The conversion between ICPC and ICD-10. Requirements for a family of classification systems in the next decade. The International Classification of Primary Care (ICPC) was developed to order medical concepts into classes that have been chosen for their relevance for family medicine. Family physicians use this to label the most prevalent conditions in their practice as well as their patients' symptoms and complaints. At the same time they do not want to be divorced from the needs of the medical community at large as these are reflected in the most recent medical nomenclature: the Tenth Revision of the International Classification of Diseases (ICD-10). A full conversion between all classes in the first and seventh component of ICPC (n = 646) with those of ICD-10 (n = 1983), with the exception of the chapter on external causes, has been prepared. It was concluded that ICD-10 at the three-digit level cannot function as a core classification for an international primary care system. Of the three-digit ICD-10 rubrics only 120 are compatible on a one to one basis with an ICPC rubric. A total of 114 three-digit ICD-10 rubrics have to be broken open into four-digit rubrics to allow at least one compatible conversion to one or more ICPC rubrics. On this basis only 25% of the diagnostic classes in ICPC can be converted to a single three- or four-digit ICD-10 rubric without lumping. The rest of ICD-10, either on the three- or on the four-digit level, has to be grouped into combinations of classes (lumping) to allow compatible conversion to the remaining rubrics of ICPC. Even though ICD-10 cannot serve as a core classification for primary care, a technical conversion between ICPC and ICD-10 is practically always possible which allows primary care physicians to implement ICD-10 as a contemporary nomenclature within the classification structure of ICPC.


Minimum basic data set in general practice: definitions and coding. A minimum basic data set (MBDS) was designed and used in general practice. The most important purpose and need for establishing such a data set is to assist in the provision of patient care. MBDS is designed to meet common needs for many general practitioners. Minimum means that individual programmes or the GP are free to collect as much additional data as they may wish. The MBDS includes two kinds of data: permanent and dynamic/encounter items. Permanent items are: patient data (identification, problem list, chronic treatments, source of payment) and GP data (professional characteristics). Dynamic/encounter items are: date, place, reasons for encounter, problems, process and prior encounter status.


Computerization of general practices and quality control. Blood glucose regulation in type 2 diabetics investigated in the Registration Network family practices. The extent to which computerized medical administration facilitates quality control was studied using as an example the quality of blood glucose regulation in diabetics supervised by general practitioners in 11 computerized practices. Systematic use of the general practice computer rapidly provided an unequivocal answer that 37% of such patients were not regulated in accordance with the guidelines for type 2 diabetes mellitus of the Dutch College of General Practitioners. The extra workload for the participating general practitioners was minimal. Automated recording of problem lists, as applied in the general practices belonging to the Registration Network, facilitates access to data on chronic diseases and risk factors for purposes of research, quality control and quality assessment.


General practitioners' attitudes towards future developments in practice computing--a representative survey in the north of Germany. A postal questionnaire was sent to a random sample of general practitioners in Lower Saxony, Germany to assess how general practitioners regard newly developed but not yet implemented options of practice computers and future applications such as expert systems and information retrieval systems. Replies were received from 276 (response rate 73.6%) general practitioners. Replying doctors were younger (P < 0.05) but they did not differ by sex, practice location, type of vocational training and grade of computerization from non-responding GPs. Twenty-eight per cent of the practices were computerized. Doctors who currently used a computer and those who intended to do so within the next 5 years were significantly younger than those doctors who did not intend to computerize their practice (P < 0.001). Female doctors were less willing to buy a computer than their male colleagues (P < 0.05). A computerized drug database and a medical library ranked best from 8 options given. Overall attitudes to all features were positive, except for an expert system giving criteria for referrals to specialist care. Doctors already working with a computer and those intending to buy one were significantly more positive about future options for computers than those doctors who do not intend to use a computer (P < 0.01). GPs' attitudes about new features of practice computers in general were positive but even more so about those options which are already available. Referral to a specialist seems to be a crucial point for GPs--they apparently do not want to be guided by a computer here.(ABSTRACT TRUNCATED AT 250 WORDS)


Cloning and expression in Escherichia coli of a phoA gene encoding a phosphate-irrepressible alkaline phosphatase of Zymomonas mobilis. The Zymomonas mobilis phoA gene, encoding a phosphate-irrepressible alkaline phosphatase (ZAPase), was cloned and its expression was studied in phoA mutants of Escherichia coli. The ZAPase was recovered in the soluble fraction of E. coli. The enzyme was synthesized constitutively and its synthesis not repressed by phosphate, unlike the phoA gene of E. coli. The phoA gene of Z. mobilis was mutagenized by Mini Mu PR13 and the mutated gene crossed into Z. mobilis in order to obtain phoA mutants by reverse genetics. Although Z. mobilis mutants with Mini Mu PR13 integrated in the chromosome were obtained, none had an allele replacement for none was defective in ZAPase.


Biosynthesis of modified peptidoglycan precursors by vancomycin-resistant Enterococcus faecium. In the presence of bacitracin, vancomycin-resistant Enterococcus faecium (vanA phenotype) accumulate UDP-N-acetylmuramyl(UDP-Mur-NAc)-tetrapeptide and a UDP-MurNAc-depsipentapeptide containing lactate substituted for the carboxy-terminal-D-alanine residue. In an in vitro peptidoglycan polymerization assay, the modified precursors function and confer resistance to vancomycin.


Urea-hydrolysis-dependent citrulline synthesis by Ureaplasma urealyticum. Some of the ammonia produced by hydrolysis of urea by Ureaplasma urealyticum is channelled into an anabolic pathway with resultant 'de novo' synthesis of citrulline. The organism appears to possess ornithine carbamoyltransferase and carbamoyl phosphate synthetase or some modified form of these enzymes.


Ergosterol levels in two L-methionine-enriched mutants of the methylotrophic yeast Candida boidinii ICCF26. Two L-methionine-enriched mutants, SN-78 and SE-57, were isolated in a sulphur-deficient medium from the methylotrophic yeast Candida boidinii ICCF26. No significant differences were detected between the L-cysteine pools of the mutants and the wild-type. In mutant strain SE-57, S-adenosylmethionine and ergosterol levels were higher than in the wild-type strain, while in the other mutant, SN-78, the levels were lower. The evidence presented would suggest that, in both the mutants and the wild-type used in this study, S-adenosylmethionine was of key importance for the accumulation of L-methionine and ergosterol.


Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR. An oligonucleotide primer (CgInt), synthesised from the variable internally transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) of Collectotrichum gloeosporioides was used for PCR with primer ITS4 (from a conserved sequence of the rDNA) to amplify a 450-bp fragment from the 25 C. gloeosporioides isolates tested. This specific fragment was amplified from as little as 10 fg of fungal DNA. A similar sized fragment was amplified from DNA extracted from C. gloeosporioides-infected tomato tissue. RAPD analysis divided 39 C. gloeosporioides isolates into more than 12 groups linked to host source and geographic origin. Based on the results obtained, the potential of PCR for detection and differentiation of C. gloeosporioides is discussed.


An FNR-dependent promoter from Escherichia coli is active and anaerobically inducible in Paracoccus denitrificans. A transcriptional fusion of a synthetic FNR-dependent promoter derived from Escherichia coli has been introduced into Paracoccus denitrificans on a broad-host-range plasmid. The patterns of expression of beta-galactosidase from this fusion and from a control which is not regulated by FNR have been studied. The results indicate that P. denitrificans expresses a transcriptional regulator which has a very similar DNA-binding specificity to FNR, and responds to a similar physiological signal.


Pulsed field gel electrophoresis of representatives of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains. Using field inversion gel electrophoresis (FIGE), different Mycobacterium tuberculosis strains, such as phage prototypes, exhibit different DNA restriction patterns which are easy to compare. Virulent and avirulent variants of M. tuberculosis H37, as well as daughter strains of M. bovis BCG, display characteristic DNA profiles. BCG strains isolated from suppurative adenitis following vaccination of French patients showed patterns identical to the BCG Pasteur strain used for vaccination. These results demonstrate that FIGE of DNA restriction fragments generated by DraI represents a suitable technique for the analysis of mycobacteria at a genomic level. The DraI profiles allow the differentiation and precise identification of the BCG Pasteur, Glaxo, Russian and Japanese strains.


Analysis of genomic DNA from Chlamydia trachomatis for Dam and Dcm methylation. Chlamydia trachomatis is a Gram-negative eubacterium with a dimorphic developmental cycle and obligate intracellular growth in the eucaryotic host. The Dam transmethylase of Escherichia coli methylates at the N6 position of adenine in the sequence 5'-GATC-3' and the Dcm transmethylase adds methyl groups to the C5 position of the internal cytosines in the sequences 5'-CCWGG-3'. In contrast to E. coli, C. trachomatis DNA appears to have unmethylated Dam sites and only low level Dcm methylation.


Mineralization of 2-chloro- and 2,5-dichlorobiphenyl by Pseudomonas sp. strain UCR2. Pseudomonas sp. strain UCR2 was isolated from a multi-chemostat mating experiment between a chlorobenzoate-degrader, Pseudomonas aeruginosa strain JB2, and a chlorobiphenyl-degrader, Arthrobacter sp. strain B1Barc. Strain UCR2 differed from either of the parental organisms in that it grew on both 2-chloro- and 2,5-dichlorobiphenyl with concomitant release of chloride. Phenotypic typing by the Biolog system indicated that strain UCR2 shared greater similarity with strain JB2 (88%) than strain B1Barc (3%). In DNA:DNA hybridization experiments, genomic DNA from strain UCR2 hybridized with both strain JB2 and strain B1Barc, with the former pairing yielding a much stronger signal than the latter. In contrast, no hybridization whatsoever was observed when the parental organisms strains JB2 and B1Barc were probed against each other.


Temperature-sensitive mutants of the Mycobacterium plasmid pAL5000. Two plasmids were isolated as thermosensitive replicons following in vitro mutagenesis of pB4, a pAL5000 derivative mycobacteria/Escherichia coli shuttle plasmid. Plasmids pCG59 and pCG63 replicate at 30 degrees C but not at 39 degrees C. This will allow their utilisation for transposon delivery, site-specific integration, or allele exchange.


Dimorphism in Itersonilia perplexans: yeast and hyphal phases differ in their sensitivity to mycocins produced by tremellaceous yeasts. The monokaryotic yeast phase of the heterobasidiomycete Itersonilia perplexans, unlike the hyphal phase, was found to be sensitive to mycocins produced by killer strains of Cryptococcus humicola, Cr. laurentii, Cystofilobasidium bisporidii and Rhodotorula fujisanense. Both the yeast and hyphal phases wer resistant to mycocins of Cr. podzolicus, Filobasidium capsuligenum, Rhodotorula glutinis, Rh. mucilaginosa, Rh. pallida, Sporidiobolus johnsonii, Sb. pararoseus and Sporobolomyces alborubescens. The different sensitivity patterns of yeast and hyphal phases are probably caused by biochemical differences in the cell walls.


Enterotoxicity and immunological properties of two mutant forms of Escherichia coli STIp with lysine or arginine substituted for the asparagine residue at position 11. Two variants of Escherichia coli heat-stable enterotoxin Ip, in which the amino acid residue at position 11 was substituted with lysine or arginine, were purified to near homogeneity from the culture supernatants of toxin-producing mutant strains. Neither the purified heat-stable enterotoxin Ip(Lys-11) nor the purified heat-stable enterotoxin Ip(Arg-11) showed a positive response in the suckling mouse assay or in the mouse intestinal loop assay. Furthermore, live bacteria producing these mutant heat-stable Ip enterotoxins did not cause fluid accumulation in mouse intestinal loops, in contrast to bacteria producing native heat-stable enterotoxin Ip. Nevertheless, antisera raised against both heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) neutralized the enterotoxic activity of native heat-stable enterotoxin Ip. These results demonstrate that heat-stable enterotoxin Ip(Lys-11) and heat-stable enterotoxin Ip(Arg-11) lose enterotoxicity but retain epitopes which are common to native heat-stable enterotoxin Ip.


Characterization of two forms of hemagglutinin/protease produced by Vibrio cholerae non-O1. Two forms (34 kDa and 32 kDa) of hemagglutinin/protease produced by Vibrio cholerae non-O1 were characterized. The hemagglutinin/protease purified by immunoaffinity column chromatography using a monoclonal antibody was essentially a 34-kDa form. By incubation of the purified 34-kDa form at 37 degrees C, it was processed (autodigested) to the 32-kDa form. The N-terminal 20 amino acid sequences of both the 34- and 32-kDa forms were identical, suggesting that proteolytic processing at the C-terminal region of the 34-kDa hemagglutinin/protease resulted in the 32-kDa form. With this shift, protease activity increased, but hemagglutinating activity decreased, suggesting that the C-terminal region of the hemagglutinin/protease is related to hemagglutinating activity.


Mycobacterium avium rough-to-smooth colony conversion resulting from growth in Tween 80 without presence of type-specific glycopeptidolipid antigens. Growth of a Mycobacterium avium complex serotype 20 rough-colony variant in 1.0% Tween 80 resulted in a smooth-colony morphological conversion that was reversible upon removal of Tween and was not associated with the presence of serotype-specific glycopeptidolipid antigens. Electron microscopic examination suggested the role of an outer layer in the Tween-related morphological modification.


Antibody-mediated uncoating of adenovirus in vitro. In a virus destabilization assay in vitro it was demonstrated that exposure of adenovirus to proteins will non-specifically protect the virus from being uncoated following transfer to low pH and hypotonic conditions. Such uncoating was also fully inhibited upon pretreatment of virus with 0.05% of the non-ionic detergent polyoxyethylenesorbitan monolaurate (Tween 20). However, in the presence of low concentrations of Tween 20 it was shown that monospecific immunoglobulins, directed against the fiber antigen and polyspecific antibodies produced in response to intact virions, were able to overcome the detergent-protecting effect of uncoating. Immunoglobulins directed towards the remaining outer-capsid components, the hexon, the penton base and the protein IIIa, revealed no such effects. The antifiber-mediated uncoating was paralleled by an aggregation of the virions. The data suggest that the virion-stabilizing effect of salt is enhanced by the hydrophobic action of a non-ionic detergent. Under these conditions the interaction between antifiber antibodies and fibers of the virion will trigger a destabilization of the virion upon transfer to low pH and hypotonic conditions.


N-acetyl-D-glucosamine-specific lectin purified from Vibrio cholerae 01. An N-acetyl-D-glucosamine-specific cell associated hemagglutinin (HA) was isolated and purified from a strain of Vibrio cholerae 01 by chitin affinity chromatography followed by separation on Bio Gel P-150. A single stained protein band of 47 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was observed with the purified HA. HA-antisera produced a single precipitin band against the purified HA in an immunodiffusion test without exhibiting any reactivity towards purified lipopolysaccharide (LPS). Purified HA, used as solid-phase antigen in an enzyme-linked immunosorbent assay (ELISA), reacted strongly with HA-antisera but cross-reacted negligibly with antisera raised against purified LPS. Hemagglutinating activity of the purified HA was highly sensitive to N-acetyl-D-glucosamine. The immunogold-labelling method using HA-antisera confirmed the location of the HA on the surface of the bacterial cells. The HA-antisera reacted with a protein component of the homologous outer membrane preparation. A significant inhibition was observed in the adhesive capability of the V. cholerae 01 strain to isolated rabbit intestinal epithelial cells (RIEC) in vitro when the later were pre-treated with the purified HA.


Interrelations between populations of methanogenic archaea and sulfate-reducing bacteria in the human colon. In humans, CH4 is produced in the colon by methanogenic archaea and is detected in breath samples from approximately 50% of healthy adults, identified as CH4-excretors. Methanogenesis and sulfate reduction have been described as two mutually exclusive processes, potentially regulated by sulfate availability. To determine whether microbial population balances reflected these apparently co-regulated activities, we compared sulfate-reducing bacteria, methanogenic archaea, sulfate and sulfide concentrations in faeces of 10 CH4-excretors (CH4+) and 9 non-CH4-excretors (CH4-). The mean +/- SE of the logarithm of methanogenic archaea per gram wet weight were 9.0 +/- 0.2 and 4.0 +/- 0.7 for CH4+ and CH4-, respectively (P < 0.001). Sulfate-reducing bacterial counts were 6.5 +/- 0.1 and 7.3 +/- 0.2, respectively (P < 0.001). Fecal sulfate and sulfide concentrations did not differ between groups. These results suggest that a competitive interrelation between methanogenic archaea and sulfate-reducing bacteria occurs in the human colon. However, it does not lead to a complete exclusion of the two populations.


Purification and partial characterization of two azoreductases from Shigella dysenteriae type 1. Two azoreductases (I and II) were purified to homogeneity from extracts of Shigella dysenteriae (type 1). Azoreductase I was a dimer of identical subunits of M(r) 28,000, whereas azoreductase II was a monomer of 11,000 M(r). Both were flavoproteins, each containing 1 mol of FMN per mol enzyme. Both NADH and NADPH functioned as electron donors for the azoreductases. Azoreductase I used Ponceau SX, Tartrazine, Amaranth and Orange II as substrates. Azoreductase II utilized all the dyes except Amaranth.


Binding of bovine lactoferrin to Streptococcus agalactiae. Bovine lactoferrin is an iron-binding protein present in mammary gland secretions. The exposure of Streptococcus agalactiae to bovine lactoferrin resulted in the binding of this protein to all the 12 strains of bovine origin tested, and also, although to a lesser degree, to the five tested strains of human origin. The interaction of lactoferrin with one high-binding bovine strain (24/60, the prototype NT/X strain) was studied. Binding was time-dependent, dose-dependent, and saturable. The binding of lactoferrin was slightly affected by cultivation conditions, and appeared to be heat-stable. The binding of biotinylated lactoferrin was inhibited by unlabelled lactoferrin but not by bovine serum albumin.


Siderophores and related outer membrane proteins produced by pseudomonads isolated from eels and freshwater. A total of 46 environmental pseudomonads, together with six type strains, were examined for their siderophore-producing activity. All strains were able to grow under iron-limiting conditions, gave orange halos in the CAS agar assay, and produced hydroxamates, and some of them also produced phenolate-type compounds. Bioassays showed that all strains, except Pseudomonas aeruginosa, promoted growth of mutant strain Arthrobacter flavescens JG-9, deficient in hydroxamate production, and some of them promoted growth of Salmonella typhimurium enb-1, which requires enterobactin for growth. The presence of iron-regulated outer membrane proteins was observed, the molecular size of the main induced proteins ranged between 76 and 93 kDa.


Stimulation of neutrophil leukocyte chemotaxis by a cloned cytolytic enterotoxin of Aeromonas hydrophila. A cytolytic enterotoxin produced by Aeromonas hydrophila, isolate SSU, has been cloned in our laboratory. This enterotoxin lysed rabbit red blood cells, destroyed Chinese hamster ovary cells, caused fluid secretion in rat ligated ileal loops, inhibited the phagocytic function of mouse phagocytes, and was lethal to mice when injected intravenously. In this study, the effect of this cytolytic enterotoxin on the chemotaxis of human leukocytes (cell line RPMI 1788) was examined. This toxin, at concentrations from 92.5 to 370 ng/ml, significantly stimulated the chemotactic activity of human leukocytes in a dose-dependent fashion. The stimulation of leukocyte chemotaxis elicited by cytolytic enterotoxin was abolished when the toxin was neutralized, heated, or exposed to low pH values. This stimulatory effect also was inhibited by various concentrations of pertussis toxin. These results demonstrated that cytolytic enterotoxin may stimulate increased chemotaxis of human leukocytes, and suggest that human leukocytes may possess cytolytic enterotoxin receptors which may be coupled to pertussis toxin-sensitive G-protein.


Phosphate starvation affects the synthesis of outer membrane proteins in Thiobacillus ferrooxidans. The outer membrane protein (omp40) component from the chemolithoautotrophic acidophilic Thiobacillus ferrooxidans is apparently regulated by the external pH and the concentration of phosphorus. Its amino-terminal sequence showed little identity with the Escherichia coli OmpC, OmpF or PhoE porins, but was 38.5% identical to the outer membrane channel-forming protein NosA from Pseudomonas stutzeri, whose expression is also regulated environmentally. In addition, the partial amino acid sequence of T. ferrooxidans omp40 showed between 34 and 38% identity with the amino-terminal end of the small outer membrane proteins Rck and PagC from Salmonella typhimurium and OmpX from Enterobacter cloacae.


Influence of nutrient media on the chemical composition of the exopolysaccharide from mucoid and non-mucoid Pseudomonas aeruginosa. Two mucoid Pseudomonas aeruginosa strains and their non-mucoid revertants isolated from two different clinical origins (cystic fibrosis and bronchiectasis) were grown in various chemically defined media. The extracted exopolysaccharide was characterized by gas-liquid chromatography and 1H-NMR spectroscopy. The exopolysaccharide was always heterogeneous, with an alginate fraction and a neutral fraction essentially composed of glucose, galactose, rhamnose and hexosamines. The alginate composition (mannuronate/guluronate ratio and O-acetylation degree) changed according to the carbon source in nutrient media and whether the strains tested were responding differently to these environmental stimuli. In all cases, the best carbon source for the alginate production was glycerol: the two cystic fibrosis strains produced a predominantly O-acetylated alginate whereas only the mucoid bronchiectasis strain produced a polymannuronate exopolysaccharide.


The bacteriocidal effects of transition metal complexes containing the NO+ group on the food-spoilage bacterium Clostridium sporogenes. The chemical and molecular mechanism of toxicity of nitrite towards food-spoilage bacteria such as Clostridium botulinum or Clostridium sporogenes is not well understood. In order to discover the active species and explore its chemistry, a number of compounds related to nitrite were synthesized. Their bacteriocidal effects on C. sporogenes were investigated in Oxoid nutrient broth No. 2 growth medium at pH 7.0. Inhibition of cell growth, expressed as the concentration which causes 50% cell inhibition, was observed with nitrite at 10 mM, whereas [Fe4S3(NO)7]-(the anion of Roussin's black salt) and (Fe2(SCH2CH2OH)2(NO)4] (a water-soluble Roussin's red salt ester) were found to be effective at 0.001 mM and 0.005 mM, respectively, confirming previous reports that iron-sulphur-nitrosyl complexes are much more toxic to these organisms than nitrite itself. The nitroprusside anion, [Fe(CN)5NO]2- was found to be toxic at 0.030 mM and the corresponding chromium species, [Cr(CN)5NO]3-, at 0.1 mM. Therefore, on the basis of the number of NO groups present, the nitrosylcyano complexes are comparable in activity with the iron-sulphur-nitrosyl compounds. These results show that neither iron nor sulphur are essential for the bacteriostatic effect of the Roussin's type compounds. The property that all these compounds have in common is that they contain NO+. It is proposed that this is the active species responsible for the preservative effect of nitrite, and that a relationship may exist between the N-O stretching frequency, a measure of the NO+ character, and the toxicity of these NO(+)-containing complexes.


Lectin-like activity of Lactobacillus acidophilus strain JCM 1026. The lectin-like activity of the Lactobacillus acidophilus strain JCM 1026 was studied by hemagglutination and hemagglutination-inhibition assays. L. acidophilus strain JCM 1026 was found to hemagglutinate human and animal erythrocytes. Neuraminidase-treatment of human-type O erythrocytes enhanced the activity. Treatment of the bacterial cells with proteinase K reduced hemagglutinating activity significantly. Although several mono- and disaccharides did not inhibit hemagglutination, several different glycoproteins did. These data indicate that a proteinaceous lectin-like component(s) recognizing carbohydrate-containing molecules is located on the cell surface of L. acidophilus.


Structure of mycoside F, a family of trehalose-containing glycolipids of Mycobacterium fortuitum. Nuclear magnetic resonance spectroscopy, fast-atom bombardment mass spectrometry, gas chromatography-mass spectrometry, as well as chemical degradations were used to elucidate the structure of the major glycolipids of Mycobacterium fortuitum. Three main glycoconjugates were detected and their structures established as 2,3-diacyl, 2,3,4- and 2,3,6-triacyl trehalose. The characteristic infrared spectrum which led to their original designation as mycoside F, a family of glycolipids limited in distribution to M. fortuitum, was due to the nature of the fatty acyl substiuents identified primarily as 2-methyl-octadecen-2-oyl. The antigenic glycolipids typified the biovar. fortuitum, thus allowing its easy recognition from the C-mycoside glycopeptidolipid-containing biovar. peregrinum.


Regulation of pag gene expression in Bacillus anthracis: use of a pag-lacZ transcriptional fusion. The production of protective antigen (PA), the common component of the two anthrax toxins, is influenced by the environment. In order to examine factors involved in its regulation, a transcriptional fusion between the promoter region of the PA gene (pag) and the lacZ gene was constructed and introduced into Bacillus anthracis Sterne. Activity of the pag promoter was followed by measuring beta-galactosidase activities under various growth and medium conditions. Expression from the pag promoter was observed throughout exponential-phase and was maximal in early stationary phase. The activity of the pag promoter was stimulated by the addition of glucose in the medium.


Uniparental cytogamy: a novel method for bringing micronuclear mutations of Tetrahymena into homozygous macronuclear expression with precocious sexual maturity. A new method of inducing self-fertilization, uniparental cytogamy, yields homozygous germinal and somatic genotypes in the ciliate Tetrahymena thermophila. Progeny are highly fertile and show a marked tendency for precocious sexual maturity. This method is highly effective in protocols designed to generate and express nonlethal dominant or recessive mutations.


Copies of a Stellate gene variant are located in the X heterochromatin of Drosophila melanogaster and are probably expressed. Two variants of X chromosome Stellate genes responsible for crystal formation in XO male primary spermatocytes occupy different genome positions. The majority if not all of the 1250-bp Stellate genes are located at the 12E site where the Ste locus has been mapped and almost all of the 1150-bp Stellate repeats are concentrated in the distal X heterochromatin. Sequencing of Stellate genes derived from X heterochromatin reveals the preservation of their open reading frames and precise matching with some Stellate cDNAs reported earlier. At least some heterochromatic Stellate genes are suggested to be expressed and, therefore, involved in the interaction with the Y chromosome locus Su(Ste), as are the Stellate genes from 12E.


Sister-chromatid misbehavior in Drosophila ord mutants. In Drosophila males and females mutant for the ord gene, sister chromatids prematurely disjoin in meiosis. We have isolated five new alleles of ord and analyzed them both as homozygotes and in trans to deficiencies for the locus, and we show that ord function is necessary early in meiosis of both sexes. Strong ord alleles result in chromosome nondisjunction in meiosis I that appears to be the consequence of precocious separation of the sister chromatids followed by their random segregation. Cytological analysis in males confirmed that precocious disjunction of the sister chromatids occurs in prometaphase I. This is in contrast to Drosophila mei-S332 mutants, in which precocious sister-chromatid separation also occurs, but not until late in anaphase I. All three of the new female fertile ord alleles reduce recombination, suggesting they affect homolog association as well as sister-chromatid cohesion. In addition to the effect of ord mutations on meiosis, we find that in ord2 mutants chromosome segregation is aberrant in the mitotic divisions that produce the spermatocytes. The strongest ord alleles, ord2 and ord5, appear to cause defects in germline divisions in the female. These alleles are female sterile and produce egg chambers with altered nurse cell number, size, and nuclear morphology. In contrast to the effects of ord mutations on germline mitosis, all of the alleles are fully viable even when in trans to a deficiency, and thus exhibit no essential role in somatic mitosis. The ord gene product may prevent premature sister-chromatid separation by promoting cohesion of the sister chromatids in a structural or regulatory manner.


Evidence for intrinsic differences in the formation of chromatin domains in Drosophila melanogaster. The results of an investigation into intrinsic differences in the formation of two different heterochromatic domains are presented. The study utilized two different position effect variegation mutants in Drosophila melanogaster for investigating the process of compacting different stretches of DNA into heterochromatin. Each stretch of DNA encodes for a gene that affects different aspects of bristle morphology. The expression of each gene is prevented when it is compacted into heterochromatin thus the genes serve as effective reporter systems to monitor the spread of heterochromatin. Both variegating mutants are scored in the same cell such that environmental and genetic background differences are unambiguously eliminated. Any differences observed in the repression of the two genes must therefore be the result of intrinsic differences in the heterochromatic compaction process for the two stretches of DNA. Studies of the effects different enhancers of variegation have upon the compaction of the two genes indicate each compaction event occurs independently of the other, and that different components are involved in the two processes. These results are discussed with regard to spreading heterochromatin and the role this process may play in regulating gene expression.


Rearrangement of upstream regulatory elements leads to ectopic expression of the Drosophila mulleri Adh-2 gene. The Adh-2 gene of Drosophila mulleri is expressed in the larval fat body and the adult fat body and hindgut, and a 1500-bp element located 2-3 kb upstream of the Adh-2 promoter is necessary for maximal levels of transcription. Previous work demonstrated that deletion of sequences between this upstream element and the Adh-2 promoter results in Adh-2 gene expression in a novel larval tissue, the middle midgut. In this study we show that the upstream element possesses all of the characteristics of a transcriptional enhancer: its activity is independent of orientation, it acts on a heterologous promoter, and it functions at various positions both 5' and 3' to the Adh-2 gene. Full enhancer function can be localized to a 750-bp element, although other regions possess some redundant activity. The ectopic expression pattern is dependent on the proximity of at least two sequence elements. Thus, tissue-specific transcription can involve complex proximity-dependent interactions among combinations of regulatory elements.


An examination of the effects of double-strand breaks on extrachromosomal recombination in mammalian cells. We studied the effects of double-strand breaks on intramolecular extrachromosomal homologous recombination in mammalian cells. Pairs of defective herpes thymidine kinase (tk) sequences were introduced into mouse Ltk- cells on a DNA molecule that also contained a neo gene under control of the SV40 early promoter/enhancer. With the majority of the constructs used, gene conversions or double crossovers, but not single crossovers, were recoverable. DNA was linearized with various restriction enzymes prior to transfection. Recombination events producing a functional tk gene were monitored by selecting for tk-positive colonies. For double-strand breaks placed outside of the region of homology, maximal recombination frequencies were measured when a break placed the two tk sequences downstream from the SV40 early promoter/enhancer. We observed no relationship between recombination frequency and either the distance between a break and the tk sequences or the distance between the tk sequences. The quantitative effects of the breaks appeared to depend on the degree of homology between the tk sequences. We also observed that inverted repeats recombined as efficiently as direct repeats. The data indicated that the breaks influenced recombination indirectly, perhaps by affecting the binding of a factor(s) to the SV40 promoter region which in turn stimulated or inhibited recombination of the tk sequences. Taken together, we believe that our results provide strong evidence for the existence of a pathway for extrachromosomal homologous recombination in mammalian cells that is distinct from single-strand annealing. We discuss the possibility that intrachromosomal and extrachromosomal recombination have mechanisms in common.


A genetic model for control of hypertriglyceridemia and apolipoprotein B levels in the Johns Hopkins colony of St. Thomas Hospital rabbits. The St. Thomas Hospital (STH) rabbit has been previously shown to have a Mendelian form of hypertriglyceridemia, accompanied by accelerated atherosclerosis, and these animals may serve as a useful model for human dyslipoproteinemia syndromes. Here we describe the establishment of a new colony of these STH animals, and present genetic analysis of triglyceride (TG) and apolipoprotein B (apoB) levels. Segregation analysis of TG in 39 STH animals and 24 controls gave evidence of Mendelian segregation for an allele leading to both elevated TG levels and increased variability in these levels. Predicted means from the most parsimonious model for the Johns Hopkins STH colony were quite similar to that seen in the original London colony, and this model accounted for 80% of the variation in TG seen in the sample. This hypertriglyceridemia locus indirectly influenced the mean apoB levels in these rabbits, and segregation analysis of mean apoB levels suggested a second locus controlling apoB levels. Analysis of residual apoB levels (adjusted for predicted effects of the hypertriglyceridemia locus) revealed clearer evidence for a second locus controlling mean apoB levels in this colony. Arguments for two distinct genetic mechanisms operating in these STH animals are presented.


Patterns of interallelic divergence at the rabbit b-locus of the immunoglobulin light chain constant region are in agreement with population genetical evidence for overdominant selection. Population studies at the b-locus of the "constant" regions of the rabbit immunoglobulin kappa 1 light chain (c kappa 1) revealed patterns of gene diversity resembling those that mark the peculiar nature of the major histocompatibility complex, such as large number of alleles, high heterozygosity levels, consistent excess of heterozygous individuals and long allele coalescence times. This paper documents the evolutionary patterns at the b-locus as inferred from DNA sequence comparisons. Among alleles, synonymous substitutions outnumbered expectations for neutral alleles by an order of magnitude. They were distributed randomly throughout the c kappa 1 coding region while interallelic amino acid differences did cluster into segments overlapping with the regions exposed to the solvent. Within these regions, acceptance rates of mutation at amino acid replacement sites were even higher than those at synonymous sites (dr/ds = 1.6-3.0), while in the intervals between these regions the opposite was found (dr/ds approximately 0.3). Under the assumption that allelic variation is adaptive at the molecular surface, the divergence patterns at the b-locus are therefore very similar to those reported for the major histocompatibility complex. An analysis at the quasi silent bas-locus (c kappa 2), which is linked to the b-locus, and comparisons among genes of the "variable" region of the kappa 1 light chains (v kappa 1), revealed patterns of divergence which differed markedly from those observed at the c kappa 1 constant regions. It is suggested that allelic variability at immunoglobulin constant regions can be due to mechanisms similar to those enhancing diversity at histocompatibility loci.


Length polymorphisms of simple sequence repeat DNA in soybean. The objective of this work was to ascertain the presence and degree of simple sequence repeat (SSR) DNA length polymorphism in the soybean [Glycine max (L.) Merr.]. A search of GenBank revealed no (CA)n or (GT)n SSRs with n greater than 8 in soybean. In contrast, 5 (AT)n and 1 (ATT)n SSRs with n ranging from 14 to 27 were detected. Polymerase chain reaction (PCR) primers to regions flanking the six SSR loci were used in PCR amplification of DNA from 43 homozygous soybean genotypes. At three loci, amplification produced one PCR product per genotype and revealed 6, 7 and 8 product length variants (alleles) at the three loci, respectively. F1 hybrids between parents carrying different alleles produced two PCR products identical to the two parents. Codominant segregation of alleles among F2 progeny was demonstrated at each locus. A soybean DNA library was screened for the presence of (CA/GT)n SSRs. Sequencing of positive clones revealed that the longest such SSR was (CA)9. Thus, (CA)n SSRs with n of 15 or more are apparently much less common in soybean than in the human genome. In contrast to humans, (CA)n SSRs will probably not provide an abundant source of genetic markers in soybean. However, the apparent abundance of long (AT)n sequences should allow this SSR to serve as a source of highly polymorphic genetic markers in soybean.


Population genetics of polymorphism and divergence. Frequencies of mutant sites are modeled as a Poisson random field in two species that share a sufficiently recent common ancestor. The selective effect of the new alleles can be favorable, neutral, or detrimental. The model is applied to the sample configurations of nucleotides in the alcohol dehydrogenase gene (Adh) in Drosophila simulans and Drosophila yakuba. Assuming a synonymous mutation rate of 1.5 x 10(-8) per site per year and 10 generations per year, we obtain estimates for the effective population size (N(e) = 6.5 x 10(6)), the species divergence time (tdiv = 3.74 million years), and an average selection coefficient (sigma = 1.53 x 10(-6) per generation for advantageous or mildly detrimental replacements), although it is conceivable that only two of the amino acid replacements were selected and the rest neutral. The analysis, which includes a sampling theory for the independent infinite sites model with selection, also suggests the estimate that the number of amino acids in the enzyme that are susceptible to favorable mutation is in the range 2-23 at any one time. The approach provides a theoretical basis for the use of a 2 x 2 contingency table to compare fixed differences and polymorphic sites with silent sites and amino acid replacements.


The effects of selection on linkage analysis for quantitative traits. The effects of within-sample selection on the outcome of analyses detecting linkage between genetic markers and quantitative traits were studied. It was found that selection by truncation for the trait of interest significantly reduces the differences between marker genotype means thus reducing the power to detect linked quantitative trait loci (QTL). The size of this reduction is a function of proportion selected, the magnitude of the QTL effect, recombination rate between the marker locus and the QTL, and the allele frequency of the QTL. Proportion selected was the most influential of these factors on bias, e.g., for an allele substitution effect of one standard deviation unit, selecting the top 80%, 50% or 20% of the population required 2, 6 or 24 times the number of progeny, respectively, to offset the loss of power caused by this selection. The effect on power was approximately linear with respect to the size of gene effect, almost invariant to recombination rate, and a complex function of QTL allele frequency. It was concluded that experimental samples from animal populations which have been subjected to even minor amounts of selection will be inefficient in yielding information on linkage between markers and loci influencing the quantitative trait under selection.


Prediction of selection response for threshold dichotomous traits. This paper presents a formula to predict expected response to one generation of truncation selection for a dichotomous trait under polygenic additive inheritance. The derivation relies on the threshold liability concept and on the normality assumption of the joint distribution of additive genetic values and their predictors used as selection criteria. This formula accounts for asymmetry of response when both the prevalence of the trait and the selection rate differ from 1/2 via a bivariate normal integral term. The relationship with the classical formula R = iota rho sigma G is explained with a Taylor expansion about a zero value of the correlation factor. Properties are illustrated with an example of sire selection based on progeny test performance which shows a departure from usual predictions up to 15-20% at low (0.05) or high (0.95) selection rates. Univariate approximations and extensions to several paths of genetic change are also discussed.


Heterozygote advantage and the evolution of a dominant diploid phase. The life cycle of eukaryotic, sexual species is divided into haploid and diploid phases. In multicellular animals and seed plants, the diploid phase is dominant, and the haploid phase is reduced to one, or a very few cells, which are dependent on the diploid form. In other eukaryotic species, however, the haploid phase may dominate or the phases may be equally developed. Even though an alternation between haploid and diploid forms is fundamental to sexual reproduction in eukaryotes, relatively little is known about the evolutionary forces that influence the dominance of haploidy or diploidy. An obvious genetic factor that might result in selection for a dominant diploid phase is heterozygote advantage, since only the diploid phase can be heterozygous. In this paper, I analyze a model designed to determine whether heterozygote advantage could lead to the evolution of a dominant diploid phase. The main result is that heterozygote advantage can lead to an increase in the dominance of the diploid phase, but only if the diploid phase is already sufficiently dominant. Because the diploid phase is unlikely to be increased in organisms that are primarily haploid, I conclude that heterozygote advantage is not a sufficient explanation of the dominance of the diploid phase in higher plants and animals.


Using markers in gene introgression breeding programs. We investigate the use of markers to hasten the recovery of the recipient genome during an introgression breeding program. The effects of time and intensity of selection, population size, number and position of selected markers are studied for chromosomes either carrying or not carrying the introgressed gene. We show that marker assisted selection may lead to a gain in time of about two generations, an efficiency below previous theoretical predictions. Markers are most useful when their map position is known. In the early generations, it is shown that increasing the number of markers over three per non-carrier chromosome is not efficient, that the segment surrounding the introgressed gene is better controlled by rather distant markers unless high selection intensity can be applied, and that selection on this segment first can reduce the selection intensity available for selection on non-carrier chromosomes. These results are used to propose an optimal strategy for selection on the whole genome, making the most of available material and conditions (e.g., population size and fertility, genetic map).


Maximum likelihood mapping of quantitative trait loci using full-sib families. A maximum likelihood method is presented for the detection of quantitative trait loci (QTL) using flanking markers in full-sib families. This method incorporates a random component for common family effects due to additional QTL or the environment. Simulated data have been used to investigate this method. With a fixed total number of full sibs power of detection decreased substantially with decreasing family size. Increasing the number of alleles at the marker loci (i.e., polymorphism information content) and decreasing the interval size about the QTL increased power. Flanking markers were more powerful than single markers. In testing for a linked QTL the test must be made against a model which allows for between family variation (i.e., including an unlinked QTL or a between family variance component) or the test statistic may be grossly inflated. Mean parameter estimates were close to the simulated values in all situations when fitting the full model (including a linked QTL and common family effect). If the common family component was omitted the QTL effect was overestimated in data in which additional genetic variance was simulated and when compared with an unlinked QTL model there was reduced power. The test statistic curves, reflecting the likelihood of the QTL at each position along the chromosome, have discontinuities at the markers caused by adjacent pairs of markers providing different amounts of information. This must be accounted for when using flanking markers to search for a QTL in an outbred population.


Chi enhances heteroduplex DNA levels during recombination. The major pathway of homologous recombination in Escherichia coli, the RecBCD pathway, is stimulated by Chi sites. To determine whether Chi enhances an early or late step in recombination, we measured formation of heteroduplex DNA (hDNA) in extracts of lambda-infected E. coli. Chi elevated hDNA levels in these extracts, supporting a role for Chi early (before hDNA formation) in recombination. RecA protein and RecBCD enzyme were both necessary for detection of hDNA, indicating that they, too, act early. Analysis of a panel of recBCD mutants indicated that Chi-nicking activity was needed for Chi's stimulation of hDNA formation. These results support a previously proposed model of recombination. Further results suggested that RecBCD enzyme has an additional role late in recombination.


Rhizobium meliloti genes involved in sulfate activation: the two copies of nodPQ and a new locus, saa. The nitrogen-fixing symbiont Rhizobium meliloti establishes nodules on leguminous host plants. Nodulation (nod) genes used for this process are located in a cluster on the pSym-a megaplasmid of R. meliloti. These genes include nodP and nodQ (here termed nodPQ), which encode ATP sulfurylase and APS kinase, enzymes that catalyze the conversion of ATP and SO(4)2- into the activated sulfate form 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in cysteine synthesis. In Rhizobium, PAPS is also a precursor for sulfated and N-acylated oligosaccharide Nod-factor signals that cause symbiotic responses on specific host plants such as alfalfa. We previously found a highly conserved second copy of nodPQ in R. meliloti. We report here the mapping and cloning of this second copy, and its location on the second megaplasmid, pSym-b. The function of nodP2Q2 is equivalent to that of nodP1Q1 in complementation tests of R. meliloti and Escherichia coli mutants in ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase. Mutations in nodP2Q2 do not have as severe an effect on symbiosis or plant host range as do those in nodP1Q1, however, possibly reflecting differences in expression and/or channeling of metabolites to specific enzymes involved in sulfate transfer. Strains mutated or deleted for both copies of nodQ are severely defective in symbiotic phenotypes, but remain prototrophic. This suggests the existence in R. meliloti of a third locus for ATP sulfurylase and APS kinase activities. We have found a new locus saa (sulfur amino acid), which may also encode these activities.


The influence of local DNA sequence and DNA repair background on the mutational specificity of 1-nitroso-8-nitropyrene in Escherichia coli: inferences for mutagenic mechanisms. We have examined the mutational specificity of 1-nitroso-8-nitropyrene (1,8-NONP), an activated metabolite of the carcinogen 1,8-dinitropyrene, in the lacI gene of Escherichia coli strains which differ with respect to nucleotide excision repair (+/- delta uvrB) and MucA/B-mediated error-prone translesion synthesis (+/- pKM101). Several different classes of mutation were recovered, of which frameshifts, base substitutions, and deletions were clearly induced by 1,8-NONP treatment. The high proportion of point mutations (> 92%) which occurred at G.C sites correlates with the percentage of 1,8-NONP-DNA adducts which occur at the C(8) position of guanine. The most prominent frameshift mutations were -(G.C) events, which were induced by 1,8-NONP treatment in all strains, occurred preferentially in runs of guanine residues, and whose frequency increased markedly with the length of the reiterated sequence. Of the base substitution mutations G.C-->T.A transversions were induced to the greatest extent by 1,8-NONP. The distribution of the G.C-->T.A transversions was not influenced by the nature of flanking bases, nor was there a strand preference for these events. The presence of plasmid pKM101 specifically increased the frequency of G.C-->T.A transversions by a factor of 30-60. In contrast, the -(G.C) frameshift mutation frequency was increased only 2-4-fold in strains harboring pKM101 as compared to strains lacking this plasmid. There was, however, a marked influence of pKM101 on the strand specificity of frameshift mutation; a preference was observed for -G events on the transcribed strand. The ability of the bacteria to carry out nucleotide excision repair had a strong effect on the frequency of all classes of mutation but did not significantly influence either the overall distribution of mutational classes or the strand specificity of G.C-->T.A transversions and -(G.C) frameshifts. Deletion mutations were induced in the delta uvr, pKM101 strain. The endpoints of the majority of the deletion mutations were G.C rich and contained regions of considerable homology. The specificity of 1,8-NONP-induced mutation suggests that DNA containing 1,8-NONP adducts can be processed through different mutational pathways depending on the DNA sequence context of the adduct and the DNA repair background of the cell.


Donor locus selection during Saccharomyces cerevisiae mating type interconversion responds to distant regulatory signals. Mating type interconversion in homothallic strains of the yeast Saccharomyces cerevisiae results from directed transposition of a mating type allele from one of the two silent donor loci, HML and HMR, to the expressing locus, MAT. Cell type regulates the selection of the particular donor locus to be utilized during mating type interconversion: MATa cells preferentially select HML alpha and MAT alpha cells preferentially select HMRa. Such preferential selection indicates that the cell is able to distinguish between HML and HMR during mating type interconversion. Accordingly, we designed experiments to identify those features perceived by the cell to discriminate HML and HMR. We demonstrate that discrimination does not derive from the different structures of the HML and HMR loci, from the unique sequences flanking each donor locus nor from any of the DNA distal to the HM loci on chromosome III. Moreover, we find that the sequences flanking the MAT locus do not function in the preferential selection of one donor locus over the other. We propose that the positions of the donor loci on the left and right arms of chromosome III is the characteristic utilized by the cell to distinguish HML and HMR. This positional information is not generated by either CEN3 or the MAT locus, but probably derives from differences in the chromatin structure, chromosome folding or intranuclear localization of the two ends of chromosome III.


Spontaneous amplification of the ADH4 gene in Saccharomyces cerevisiae. Five spontaneous amplifications of the ADH4 gene were identified among 1,894 antimycin A-resistant mutants isolated from a diploid strain after growth at 15 degrees. Four of these amplifications are approximately 40-kb linear extrachromosomal palindromes carrying telomere homologous sequences at each end similar to a previously isolated amplification. ADH4 is located at the extreme left end of chromosome VII, and the extrachromosomal fragments appear to be the fusion of two copies of the end of this chromosome. The fifth amplification is a chromosomal amplification carrying an extra copy of ADH4 on both homologs of chromosome VII. These results suggest that the ADH system can be used to study amplification in Saccharomyces cerevisiae.


The CCR4 protein from Saccharomyces cerevisiae contains a leucine-rich repeat region which is required for its control of ADH2 gene expression. The CCR4 gene from Saccharomyces cerevisiae is required for the transcription of the glucose-repressible alcohol dehydrogenase (ADH2). Mutations in CCR4 also suppress the transcription at the ADH2 and his4-912delta loci caused by defects in the SPT10 (CRE1) and SPT6 (CRE2) genes. The CCR4 gene was mapped to the left arm of chromosome I and cloned by complementation of function using previously isolated segments of chromosome I. DNA sequence analysis of the cloned gene defined CCR4 as a 2511 bp open reading frame that would encode a polypeptide of 837 amino acids. The CCR4 mRNA was found to be 2.8 kb in size and Western analysis identified CCR4 as a 95,000 D protein. Disruption of the CCR4 gene resulted in reduced levels of ADH2 expression under both glucose and ethanol growth conditions and in temperature sensitive growth on nonfermentative medium, phenotypes essentially indistinguishable from previously identified mutations in CCR4. The amino terminus of the CCR4 protein was found to be rich in glutamine residues similar to a number of genes which are required for transcription. More importantly, CCR4 showed similarity to a diverse set of proteins sharing a leucine-rich tandem repeat motif, the presence of which has been implicated in mediating protein-protein interactions. Deletions of several of the five leucine-rich repeats in CCR4 were shown to produce nonfunctional proteins indicating the importance of the repeats to CCR4 activity. This leucine-rich repeat region may mediate the contact CCR4 makes with another factor.


Isolation and characterization of two Saccharomyces cerevisiae genes encoding homologs of the bacterial HexA and MutS mismatch repair proteins. Homologs of the Escherichia coli (mutL, S and uvrD) and Streptococcus pneumoniae (hexA, B) genes involved in mismatch repair are known in several distantly related organisms. Degenerate oligonucleotide primers based on conserved regions of E. coli MutS protein and its homologs from Salmonella typhimurium, S. pneumoniae and human were used in the polymerase chain reaction (PCR) to amplify and clone mutS/hexA homologs from Saccharomyces cerevisiae. Two DNA sequences were amplified whose deduced amino acid sequences both shared a high degree of homology with MutS. These sequences were then used to clone the full-length genes from a yeast genomic library. Sequence analysis of the two MSH genes (MSH = mutS homolog), MSH1 and MSH2, revealed open reading frames of 2877 bp and 2898 bp. The deduced amino acid sequences predict polypeptides of 109.3 kD and 109.1 kD, respectively. The overall amino acid sequence identity with the E. coli MutS protein is 28.6% for MSH1 and 25.2% for MSH2. Features previously found to be shared by MutS homologs, such as the nucleotide binding site and the helix-turn-helix DNA binding motif as well as other highly conserved regions whose function remain unknown, were also found in the two yeast homologs. Evidence presented in this and a companion study suggest that MSH1 is involved in repair of mitochondrial DNA and that MSH2 is involved in nuclear DNA repair.


HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation. Genetic and biochemical evidence shows that a single nuclear gene HTS1 encodes both the mitochondrial and cytoplasmic histidyl-tRNA synthetases (Hts). The gene specifies two messages, one with two in-frame ATGs (-60 and +1) and another with only the downstream ATG (+1). We have made a new set of mutations that enables us to express only the mitochondrial or the cytoplasmic form and compared the subcellular distribution of the Hts1 protein in these mutants and wild type, using an antibody that interacts with both the mitochondrial and cytoplasmic Hts1 as well as Hts1::LacZ fusions. Mutations in the upstream ATG (-60) or frameshift mutations in the presequence affect only the mitochondrial enzyme and not the cytoplasmic enzyme. Mutations in the downstream ATG (+1 ATG to ATC) destroy the function of the cytosolic enzyme, but do not affect the function of the mitochondrial enzyme. Overexpression of this construct restores cytoplasmic function. Cells expressing a truncated form of Hts containing a deletion of the first 20 amino-terminal residues (Htsc) produce a functional cytoplasmic enzyme, which does not provide mitochondrial function. Overexpression of this truncated cytoplasmic protein provides mitochondrial function and produces detectable levels of the synthetase in the mitochondrion. These experiments suggest that Hts1 contains two domains that together allow efficient localization of Htsm to the mitochondrion: an amino-terminal presequence in the mitochondrial precursor that is likely cleaved upon delivery to the mitochondrion and a second amino-terminal sequence (residues 21-53) present in both the precursor and the cytoplasmic form. Neither one by itself is sufficient to act as an efficient mitochondrial targeting signal. Using our antibody we have been able to detect a protein of increased molecular mass that corresponds to that of the predicted precursor. Taken together these studies show that the specificity of compartmentation of the Hts protein depends upon both the primary sequence and the concentration of the protein in the cell.


Loss of N-myc function results in embryonic lethality and failure of the epithelial component of the embryo to develop. myc genes are thought to function in the processes of cellular proliferation and differentiation. To gain insight into the role of the N-myc gene during embryogenesis, we examined its expression in embryos during postimplantation development using RNA in situ hybridization. Tissue- and cell-specific patterns of expression unique to N-myc as compared with the related c-myc gene were observed. N-myc transcripts become progressively restricted to specific cell types, primarily to epithelial tissues including those of the developing nervous system and those in developing organs characterized by epithelio-mesenchymal interaction. In contrast, c-myc transcripts were confined to the mesenchymal compartments. These data suggest that c-myc and N-myc proteins may interact with different substrates in performing their function during embryogenesis and suggest further that there are linked regulatory mechanisms for normal expression in the embryo. We have mutated the N-myc locus via homologous recombination in embryonic stem (ES) cells and introduced the mutated allele into the mouse germ line. Live-born heterozygotes are under-represented but appear normal. Homozygous mutant embryos die prenatally at approximately 11.5 days of gestation. Histologic examination of homozygous mutant embryos indicates that several developing organs are affected. These include the central and peripheral nervous systems, mesonephros, lung, and gut. Thus, N-myc function is required during embryogenesis, and the pathology observed is consistent with the normal pattern of N-myc expression. Examination of c-myc expression in mutant embryos indicates the existence of coordinate regulation of myc genes during mouse embryogenesis.


Embryonic lethality in mice homozygous for a targeted disruption of the N-myc gene. The N-myc gene encodes a putative transcription factor that is thought to function in the regulation of gene expression during cell differentiation and/or growth. To examine the role of N-myc during development, we have used targeted mutagenesis in embryonic stem cells to produce a mouse line that carries an N-myc null allele. Mice homozygous for the mutation died between 10.5 and 12.5 days of gestation. Histological analysis of mutant embryos revealed that organs and tissues expected at these stages of development were present. However, multiple defects were observed, primarily in tissues and organs that normally express N-myc. In particular, mutant hearts were underdeveloped, often retaining the S-shape more typical of 9-day-old embryos. In addition, cranial and spinal ganglia were reduced in size and/or cellularity. Most of the noted defects were more consistent with a role of N-myc in proliferation of precursor populations than with a block in differentiation per se, at least at these early stages. These results demonstrate that N-myc plays an essential role during development and clearly confirm that N-myc has a physiological function that is distinct from that of the other myc-family genes.


Mechanism of transcriptional antirepression by GAL4-VP16. Promoter- and enhancer-binding factors appear to function by facilitating the transcription reaction as well as by counteracting chromatin-mediated repression (antirepression). We have examined the mechanism by which a hybrid activator, GAL4-VP16, is able to counteract histone H1-mediated repression by using both H1-DNA complexes and reconstituted H1-containing chromatin templates. The GAL4 DNA binding domain alone was sufficient to disrupt local H1-DNA interactions, but a transcriptional region was additionally necessary for antirepression. GAL4-VP16-mediated antirepression required an auxiliary factor, denoted as a co-antirepressor, which was partially purified from Drosophila embryos. We have found that the co-antirepressor activity was sensitive to digestion with RNase A. Moreover, total RNA from Drosophila embryos could partially substitute for the co-antirepressor fraction, which indicated that the co-antirepressor may function as a histone acceptor ("histone sink"). These findings suggest a model for gene activation in which sequence-specific transcription factors disrupt H1-DNA interactions at the promoter to facilitate transfer of H1 to a histone acceptor, which then allows access of the basal transcription factors to the DNA template.


Initiation on chromatin templates in a yeast RNA polymerase II transcription system. Templates were prepared with either the TATA box or transcription start sites of the yeast CYC1 promoter in a nucleosome. In both cases, initiation in an unfractionated yeast RNA polymerase II transcription system was abolished by the nucleosome. The inhibition appeared to be relieved by the activator protein Gal4-VP16 binding to a site upstream of the promoter. Inhibition was not relieved, however, in a transcription system reconstituted from purified components, indicating a requirement for additional factors for the effect of Gal4-VP16.


Evidence that SNF2/SWI2 and SNF5 activate transcription in yeast by altering chromatin structure. Changes in chromatin structure have frequently been correlated with changes in transcription. However, the cause-and-effect relationship between chromatin structure and transcription has been hard to determine. In addition, identifying the proteins that regulate chromatin structure has been difficult. Recent evidence suggests that a functionally related set of yeast transcriptional activators (SNF2/SWI2, SNF5, SNF6, SWI1, and SWI3), required for transcription of a diverse set of genes, may affect chromatin structure. We now present genetic and molecular evidence that at least two of these transcriptional activators, SNF2/SWI2 and SNF5, function by antagonizing repression mediated by nucleosomes. First, the transcriptional defects in strains lacking these SNF genes are suppressed by a deletion of one of the two sets of genes encoding histones H2A and H2B, (hta1-htb1) delta. Second, at one affected promoter (SUC2), chromatin structure is altered in snf2/swi2 and snf5 mutants, and this chromatin defect is suppressed by (hta1-htb1) delta. Finally, analysis of chromatin structure at a mutant SUC2 promoter, in which the TATA box has been destroyed, demonstrates that the differences in SUC2 chromatin structure between SNF5+ and snf5 mutant strains are not simply an effect of different levels of SUC2 transcription. Thus, these results strongly suggest that SNF2/SWI2 and SNF5 cause changes in chromatin structure and that these changes allow transcriptional activation.


Drosophila C/EBP: a tissue-specific DNA-binding protein required for embryonic development. Recently, we reported the cloning of the Drosophila melanogaster homolog of the vertebrate CCAAT/enhancer-binding protein (C/EBP). Here, we describe studies of the DNA-binding and dimerization properties of Drosophila C/EBP (DmC/EBP), as well as its tissue distribution, developmental regulation, and essential role in embryonic development and conclude that it bears functional as well as structural similarity to mammalian C/EBP. DmC/EBP contains a basic region/leucine zipper (bZIP) DNA-binding domain very similar to that of mammalian C/EBP and the purified C/EBPs bound to DNA with the same sequence specificity. Among the DNA sequences that DmC/EBP bound with high affinity was a conserved site within the promoter of the DmC/EBP gene itself. In vitro, DmC/EBP and mammalian C/EBP specifically formed functional heterodimers; however, as we found no evidence for a family of DmC/EBPs, DmC/EBP may function as a homodimer in vivo. The DmC/EBP protein was expressed predominantly during late embryogenesis in the nuclei of a restricted set of differentiating cell types, such as the lining of the gut and epidermis, similar to the mammalian tissues that express C/EBP. We have characterized mutations in the DmC/EBP gene and found that deleting the gene caused late embryonic lethality. Embryos that lack C/EBP die just before or just upon hatching. The lethal phenotype of C/EBP mutants can be rescued with the cloned C/EBP gene introduced by P-element-mediated germ-line transformation. The strict requirement for C/EBP during Drosophila embryogenesis, coupled with its structural and functional similarities to mammalian C/EBP, provides a useful genetic system in which to study the role of C/EBP in development.


Pumilio is essential for function but not for distribution of the Drosophila abdominal determinant Nanos. The Drosophila gene pumilio is expressed maternally, and its function is essential during early embryogenesis for the formation of abdominal segments. Our molecular analysis reveals that pumilio is a large gene that encodes a protein of 160 kD whose RNA is enriched at the posterior pole of the egg. As with pumilio, the maternal effect gene nanos is specifically required for abdomen formation. The Nanos protein is expressed in a posterior-to-anterior concentration gradient in the developing embryo. Previous experiments demonstrated a genetic interaction between pumilio and nanos, and led to the suggestion that pumilio is required for the proper spatial distribution of the Nanos protein. Here, we show that the expression and distribution of nanos RNA and protein in embryos derived from pumilio mutant females are indistinguishable from wild type. We conclude that abdomen formation depends both on Nanos activity, spreading from the localized posterior source, and on Pumilio activity, present throughout the embryo.


Transcription factors junB and c-jun are selectively up-regulated and functionally implicated in fibrosarcoma development. Bovine papillomavirus transgenic mice develop skin tumors arising from dermal fibroblasts in a process comprised of three distinctive stages: mild and aggressive fibromatoses, and fibrosarcoma. In both tissue biopsies and derivative cell lines, the proto-oncogenes junB and c-jun are induced in the latter two stages, in contrast to junD and fos. Fibrosarcoma cell lines have increased AP-1 DNA-binding activity. Overexpression of junB or c-jun by transfection into the mild fibromatosis stage elicited changes in cell shape and anchorage independence, whereas junD did not. Similar transfections of normal skin fibroblasts had no effect. Thus, junB and c-jun represent progression factors whose activities are necessary at an intermediate stage of tumor development, subsequent to the initiation of aberrant proliferation.


The proto-oncogene bcl-3 encodes an I kappa B protein. The bcl-3 gene product, overexpressed in chronic lymphocytic leukemia (CLL) patients with the translocation t(14;19), is a member of the I kappa B family. The bcl-3 protein is able to inhibit the DNA binding and trans-activation of authentic NF-kappa B heterodimers p50-p65 and p49-p65, as well as p50 and p49 homodimers. The bcl-3 protein does not inhibit either the DNA-binding activity of the Rel protein or its ability to trans-activate genes linked to the kappa B site. A human 37-kD protein (I kappa B alpha), identified previously as a member of the I kappa B family, is also unable to inhibit DNA-binding activity of the Rel protein. However, unlike bcl-3, the 37-kD (I kappa B alpha) protein has no effect on the DNA-binding activity of p50 or p49 homodimers. Two dimensional phosphotryptic peptide maps of the human bcl-3 and the human 37-kD (I kappa B alpha) proteins reveal that the phosphopeptides from the 37-kD (I kappa B alpha) protein are nested within the bcl-3 protein. Furthermore, bcl-3 antisera immunoprecipitates an in vitro-radiolabeled 37-kD (I kappa B alpha) protein. Proteins of 56 and 38 kD can be identified in HeLa cells stimulated with PMA and immunoprecipitated with bcl-3 antisera. Comparison of tryptic peptide maps of the bcl-3 protein synthesized in vitro, and p56 and p38 from HeLa cells, shows that they are all structurally related. Removal of the amino-terminal sequences of the bcl-3 protein generates a protein that inhibits the DNA binding of the p50-p65 heterodimer but, like the 37-kD (I kappa B alpha) protein, is no longer able to inhibit the binding of the p50 and p49 homodimers with kappa B DNA. We propose that the bcl-3 and 37-kD (I kappa B alpha) proteins are related and are members of the I kappa B family.


Serine-to-alanine substitutions at the amino-terminal region of phytochrome A result in an increase in biological activity. We have used a tobacco transgenic plant system to assay the structure/function relationship of phytochrome A (phyA), a plant photoreceptor. The amino terminus of phyA from different plant species is very rich in serine residues. To investigate whether these serine residues are required for phytochrome function, the first 10 serine codons encoding amino acid residues 2-4, 10-14, 19, and 20 in the amino-terminal domain of the rice phyA gene (phyA) were changed to alanine codons. The mutant (S/A phyA), as well as the wild-type phyA cDNA, was placed under the control of the 35S promoter, and the chimeric genes were transferred into the tobacco genome by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing either wild-type or S/A phyA showed similar phenotypic alterations, including dwarfism and dark-green leaves. However, hypocotyl elongation experiments revealed that transgenic seedlings expressing S/A phyA showed a higher amplitude of the red light response with respect to the inhibition of hypocotyl elongation. The observed difference is not correlated with expression levels of the transgene. The chromophore is attached to the mutant phyA apoprotein (PHY A), and the mutant photoreceptor is photoreversible, giving a difference spectrum indistinguishable from that of the rice phyA. Our results indicate that the S/A mutant has a higher biological activity as compared with the wild-type rice phyA.


Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora. The surface of many fungal spores is covered by a hydrophobic sheath termed the rodlet layer. We have determined that the rodlet protein of Neurospora crassa is encoded by a cloned gene designated bli-7, and that bli-7 is identical to the known gene eas (easily wettable). Using eas DNA as a probe we show that eas mRNA is abundant in illuminated mycelia and conidiophores but is not detectable or is barely detectable in dark-grown mycelia, mature macroconidia, microconidia, and ascospores. Mutations in the genes acon-2, acon-3, and fl block early conidiophore development; of these, only fl prevents normal eas transcription. The EAS protein is homologous to the rodlet protein (RodA) of Aspergillus nidulans, and the hydrophobins of Schizophyllum commune. eas is the first cloned conidiation (con) gene of N. crassa that is associated with a phenotypic alteration.


The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer. The Neurospora crassa clock-controlled gene (ccg-2) is transcriptionally activated by the circadian clock in a time-of-day-specific manner. Transcript and sequence analyses of ccg-2 reveal that the predicted Ccg-2 polypeptide bears significant similarity to a class of low-molecular-weight, cysteine-rich, hydrophobic proteins (hydrophobins), first identified in Schizophyllum, and including the product of the developmentally regulated Aspergillus gene, rodletless, required for spore surface rodlets. Allelism between ccg-2 and easily wettable (eas) (one of the first developmental genetic loci identified in Neurospora) was predicted on the basis of this similarity, their close genetic linkage, and previous findings demonstrating that eas mutants lack rodlets. In this study allelism is confirmed experimentally by showing that (1) transformation of an eas mutant strain with ccg-2 DNA results in phenotypic complementation, including restoration of surface rodlets, (2) inactivation of the ccg-2 gene, by RIP, results in an eas phenotype including loss of rodlet fascicles, and (3) the original eas strain has dramatically reduced levels of ccg-2 mRNA. Thus, the clock-controlled ccg-2 gene encodes an integral component of fungal asexual spores important for spore dispersal. The dramatic reduction of ccg-2 expression in the eas mutant has no apparent effect on the normal operation of the circadian clock, confirming that there is no feedback of this clock output on the oscillator itself. These data, in conjunction with the previous observation that ccg-2 is light induced, serve to focus attention on the dual interacting role of light and the circadian clock in the regulation of fungal spore development.


A temporally controlled sigma-factor is required for polar morphogenesis and normal cell division in Caulobacter. The transcription of many spatially and temporally controlled flagellar structural genes in Caulobacter requires the RNA polymerase sigma 54 subunit. Like flagellar biogenesis, stalk formation is an asymmetric polar morphogenesis that occurs once each cell cycle in response to internal cell cycle signals. We have isolated the sigma 54 gene (rpoN) and describe here a novel role for this alternative sigma-factor in cell differentiation: It is required for the biogenesis of both polar structures, and the disruption of the rpoN gene results in aberrant cell division. Surprisingly, the transcription of rpoN is temporally regulated during the cell cycle; it increases 10-fold commensurate with stalk formation and just before the onset of flagellar gene expression. These results suggest that sigma 54 abundance responds to cell cycle cues and is involved in the global timing of the central events of Caulobacter development, whereas the transcriptional activators of sigma 54-dependent promoters are responsible for the refined control of the expression of individual or small groups of genes required for each specific event.


Transcriptional control via translational repression by c4 antisense RNA of bacteriophages P1 and P7. The c4 repressors of bacteriophages P1 and P7 are antisense RNAs that inhibit antirepressor synthesis. This antisense inhibition is unusual in that the c4 repressor and the repressed genes orfx and ant are cotranscribed in that order from the same promoter, and c4 RNA is processed from a precursor RNA. Here, we show that c4 RNA directly represses translation of orfx, a small open reading frame, to which ant is translationally coupled. This translational repression blocks ant transcription via a rho-dependent terminator. Thus, c4 RNA controls expression of the ant gene by a novel indirect mechanism combining translational repression, translational coupling, and rho-dependent termination.


Biphasic effect of Max on Myc cotransformation activity and dependence on amino- and carboxy-terminal Max functions. In Ras cotransformation assays, Max exhibited a biphasic effect on Myc transformation activity. Cotransfection of low levels of Max expression plasmid stimulated Myc transformation activity, but cotransfection of high levels suppressed it. Mutations in the functionally undefined Max amino- and carboxy-terminal regions outside of the B/HLH/LZ motif partly separated these activities, suggesting various modes of Max regulation. We demonstrate that the Max protein is a nuclear protein in vivo and identify a carboxy-terminal region similar to nuclear localization signals whose integrity is necessary for efficient localization. Two mutants that delete amino- or carboxy-terminal consensus signals for casein kinase II (CKII) exhibited altered gel mobility and DNA-binding potential in vitro and showed modified transforming potential in the Ras cotransformation assay, suggesting that CKII or a CKII-related enzyme may regulate Max function in vivo. Our data suggest that both the ratio of Myc/Max hetero-oligomers to Max homo-oligomers and Max-specific regulation can contribute to determining the biological activity of Myc in vivo.


Putting the heat on sex determination.

Putting the heat on sex determination. Sex determination and differentiation are inherently fascinating to both layperson and geneticist. Major advances have accelerated interest in the molecular genetic events mediating these processes in nematodes, flies, mice and humans. Far less attention has been paid to those organisms, particularly reptiles, where sex is determined by environmental cues. However, recent experimental evidence suggests that the two modes of sex determination may not only share common genetic elements, but may also be regulated by similar mechanisms. We argue that the ability to manipulate sex by temperature provides a particularly suitable model for exploring the molecular basis of this fundamental biological process.


Evolution of pericentromeric heterochromatin of human X chromosome. An unusual large heterochromatic segment around the pericentromeric region of the X-chromosome is reported. In normal circumstances, the pericentromeric region of the X-chromosome is negative by the restriction endonuclease AluI/Giemsa technique. However, this unusual X-chromosome was found to have AluI resistant (positive) chromatin. The evolution of extra heterochromatin is a postzygotic event as substantiated by the presence of a normal cell line.


Nucleotide sequence analysis of a mouse Y chromosomal DNA fragment containing Bkm and LINE elements. The strong suppression of crossing-over between the X and Y chromosomes permits rapid accumulation of repetitive sequences in the Y chromosome. To gain insight into the mechanism responsible for the sequence amplification, it is essential to characterize Y chromosomal repetitive sequences at the molecular level. Here, we report the entire nucleotide sequence (3,902bp) of AC11, a mouse sequence that is repeated 300 times in the Y chromosome. AC11 is AT rich (32.8% GC), and contains many short poly(A) sequences. In addition, it has Bkm and LINE sequences as well as a Y chromosome-specific sequence. The Bkm sequence consists of typical (GATA) and (GACA) repeating units, whereas the LINE sequence deviates considerably from other mouse LINE sequences (71-76% identity) and may be considered atypical. The Y chromosome-specific region seems to be unique and does not identify similar sequences in the GenBank library. The information obtained from the nucleotide sequence should form the foundation to study the evolutionary processes through which AC11-related sequences have accumulated in the mouse Y chromosome.


Accumulation of amyloid precursor protein and beta-protein immunoreactivities in axons injured by cerebral infarct. To determine the distribution of amyloid precursor protein (APP), monoclonal antibodies against APP45-62 (APP1-28-9) and beta1-17 (4A18 and 4A61) were produced. In the sections of Alzheimer's disease (AD) brain, APP1-28-9 was reactive with neurites around senile plaques and a few neurons but not with amyloid cores. This antibody also immunostained the axons in the ischemic lesions of brain tissues from cases with cerebral infarct. 4A18 and 4A61 were reactive with amyloid plaques but not with neurites and neurons. The latter two antibodies also immunostained axons in ischemic lesions. These findings suggest that APP, transported by the fast axonal flows, accumulated in the injured axons in the central nervous system. The beta immunoreactivity appearing in those axons may provide a clue to the mechanism of amyloidogenesis.


Alzheimer's disease beta-amyloid precursor protein in rat neural cells in culture. Immunochemical studies were performed on Alzheimer's beta-amyloid precursor protein (APP) in rat brain and cultured rat neural cells. Multiple APP subtypes were detected on immunoblots of brain homogenate with several antisera specific for subsequences of APP. In rat neural cell cultures, it was demonstrated that the composition of APP subtypes differed among cell types and subcellular fractions, and that APP subtypes in PC12h cells varied in their heparin binding affinity, suggesting distinct functional roles for different APP subtypes. Compatible with the possible role of APP in cell-matrix interaction, an increase in oligodendroglial APP was observed following their attachment onto poly-L-lysine substratum.


Circulating suppressing factor for the muscarinic acetylcholine receptor in patients with senile dementia of the Alzheimer type. Circulating suppressing factor for the binding of quinuclidinyl benzilate (QNB), an antagonist for the muscarinic acetylcholine receptor, to the synaptic membranes was evaluated in 48 patients with senile dementia of the Alzheimer type (SDAT), in 17 patients with the vascular type dementia (VTD) and in 11 nondemented elderly subjects (NE). The mean suppression rate on the binding in the SDAT group was significantly greater than that in the NE group, although that in the VTD group was similar to that in the NE group. Moreover, the percent QNB binding was significantly (p < 0.05) correlated with the score of the mini-mental state in the SDAT group. The circulating suppressing factor may participate in the pathogenesis of SDAT.


Production of endothelin-1 in vascular endothelial cells is regulated by factors associated with vascular injury. We studied the effects of factors associated with vascular injury on the production of endothelin-1 (ET-1) in cultured porcine aortic endothelial cells. ET-1 mRNA expression and peptide production in endothelial cells were increased by thrombin, transforming growth factor beta 1 (TGF beta 1), interleukin-1 (IL-1) and tumor necrotizing factor alpha (TNF alpha). The analysis of the enhancer/promoter region of the human ET-1 gene showed that the ET-1 gene transcription is regulated in a cell-specific manner and is activated by thrombin, TGF beta 1 and IL-1. These results suggest that the production of ET-1 in endothelial cells is regulated by factors associated with platelet aggregation, macrophage infiltration and the formation of atherosclerotic lesions.


Expression of glucose transporter isoforms with aging. To elucidate the cellular mechanisms for impairment of glucose metabolism associated with aging, the facilitative glucose transporter protein and mRNA were studied in various tissues of young (7-week-old) and aged (20-month-old) rats. GLUT4 glucose transporter protein, a major glucose transporter isoform in the insulin-responsive tissues, was selectively decreased in the epididymal fat tissues of the aged rats compared with the young rats. This decrease is likely to be due to a decrease in protein synthesis rather than in protein stability, since GLUT4 mRNA per unit cellular total RNA was also decreased. GLUT4 mRNA in the skeletal muscle was rather increased in spite of the decreased level of GLUT4 protein in the aged rats, suggesting that the translational efficiency and/or stability of GLUT4 protein is decreased in the skeletal muscle of the aged rats compared with the young rats. In contrast to these alterations in GLUT4 expression, no apparent decrease in the GLUT1 protein amount was observed in the fat tissues, skeletal muscle and brain of the aged rats compared with the young rats. Thus, the tissue and isoform-specific alterations in glucose transporter expression are associated with aging and may contribute to impairment of glucose metabolism observed with aging.


Human platelet-derived transforming growth factor-beta stimulates synthesis of glycosaminoglycans in cultured porcine aortic endothelial cells. Human platelet-derived transforming growth factor-beta (TGF-beta) stimulated the incorporation of [35S]sulfate into glycosaminoglycans (GAGs) both in the medium and on the cell surface of cultured aortic endothelial cells in a dose- and time-dependent manner. TGF-beta inhibited the suppression of GAG synthesis by other cytokines. TGF-beta increased the protein synthesis, while reducing the DNA synthesis by endothelial cells. The proportion of anticoagulant heparan sulfate in total GAGs was reduced. Thus, TGF-beta affected the endothelial GAG metabolism both quantitatively and qualitatively.


Genetic and environmental factors of bone mineral density indicated in Japanese twins. To evaluate the effects of genetic and environmental factors on the bone mass, we determined the bone mineral density (BMD) in the total body, the lumbar spine and in the femoral neck in 23 Japanese twin pairs including 21 monozygotic (MZ) pairs, applying dual-energy X-ray absorptiometry. In MZ pairs aged 20-49 years, highly significant intraclass correlation coefficients, ranging from 0.775 to 0.926, were observed at several sites, including the lumbar spine and the femoral neck, which suggests considerable contributions of genetic factors to the BMD. Neither intraclass correlation coefficients nor intrapair differences were found to change with increasing age in the present analysis. In female MZ pairs, exercises both at the present time and in the past were correlated with the BMD at several sites as well as body mass index in multiple regression analyses.


Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage. The stable free radical Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) has been shown to protect against X-ray-induced cytotoxicity and hydrogen peroxide- or xanthine oxidase-induced cytotoxicity and mutagenicity. The ability of Tempol to protect against X-ray- or neocarzinostatin (NCS)-induced mutagenicity or DNA double-strand breaks (dsb) was studied in Chinese hamster cells. Tempol (50 mM) provided a protection factor of 2.7 against X-ray-induced mutagenicity in Chinese hamster ovary (CHO) AS52 cells, with a protection factor against cytotoxicity of 3.5. Using the field inversion gel electrophoresis technique of measuring DNA dsb, 50 mM Tempol provides a threefold reduction in DNA damage at an X-ray dose of 40 Gy. For NCS-induced damage, Tempol increased survival from 9% to 80% at 60 ng/mL NCS and reduced mutation induction by a factor of approximately 3. DNA dsb were reduced by a factor of approximately 7 at 500 ng/mL NCS. Tempol is representative of a class of stable nitroxide free radical compounds that have superoxide dismutase-mimetic activity, can oxidize metal ions such as ferrous iron that are complexed to DNA, and may also detoxify radiation-induced organoperoxide radicals by competitive scvenging. The NCS chromophore is reduced by sulfhydryls to an active form. Electron spin resonance (ESR) spectroscopy shows that 2-mercaptoethanol-activated NCS reacts with Tempol 3.5 times faster than does unactivated NCS. Thus, Tempol appears to inactivate the NCS chromophore before a substantial amount of DNA damage occurs.


MDA, oxypurines, and nucleosides relate to reperfusion in short-term incomplete cerebral ischemia in the rat. Short-term incomplete cerebral ischemia (5 min) was induced in the rat by the bilateral clamping of the common carotid arteries. Reperfusion was obtained by removing carotid clamping and was carried out for the following 10 min. Animals were sacrificed either at the end of ischemia or reperfusion. Controls were represented by a group of sham-operated rats. Peripheral venous blood samples were withdrawn from the femoral vein from rats subjected to cerebral reperfusion 5 min before ischemia, at the end of ischemia, and 10 min after reperfusion. Neutralized perchloric acid extracts of brain tissue were analyzed by a highly sensitive high-performance liquid chromatography (HPLC) method for the direct determination of malondialdehyde, oxypurines, nucleosides, nicotinic coenzymes, and high-energy phosphates. In addition, plasma concentrations of malondialdehyde, hypoxanthine, xanthine, inosine, uric acid, and adenosine were determined by the same HPLC technique. Incomplete cerebral ischemia induced the appearance of a significant amount (8.05 nmol/g w.w.; SD = 2.82) of cerebral malondialdehyde (which was undetectable in control animals) and a decrease of ascorbic acid. A further 6.6-fold increase of malondialdehyde (53.30 nmol/g w.w.; SD = 17.77) and a 18.5% decrease of ascorbic acid occurred after 10 min of reperfusion. Plasma malondialdehyde, which was present in minimal amount before ischemia (0.050 mumol/L; SD = 0.015), significantly increased after 5 min of ischemia (0.277 mumol/L; SD = 0.056) and was strikingly augmented after 10 min of reperfusion (0.682 mumol/L; SD = 0.094). A similar trend was observed for xanthine, uric acid, inosine, and adenosine, while hypoxanthine reached its maximal concentration after 5 min of incomplete ischemia, being significantly decreased after reperfusion. From the data obtained, it can be concluded that tissue concentrations of malondialdehyde and ascorbic acid, and plasma levels of malondialdehyde, oxypurines, and nucleosides, reflect both the oxygen radical-mediated tissue injury and the depression of energy metabolism, thus representing early biochemical markers of short-term incomplete brain ischemia and reperfusion in the rat. In particular, these results suggest the possibility of using the variation of malondialdehyde, oxypurines, and nucleosides in peripheral blood as a potential biochemical indicator of reperfusion damage occurring to postischemic tissues.


Breath ethane: a specific indicator of free-radical-mediated lipid peroxidation following reperfusion of the ischemic liver. A major component of the organ injury mediated by toxic oxidants, such as seen following reperfusion of the ischemic liver, is due to the peroxidation of polyunsaturated fatty acids, especially of cell membranes. We utilized the measurement of exhaled breath ethane, a metabolic product unique to oxidant-mediated lipid peroxidation, as a noninvasive indicator of this process in swine liver subjected to warm ischemia/reperfusion. Under rigorously controlled anesthesia conditions, pig livers were subjected to 2 h of warm total ischemia, followed by reperfusion in situ. Expired air was collected and its ethane content quantitated by a novel gas chromatographic technique. The time course of breath ethane generation correlated closely with the appearance of hepatocellular injury as measured by impairment of Factor VII generation and other measures of liver integrity. Moreover, the administration of the specific superoxide free radical scavenger, superoxide dismutase (SOD), significantly attenuated both the elaboration of ethane and the hepatocellular injury. These findings not only provide confirmation of the previously reported link between hepatocellular injury by free radicals generated at reperfusion, but also establish the use of expired breath ethane analysis as a sensitive, specific, and noninvasive indicator of the injury process in real time.


The inhibitory effects of 21 mimics of superoxide dismutase on luminol-mediated chemiluminescence emitted from PMA-stimulated polymorphonuclear leukocyte. Four groups comprising 21 superoxide dismutase (SOD) mimics synthesized by us were comparatively studied for their inhibitory effects on luminol-mediated chemiluminescence emitted from phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocyte (PMNL). Among these groups, 20-membered macrocyclic bicopper(II) complexes and 13-membered macrocyclic dioxotetramine copper(II) complexes exhibited relatively higher activities of scavenging reactive oxygen species (ROS) produced by PMA-stimulated PMNL as compared with polyamine Cu(II)-Zn(II) complexes and copper(II) complexes of bis-shiff-base. Moreover, distinctly different effects of SOD mimics in the biological system have been found even in the same group. It is suggested that the biological effects of some SOD mimics are related to their structures.


Fenton reagents may not initiate lipid peroxidation in an emulsified linoleic acid model system. This study includes two parts. First, the Fe2+ autooxidation and chelation processes in the presence of the chelators ethylenediaminetetraacetic acid (EDTA) and diethylenetriamine pentaacetic acid (DTPA) were studied by measuring UV light absorbance alterations. Competition for Fe3+ between chelators and water or phosphate buffer (PB) ions was confirmed. The addition of EDTA or DTPA to Fe3+ in water or PB only slowly turned the water/PB-Fe3+ complexes to EDTA-Fe3+ or DTPA-Fe3+ complexes. In the second part of this study, the initiation mechanisms of Tween 20 emulsified linoleic acid peroxidation under stimulation by chelator-Fe-O2 complexes were studied by measuring changes in UV light absorbance following diene conjugation. Fe3+ in the presence of EDTA or DTPA did not stimulate diene conjugation. Fe2+ (0.10 mM) and EDTA (0.11 mM) stimulated diene conjugation of the linoleic acid emulsion, but only after apparent Fe2+ autooxidation. Fe2+ and DTPA, as well as premixed DTPA-Fe2+ complex, resulted in very fast diene conjugation in a wide range of concentrations. A nonlinear, mainly square root relation between Fe2+ concentration and peroxidation rate was noted. Superoxide dismutase (SOD), catalase, and mannitol did not prevent the lipid peroxidation. H2O2 substantially decreased the DTPA-Fe2+ stimulated, otherwise rapid, diene conjugation but slightly enhanced the slower one stimulated by EDTA-Fe2+. Without ambient oxygen, Fenton reagents did not result in .H abstraction-related diene conjugation. The findings suggest that .OH resulting from Fenton reagents may not be the main cause for the initiation of peroxidation in this model system. Furthermore, a study with different combinations of Fe2+ and Fe3+ did not support the Fe2+/Fe3+ (1:1) optimum ratio hypothesis. We therefore conclude that perferryl ions or chelator-Fe-O2 complexes may be responsible for the first-chain initiation of lipid peroxidation, at least in this model system.