complementarity determining regions (CDRs). Two of the MAbs, NC10.10 and NC10.8, showed fluorescence quenching and hypsochromic (blue) shifts in the emission maxima upon complexation with the TGS ligand. Experiments with three other MAbs, NC10.1, NC6.8 and NC2.3, revealed only monotonic fluorescence quenching. The association constants obtained by spectroscopic techniques for the different MAb-TGS complexes were found to be comparable with those determined using a conventional RIA. The thermodynamic parameters of the MAb-TGS complexation were also examined. The intermolecular complexation was found to be exothermic for four of the five MAbs in this study. However, MAb NC2.3 was found to be an exception, in that it was associated with a small positive enthalpic change. This type of spectrofluorimetric analysis can aid in the identification of interactive residues and molecular dynamics involved in TGS recognition by this set of MAb. Such information may prove useful in understanding the molecular recognition motifs responsible for the intense taste properties of high potency guanidine sweeteners.


Anthropological survey on north Cameroon peopling: I. GC subtyping (with a brief overview on the GC distribution in sub-Saharan Africa). Group-specific component (GC) polymorphism was investigated in 1324 subjects belonging to 12 ethnic groups from North Cameroon including Daba, Fali, Fulbe, Kanuri, Mada, Mafa, Mandara, Mundang, Uldeme, Podokwo, Tali, and Tupuri. The GC*1F frequency ranged from 0.785 among Fulbe to 0.847 among Mundang, that of GC*1S from 0.071 among Mafa to 0.125 among Fulbe and that of GC*2 from 0.035 among Uldeme to 0.092 among Tupuri. The results obtained are discussed in the light of the knowledge about the distribution and the possible selective relevance of GC polymorphism.


Distribution of some erythrocytary enzymes in the Piaroa Indians of Venezuela. Eight enzyme gene markers were studied in a sample of Piaroa Indians of Venezuela. ADA, DIA, PGD and AK markers seemed to be non-polymorphic whereas the following markers were found to be polymorphic: ACP1, where the only common electrophoretic alleles present were ACP1*A and ACP1*B (.955); ESD (ESD*1 = .781); PGM1 (PGM1*1 = .736) for which the subtypes (PGM1*1S = .546; PGM1*1F = .190; PGM1*2S = .140; PGM1*2F = .124) were also tested, and GLO1 (GLO1*1 = .347). Some showed a certain heterogeneity in distribution within the Piaroa population. The Piaroa Indians turned out to be different from other ethnic groups living in the same territory.


Mitochondrial DNA studies in the South African Indian population. The polymorphisms of mitochondrial DNA (mtDNA) for the restriction enzymes HpaI, BamHI, HaeII, MspI, AvaII, and HincII were studied in a sample of 147 unrelated Indian individuals from South Africa, who were subdivided according to religion and language into four groups, namely, Tamil, Hindi, Gujerati and Moslem. They were found to be monomorphic with the enzymes BamHI, and HaeII, and little variation was observed with the enzymes MspI and HincII. Six individuals were found to contain the non-Caucasoid HpaI morph, HpaI-3, which is found more commonly in indigenous African populations. This suggests that some flow of maternal genes from indigenous African populations into the Indian population may have occurred. Despite these interactions, Indians in South Africa display very little mtDNA variation (F = 0.77) when compared with those living in Nepal (F = 0.35) and New Delhi (F = 0.51). When compared with other Caucasoid populations, Indians are more homogenous in their genetic structure, which may be attributable to the high level of inbreeding among them, due to strict caste endogamy and certain religious customs that are still practised by the majority of Indians today.


MtDNA polymorphisms among Tharus of eastern Terai (Nepal). Tharus--a population of Terai (a region with a severe malarial morbidity in the past)--can be subdivided into three main groups: Western, Central and Southern Tharus. They have usually been considered a Mongoloid population and this has been further substantiated by mtDNA findings on Central Tharus. Studies on the distribution of malaria-related genes have shown an extremely high frequency (0.8) of the alpha-thal gene among Western and Central Tharus. This frequency, however, unexpectedly turned out to be only 0.04 in a sample of Eastern Tharus. This raised doubts on the common notion that Tharus are a single anthropological entity. In the present investigation mtDNA markers were studied in the same sample of Eastern Tharus previously examined for the alpha-thal gene. The findings were: 1. the same three features which confirmed the classification of Central Tharus as Mongoloids (i.e., the common occurrence of HpaI-1/HincII-1 and HaeII-5 morphs, and the lack of BamHI polymorphism) were also present in this sample. Since the only neighbouring population accessible to Tharus, until recently, has been Hindu (Caucasoids), this result strongly supports the notion that Tharus are indeed a single anthropological entity; 2. two statistically significant differences between Eastern and Central Tharus--namely, a much higher HaeII morph 5 frequency among Central Tharus, and the absence in the same group of the mutation at 15.487 bp (very common among Eastern Tharus)--together with the results on alpha-tal gene, suggested that Tharu subgroups underwent an effective reproductive isolation.


Plasma protein polymorphisms in Malas and Madigas of coastal Andhra Pradesh, south India. Four plasma protein polymorphisms: Group specific Component (GC), Haptoglobin (HP), Transferrin (TF) and Caeruloplasmin (CP), have been determined in two endogamous populations (Mala and Madiga) of Visakhapatnam of Coastal Andhra Pradesh, South India. The results were compared between the two caste populations and they revealed no significant differences. The results were also compared with those available from other Andhra populations.


Haptoglobin polymorphism in Hungary.

Haptoglobin polymorphism in Hungary. The haptoglobin phenotype was determined in 2609 blood samples collected from every Hungarian county. The results of the nation-wide survey are in agreement with those of a series of previous studies in which only Budapest inhabitants were investigated. However, a statistically significant difference was found in the haptoglobin phenotype frequency and distribution of people living west and east of the Danube. A lower HP*1 frequency was observed in the inhabitants of the eastern part of Hungary when compared to those living in the western part.


Human enzyme polymorphism in the Canary Islands. V. Western Islands. Autochthonous human samples of the three westernmost islands of the Canarian Archipelago, La Palma, Gomera, and Hierro, have been analyzed for eight red cell polymorphic enzymes. Only a small intra and inter-insular differentiation exists among these three islands. However, a marked heterogeneity is found when all seven islands of the Archipelago are compared by Nei's genetic distances [1972]. Nevertheless, there is no correlation between genetic and geographic distances. Historical factors could explain the frequency similarities of some distant islands. The rare variant GD*A+Negroid allele, and the endemic GD*Gc allele, previously found in other islands, have also been detected in this survey.


Ethnicity and blood group polymorphisms in the population of Melbourne, Australia. In an investigation of the extent of genetic variation in Melbourne, Australia, blood samples were collected from 3 of the largest ethnic groups comprising the present population; 251 Australian born of Anglo-Irish descent, 270 Greek born and 239 Italian born. Each sample was analysed for 5 red cell antigen systems, ABO, MNS, RH, KEL and FY. The Australian born sample was more similar to the Italians than the Greeks except for KEL R matrix and genetic distance analyses indicated that the Greek immigrants were similar to Greeks in Greece, but that Italian immigrants to Melbourne were not as close to a Southern Italian sample as their origins would suggest.


HLA-DQa allelic frequencies detected with PCR in a variety of human populations. Polymerase chain reaction (PCR) amplification and oligonucleotide probe hybridization may be used to detect DNA polymorphisms rapidly in large samples. In this study, 475 individuals from thirteen human populations were allelotyped at the human leukocyte antigen (HLA) DQa (DQA1) locus. A 242 or 239 bp fragment was amplified from each individual's DNA. Each of six alleles was detected by hybridization to allele specific oligonucleotide probes (ASOs). Allelic frequencies varied between populations, but the measure of gene frequency variation among populations, the FST value, was relatively low. Most populations had genotypic frequencies in agreement with Hardy-Weinberg equilibrium expectations. Principal component analysis was performed on the populations, and results are presented in graphic form. The heterozygosity at this locus is high in all populations; the average (74%) is close to the theoretical maximum (83%) for a 6 allele system. It is likely that this system is affected by stabilizing selection, which makes it less than optimal for the study of random evolutionary divergence between populations.


Internal and interfacial structure of membranes studied using X-ray standing waves. The X-ray standing wave (XSW) method developed in the mid-Sixties was used then to determine the position of heavy atoms in and on crystals of silicon and germanium with sub-Angström resolution. The advent of layered synthetic microstructures, used primarily as wide-bandpass X-ray monochromators, heralded a new era in the use of XSW to study biologically relevant structures with a length scale of the order of tens of Angströms. The original measurements were performed on model membrane Langmuir-Blodgett (LB) films and served to establish the utility of the XSW approach in determining heavy-atom location in such systems with sub-Angström resolution and in tracking the heavy-atom layer as it moves during a thermotropic transition. Recent measurements show that the XSW is well defined at close to 1000 A from the XSW generating surface. Thus, the useful probing distance of XSW is of this length scale also without a compromise in resolution. In addition to the above measurements on well ordered systems the XSW method is being used to profile ion distribution 'directly' at the membrane/aqueous interface. Recent results show that the diffuse double layer can be established reversibly by suitably adjusting the pH of the aqueous phase next to a phospholipid membrane. The advantages and disadvantages of this new surface technique as applied to the study of membrane structure and interfacial phenomena are discussed.


[The dynamics of the tuberculosis endemic in Hunedoara County 1982-1992]. A retrospective study through the analysis of the main epidemiological indices of tuberculosis endemic in the Hunedoara district, in comparison with the mean level for the whole country, was performed over a period of 10 years (1982-1991) (data given by the "M.Nasta" Pneumophthisiology Institute). The main epidemiological indices analysed were: general morbidity both in adults and children, for new cases and relapses apart, mortality through tuberculosis, main bacteriological indices, prevalence of Tb cases. The data in Tb infection incidence and prevalence could not be included in the study. This study is a starting point for new epidemiological evaluations within the district, in those areas with a Tb incidence higher than the district average value, in order to define additional measures of Tb control.


[The association of tuberculosis with HIV/AIDS infection in children in Romania]. The great number of AIDS cases in children in Romania, together with the high annual risk of Tb infection, created the premises for the occurrence of a relatively great number of disease cases through HIV infection/AIDS + tuberculosis, particularly in the age-group "0-5 years". Serum positive HIV children were considered as AIDS cases when tuberculosis was also associated. Twelve cases in which the infections were concomitant, transmitted through injections, constituted an exception to the point. The 12 children serum positive for HIV showed a primary musculo-cutaneous complex on their thighs, at the very place of injections. A proportion of 50% of them showed a favourable evolution under anti-Tb treatment. Most children developed primary tuberculosis aerogenically acquired, associated with AIDS. A proportion of 59.5% of them evoluted towards severe disseminated forms (milliaria, meningitis), with many deaths, and 37.8% only showed a favorable evolution under anti-tuberculosis treatment. HIV infection in children took place predominantly between 1987-1989. Tuberculosis was associated 1-2 years later, when the switching from bacillary infection into active tuberculosis was facilitated by the progressive immunodepression which is specific for AIDS. The tuberculin test with 2 IU-PPD was positive in less advanced AIDS cases but faded in children in the final stage of the syndrome or in those with severe forms of tuberculosis. Tuberculosis finding out in children with HIV infection/AIDS is however possible; therefore, skin test reaction is compulsory in all children in this category. In children with a tuberculosis cured through specific treatment in their histories, the association of HIV infection reaching AIDS stage can lead to a Tb relapse.(ABSTRACT TRUNCATED AT 250 WORDS)


Detoxification of the crotoxin complex by gamma radiation. 1. Crotoxin, the major neurotoxin of the South American rattlesnake venom (2 mg protein/ml in 0.85% NaCl) was irradiated with a Co-60 gamma source at a dose rate of 1100 Gy/h and at doses of 250, 500, 1000, 1500 and 2000 Gy. 2. Irradiated crotoxin was analyzed for free SH-groups, protein concentration, electrophoretic profile (SDS-PAGE), 50% lethal dose (LD50) in mice, and antigenicity against crotalic antiserum by diffusion immunoassay. 3. Irradiation led to the formation of protein aggregates and solubility was reduced at doses of 1000 Gy or higher. 4. The LD50 increased about 2-fold for 1000 Gy and 3.5-fold for 1500 Gy. However, the antigenic response was not changed as judged by the capacity of irradiated protein receiving up to 1000 Gy to react with anti-Crotalus durissus terrificus venom horse serum. 5. The dose of 1000 Gy cleaved 0.95 disulfide bridges/mol and 1500 Gy cleaved 1.42 bridges/mol, indicating the importance of disulfide bond integrity for toxicity.


Proteins released from vaccinia cores are probably not involved in protein synthesis inhibition in vitro. 1. It is known that vaccinia core proteins are released into the supernatant fraction when cores are incubated under appropriate conditions. When prepared in the absence of viral transcription this fraction inhibits in vitro protein synthesis. 2. We show here that after incubation, the cores loose the capability to inhibit protein synthesis. Furthermore, we show that no inhibition of translation is observed with supernatant fractions prepared from transcribing cores. 3. SDS-PAGE analysis of the supernatant fraction of cores obtained in the presence and absence of viral transcription indicated that their protein composition is similar and that approximately 17 peptides are released from the cores and that 4 are phosphorylated. 4. We conclude that the proteins associated with vaccinia cores and released in the presence of ribonucleotides are not involved in protein synthesis inhibition.


Simplified micromethod for the HPLC measurement of diclofenac in plasma. A simple and sensitive micromethod based on HPLC is described for the measurement of diclofenac in 200 microliters plasma. A single extraction with dichloromethane in acidic medium was an essential clean-up step. Diclofenac and its internal standard (cyclohexendiphenyl propionic acid) was eluted at 3.3 and 6.5 min from a 4-micron C18 reverse-phase column using a mobile phase consisting of 0.75 M sodium acetate buffer, pH 5.0, and acetonitrile (55:45, v/v) at a flow rate of 0.9 ml/min with detection at 282 nm. The method, validated on the basis of parameters evaluated for the confidence limits of diclofenac measurements in spiked plasma, presented 1 ng/ml sensitivity, 10-10,000 ng/ml linearity, and 3.5% and 5.7% intra- and interassay precision, respectively. Peak plasma diclofenac levels ranging from 177 to 841 ng/ml and from 276 to 1008 ng/ml were obtained for two slow-release formulations. A wide range (1 ng/ml-3 micrograms/ml) was observed for plasma diclofenac levels of volunteers during a 24-h study period.


[HIV infection case finding in children hospitalized in a pediatric pneumophthisiology department]. The study deals with HIV infection in children admitted to the "Phthisiopediatrics" Department of Braşov Hospital. They represent about 1% of the hospitalized cases (5 of 564). Considering, however, the age group of interest (0-5 years), they constitute 4.6% of hospitalized cases. Four of the five children were born in 1989, and all of them show a rather rich pathological history, with many hospitalizations in pediatrics departments, treatments by injections and even transfusions (2 cases). Vertical transmission being excluded--the respective mothers were negative at testing--the iatrogenic infection seems most probable. Together with the study on AgHBs carriers, where the results were similar, the present study made us think over the elementary prophylaxis measures in current medical practice.


Effect of age and sex on glomerular filtration rate measured by 51Cr-EDTA. 1. The effect of age and sex on glomerular filtration rate (GFR) was measured by the 51Cr-EDTA radioisotopic method in 76 normal individuals (43 women and 33 men). 2. Age has a significant effect on GFR. Subjects aged 41 to 60 years have GFR values [104.5 +/- 16.5 ml min-1 (1.73 m2)-1, N = 43] lower than younger individuals aged 20 to 40 years [116.6 +/- 11.2 ml min-1 (1.73 m2)-1, N = 33]. GFR decreases after 40 years of age by approximately 6.0 ml min-1 (1.73 m2)-1 per decade. 3. GFR values in women [105.9 +/- 16.0 ml min-1 (1.73 m2)-1, N = 43] were lower when compared to men [114.8 +/- 14.3 ml min-1 (1.73 m2)-1, N = 33]. 4. We conclude that the effect of sex and age must be taken into account when establishing reference values for GFR.


[The clinical, radiological and therapeutic aspects of endotracheobronchial foreign bodies in children]. During about 30 years of activity in the Bronchology room of the Phthisiopediatrics Unit-Braşov, 87 children having inhaled a foreign body (FB) in their airways were assisted. A number of 85 of them were solved through endoscopic interventions, performed by simple or complex (with ORL and intensive care) teams. The 85 FB were mostly (55) of vegetable nature (grains of beam and maize, seeds of pumpkin and sun flower), fragments of bone (10), of metal (10), and of plastic (8). FB localisation was: tracheal (30), in the right side bronchi (31), and left ones (26). Infancy was quite often interested: 60 children under 3 years of age and 9 aged less than 1 year. Clinical-radiological manifestation of FB were grouped in: without clearcut syndrome, well tolerated; with a clinical picture of acute pneumonia; with syndrome of complete or incomplete ("with valve") obstruction. Early diagnosis and prompt endoscopy solving prevent immediate or late complications.


Fuel oxidation by insulin-dependent diabetics during late pregnancy in response to an oral glucose load. 1. To determine the oxidative response to a 50-g oral glucose challenge by diabetic women during late pregnancy under a more intensive therapeutic regimen than is conventionally employed, six normal pregnant women and ten insulin-dependent pregnant diabetic women were studied during the third trimester. Fuel (carbohydrate and lipid) oxidation rates were determined by indirect calorimetry, blood levels of substrates and C-peptide were measured directly, and glucose metabolism data (oxidation and nonoxidative metabolism) were estimated for both groups at the postabsorptive state and for the 2-h period following glucose ingestion. 2. The increases in the non-protein respiratory quotient (npRQ) and carbohydrate oxidation rates in response to glucose ingestion in the diabetic pregnant group were significantly smaller than in the normal pregnant individuals. The total amount of glucose oxidized by the diabetic pregnant group during the 2-h tests (6.1 +/- 0.6 g/m2) was significantly smaller than the oxidized by the normal pregnant group (8.3 +/- 0.4 g/m2), whereas there was more but not statistically significant lipid oxidation in the diabetic group (3.0 +/- 0.3 vs 2.6 +/- 0.1 g/m2). 3. The diabetic pregnant group not only oxidized less glucose (10.9 +/- 1.1 vs 14.1 +/- 0.8 g, P < 0.05) but more of this hexose remained in their glucose space (9.1 +/- 1.6 vs 3.2 +/- 1.1, P < 0.05) and they excreted 2.8 +/- 1.0 g into the urine. 4. The diabetic pregnant subjects had significantly lower blood levels of lactate, pyruvate and C-peptide than the normal pregnant subjects, but significantly higher blood levels of glucose, beta-hydroxybutyrate and acetoacetate. 5. The present data show that an intensive conventional therapeutic regimen during late pregnancy was not sufficient to completely normalize the glucose-processing capability of insulin-dependent diabetic patients.


Scintigraphic study of the gastrointestinal transit of a liquid meal in patients with chronic Chagas' disease. Gastric emptying and small bowel transit of a liquid meal (isotonic dextrose) were assessed by a scintigraphic technique in 16 patients with Chagas' disease involving the esophagus and/or the colon, including one case with mega-jejunum, and in 10 control subjects. Initial gastric emptying was faster in the Chagas' disease group than in controls (gastric retention at 15 min, median and (range): 52% (15-86) vs 71% (43-97), P < 0.01) although there was no significant difference in total gastric emptying (T1/2: 20 min (4-132) vs 31 min (13-280), P > 0.05). Both the front (time to reach the proximal small bowel: 2.5 min (2.5-8) vs 15 min (5-40), P < 0.01) and the bulk of the meal (geometric center values at 15 min: 1.8 (1.2-2.4) vs 1.6 (1.2-1.8), P < 0.05) travelled faster through the proximal small bowel in Chagas' disease patients than in control subjects. There was no difference between the two groups concerning the time taken by the front of the meal to arrive to the caecum (110 min (15-180) vs 90 min (60-140), P > 0.20), but a precise delineation of this region was possible in only less than half of the patients and controls. In the patient with mega-jejunum, a remarkable delay in the intestinal progression of the liquid meal was found. These results suggest that the diffuse lesion of the enteric nervous system in chronic Chagas' disease mainly affects the control of gastric emptying and leads to accelerated proximal small bowel transit of a liquid meal.(ABSTRACT TRUNCATED AT 250 WORDS)


The long-term evolution of immune deposits in passive Heymann nephritis. 1. To study the long term course of passive Heymann nephritis (PHN), 42 adult male Wistar rats were injected with rabbit anti-FX1A serum (PHN group) and 42 rats received normal rabbit serum (control group). Two animals from each group were sacrificed 2 weeks after the inoculation and 10 animals each from the control and PHN groups were sacrificed 4, 13, 25 and 53 weeks later. 2. The PHN group exhibited a significant elevation in 20-h proteinuria which lasted from the first week (control group, 9.19 +/- 0.87; PHN group, 25.3 +/- 2.66) to the 25th week (control group, 22.6 +/- 2.15; PHN group, 66.7 +/- 10.4) except for week 17. From week 29 to week 53 there was no statistical difference between the 2 groups. 3. Light microscopy showed no difference between the kidneys of PHN and control rats. Immunofluorescence microscopy in PHN rats showed granular deposition of autologous and heterologous IgG on the glomerular basement membrane (GBM), whose intensity and pattern did not change during 53 weeks of observation. 4. When examined by electron microscopy the glomeruli of PHN rats showed: a) electron-dense deposits which were initially subepithelial and homogeneous and later intramembranous, granular and often surrounded by an electron-transparent halo; b) focal thickening of the GBM at the sites of intramembranous deposits; c) effacement of podocytes located close to the deposits; d) "penetration" of the podocytes into the GBM associated with the deposits; e) presence of osmiophilic granules in the cytoplasm of the podocytes located inside the GBM similar to the granules of the deposits next to them. The association of the penetration of the podocytes into the GBM with the deposits and the presence of the osmiophilic granules inside the foot process have not been described previously in PHN.


The complement system of Calomys callosus, Rengger, 1830 (Rodentia, Cricetidae). The complement system (C) of Calomys callosus, Rengger, 1830 (Rodentia, Cricetidae), a wild reservoir for several infectious agents in Latin America, was characterized. Sera from normal adult animals lysed sheep erythrocytes (Es) previously sensitized with rabbit serum anti-Es (Ar) in the presence of veronal-buffered saline containing 0.15 mM CaCl2 and 0.5 mM MgCl2, pH 7.4, or unsensitized rabbit erythrocytes (Er) in the presence of one-half isotonic strength veronal-buffered-saline containing 2.5% glucose, 2 mM MgCl2 and 10 mM EGTA, pH 7.4. Both hemolytic curves were sigmoidal in shape, with CH50 values of 30-40 for females and 20-30 for males. C5, determined hemolytically using the intermediate cells EsArClm4m2m3m, was approximately 4.5 x 10(8)/ml and 4.0 x 10(8)/ml for females and males, respectively. Immunochemical serum analyses by double immunodiffusion or by immunoblotting using polyclonal antisera against human C1s, C1q, C2, C3, C4, C5, C8 and factors B, I and H indicated that C. callosus C components factor B, C4 and C3 cross-reacted with the corresponding human C components. Thus, C. callosus was found to contain effective classical and alternative pathways (CP, AP) and common pathways, reasonable amounts of C5 and common epitopes in the key C components, factor B, C4 and C3, which were preserved during evolution.