Public health significance of upper body adiposity for non-insulin dependent diabetes mellitus in Mexican Americans. An unfavourable body fat distribution has been associated with an increased prevalence and incidence of non-insulin dependent diabetes mellitus (NIDDM). The potential utility of assessing body fat distribution in diabetes screening, however, has not been assessed. We compared the impact of upper body fat distribution (assessed by the waist-to-hip ratio (WHR)) and body mass index (BMI) and NIDDM using the population attributable risk approach of Levin in 1965 Mexican Americans from the San Antonio Heart Study, a population-based study of diabetes and cardiovascular disease. The population attributable risk percentage (PAR%) was 52.0% for WHR compared to 43.4% for body mass index. After stratification by BMI, women with a high WHR had a PAR% of approximately 50% and men had a PAR% of 28-58%. For any given cutpoint (e.g. the 10th percentile, 20th percentile, etc.) of WHR used to screen for NIDDM, WHR had both a higher sensitivity and a lower false positive rate than the corresponding cutpoint of BMI. To evaluate the relative contribution of WHR in identifying prevalent cases of NIDDM, multiple logistic regression analyses were performed, and the number of subjects identified as being in the top 20% of the risk score distribution was compared using a model that included WHR and a model that included BMI. In men, BMI did not increase the sensitivity in detecting NIDDM subjects once age was accounted for; WHR increased the sensitivity only slightly. In women, sensitivity was enhanced modestly using both measures, although WHR again was the more sensitive method. These data suggest that WHR is a better single screening measure for NIDDM than BMI.


Resistance to insulin mediated glucose disposal in obese subjects: respective effect of lipid metabolism and glycemia. The present study was designed to assess the respective effect of altered lipid metabolism and hyperglycemia on glucose metabolism in vivo in obese subjects. Six young obese non-diabetic volunteers were studied on four occasions during hyperinsulinemic clamp, twice during euglycemia and twice during hyperglycemia, with or without the infusion of beta-pyridylcarbinol, an inhibitor of lipid metabolism. Glucose oxidation was calculated from continuous respiratory exchange measurements, and glucose storage was obtained as the difference between total glucose disposal and glucose oxidation. Two-way analysis of variance (with interaction term) demonstrated (i) a significant increase for total glucose disposal with beta-pyridylcarbinol but no significant effect of hyperglycemia and no interaction between the two treatments, and (ii) an important increase of beta-pyridylcarbinol to enhance glucose storage but no significant effect of hyperglycemia and no interaction between the two treatments. These results show that obese people, at physiological insulinemia, enhance their glucose disposal and glucose storage when lipid oxidation is artificially lowered. This suggests that enhanced lipid oxidation is related to insulin resistance in these patients. However, hyperglycemia in these patients failed to compensate for defective glucose disposal or storage.


Fluoxetine treatment of the obese diabetic. Fluoxetine, an inhibitor of serotonin re-uptake, has been shown to cause weight loss in humans and animals. In order to determine the effects in diabetic subjects, 48 male and female, obese, type 2 non-insulin dependent diabetics being treated with insulin were randomized to receive fluoxetine 60 mg or placebo once daily in double blind fashion for 24 weeks. In all subjects, this treatment was preceded by four weeks and followed by six weeks of single blind placebo washout treatment. Subjects performed daily home glucose monitoring and were given instruction in a 1200 kcal American Diabetes Association diet. Fluoxetine treated subjects who completed the trial (n = 16) lost more weight than placebo treated subjects (n = 20) (9.3 +/- 2.4 vs. 1.9 +/- 2.9 kg +/- s.e.m, P less than 0.05). Subjects in the fluoxetine group also showed a greater percentage decrease in insulin dose than those in the placebo group (46.9 +/- 7.6% vs. 19.3 +/- 7.6%, P less than 0.01). During active treatment, the change in serum glucose levels did not differ between the two groups, while glycohemoglobin fell more in fluoxetine treated subjects than in placebo treated subjects at two of four follow-up visits. These results suggest that fluoxetine may be of benefit in the treatment of obese patients with type 2 non-insulin dependent diabetes mellitus.


History of body weight and physical activity of elderly women differing in current physical activity. Development of overweight and physical activity during life was studied retrospectively in a group of physically active and a group of sedentary elderly women. The two groups of elderly women were selected based on a validated physical activity questionnaire. A previous study on their current dietary intake and nutritional status showed a 12 kg higher body weight in the sedentary group compared to the physically active group, whereas body height did not differ. In order to study the relationship between development of overweight and physical activity in these elderly women, a retrospective study was carried out in 45 subjects. Information about former physical activity was collected by means of a detailed structured interview. Information about body shape and fatness, expressed as 'weight index', was obtained using silhouettes and subjective rating of obesity of subjects compared with peers. Classification of obesity was checked by old photographs rated by interviewers, sizes of clothing and recalled body weight and height. Information was collected about the situation at age 12, 25, 40 and 55 years whereas the mean current age is 71. Weight index was statistically significantly different between the active and sedentary group from age 25 onwards (P less than 0.05). Photographs proved to be useful for a valid and objective categorization. No differences were found in physical activity scores between the groups at age 12, 25, 40 and 55 years. It is concluded that the current difference in body shape and fatness between physically active and inactive elderly women was already present at age 25 and persisted throughout their adult life.(ABSTRACT TRUNCATED AT 250 WORDS)


The prediction of total body water from body impedance in young obese subjects. Almost all formulae for the prediction of total body water (TBW) from body impedance are based on the assumption of a constant conductor configuration--i.e. a constant subject section. In this paper we report on data obtained for a group of 19 young obese subjects (relative weight greater than 120%) and 10 young normal subjects (relative weight 80-110%). In obese subjects, the application of two different formulae generated from normal children gave biased results and led to an underestimation of TBW with respect to the reference value obtained by deuterium oxide dilution. Body mass index accounted for more than 40% of the inter-individual variability, suggesting that body size was not taken sufficiently into consideration by the predictive formulae used. We have used the body surface area as the anthropometrical parameter for the prediction of TBW from body impedance. The regression formula that we propose (TBW = 1.156 x (surface area/body impedance) - 2.356; R = 0.96), although requiring further validation on external populations, seems to provide a more realistic assessment of TBW in young obese subjects. We therefore suggest that the assessment of TBW in young obese subjects requires specifically designed prediction formulae.


The metabolic fate of an oral 14C-alanine load in the obese Zucker rat. Following an oral 14C-alanine load, obese Zucker rats showed the same rate of intestinal amino acid absorption as their lean counterparts. Alanine absorption was unchanged by a 24 h starvation period. The whole-body oxidation of the absorbed tracer was lower in the fa/fa rats and was significantly decreased in both lean and obese groups when the animals were submitted to starvation. The incorporation of the 14C-tracer into 14C-lipid was significantly higher in the carcass, skeletal muscle, white adipose tissue and liver of the obese animals, while that of brown adipose tissue was decreased as compared to that of the lean rats. Starvation induced no variation in 14C-lipid incorporation in the lean (+/?) animals while it sharply decreased this parameter in the obese. The incorporation of the tracer into 14C-glycogen and 14C-protein was also increased in the liver of the obese rats while no changes in incorporation into these fractions were observed in skeletal muscle. It is concluded that dietary amino acids significantly contribute to the hyperlipogenesis found in the liver and adipose tissue of the fa/fa rats.


Adipose tissue lipolysis after long-term overfeeding in identical twins. Ten pairs of young male sedentary, non-obese, monozygotic (MZ) twins, aged 21 +/- 2 years (mean +/- s.d.), were overfed for a period of 100 days during which they ingested 4.2 MJ (1000 kcal) per day above their individual energy needs, 6 days per week. There was a mean 8.4 kg increase in body weight and the average gain in body fat reaches 5.6 kg (P less than 0.0001). A biopsy of subcutaneous abdominal fat was performed, before and after the treatment, to determine fat cell weight as well as basal and catecholamine stimulated lipolyses from collagenase isolated adipocytes. Although analysis of variance revealed an increase in abdominal fat cell weight, no significant changes were noted in basal and catecholamine-stimulated lipolyses, due to large variation among individuals, results being expressed either per cell number or corrected for cell surface area. However, significant intrapair resemblance was observed in the changes of basal and epinephrine stimulated lipolyses (ri of about 0.60 in both cases), suggesting a concordant within-pair response, despite large between-pair variation. These results support the notion that the genotype may play an important role in regulating the response of abdominal adipose cells lipolytic activity to caloric excess.


Correlations of body mass index of adult adoptees and their biological and adoptive relatives. Adult family members resemble each other in body mass index (weight/height2). Our aim was to assess the extent to which the genetic relationship can account for the resemblance in natural families. We estimated the correlations in body mass index between adult adoptees, their adoptive parents, and their biological parents and siblings and compared the correlations to those between the biological relatives. Height, current weight, and greatest ever weight were obtained for 3651 adoptees. Groups representing thin, medium weight, overweight and obese subjects were selected by current body mass index (n = 540) and similarly selected by maximum body mass index (n = 524). The heights and weights of their biological and adoptive family members were assessed. The correlation in current body mass index between the adoptees and biological mothers, fathers, and full siblings were 0.15, 0.11, and 0.23, respectively (all P less than 0.001). These correlations were compatible with those found among the biological relatives living together. The correlations exhibited no consistent gender-related pattern. There was no correlation in body mass index between the adoptees and their adoptive parents. Similar results were obtained for maximum body mass index. Moreover, the correlations between the adoptees and their biological relatives are very similar to those found in large population studies of natural families. This study suggests that the genetic relationship fully accounts for the familial resemblance in body mass index among adults.


[Hodgkin's disease in HIV infection--detection of Epstein-Barr virus DNA in tongue epithelium and lymphoma]. We present an HIV-infected patient with lymphadenopathy syndrome in whom an unusually aggressive case of Hodgkin's disease developed. Examination of tissue excised from the lymphoma and of epithelial cells scraped from the tongue of the patient revealed Epstein-Barr virus (EBV). The relationship between an enhanced replication of EBV in the epithelium of the tongue and the elevated frequency of Hodgkin's lymphomas containing EBV-DNA among HIV-infected patients is discussed.


Differentiation of reptilian neural crest cells in vitro. An attempt was made to culture neural crest cells of the turtle embryo in vitro. Trunk neural tubes from the St. 9/10 embryos were explanted in culture dishes. The developmental potency of the turtle neural crest cells in vitro was shown to be essentially similar to that of avian neural crest cells, although they seem to be more sensitive to melanocyte-stimulating hormone (MSH) stimulation. We describe conditions under which explanted neural tube gives rise to neural crest cells that differentiate into neuronal cells and melanocytes. The potency of melanocyte differentiation was found to vary according to the concentration of fetal bovine serum (FBS, from 5 to 20%). Melanization of neural crest cells cultured in the medium containing FBS and alpha-MSH was more extensive than those cultured with FBS alone, combinations of FBS and chick embryo extract, or turtle embryo extract. These culture conditions seem to be useful for the study of the developmental potency of the neural crest cells as well as for investigating local environmental factors.


Gene transfer in primary cultures of human hepatocytes. Using liposomes as the mediator of DNA transfer, we were successful in the transfection of human hepatocytes isolated from surgical samples with an E. coli beta-galactosidase gene (beta-gal). A comparison of transfection efficiency showed that of the four promoters used, cytomegalovirus (CMV) promoter yielded higher transfection efficiencies than Rous sarcoma virus (RSV), Simian virus-40 (SV-40) and human alpha-1 antitrypsin (AAT) promoters. These studies represent the first report on the successful transfection of primary cultures of human hepatocytes.


Testosterone and spermatogenesis. Identification of stage-specific, androgen-regulated proteins secreted by adult rat seminiferous tubules. The aim of this study was to identify potential androgen-regulated proteins (ARP) that might mediate the supportive effects of testosterone on spermatogenesis. Adult rats were injected with ethane dimethane sulphonate (EDS) to destroy Leydig cells and thus induce complete testosterone withdrawal. Other EDS-treated rats were injected with 25 mg testosterone esters (TE) every 3 days to maintain quantitatively normal spermatogenesis. A timeframe for the study of androgen action on spermatogenesis was deduced from enumeration of degenerating germ cells at stage VII of the spermatogenic cycle in perfusion-fixed testes from rats in the early stages (4 to 8 days) after EDS treatment. Based on this data and changes in testicular interstitial fluid volume, long seminiferous tubule segments were isolated from control rats and from EDS-treated rats (+/- TE-supplementation) at stages II-V, VI-VIII, or IX-XII, 2 days to 6 days after EDS treatment. Seminiferous tubule segments were incubated for 22 hours with 60 microCi 35S-labelled methionine. Incorporation into newly synthesized proteins in the seminiferous tubule culture medium (= secreted proteins) or in seminiferous tubule lysates (= intracellular proteins) was determined by trichloroacetic acid-precipitation followed by analysis using two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis. In control rats, incorporation of 35S-methionine into proteins secreted by isolated seminiferous tubules was more than twice as great at stages VI-VIII than at stages II-V or IX-XII. This doubling in methionine incorporation into stages VI-VIII secreted proteins was abolished, however, 4 days after EDS treatment (when germ cell degeneration at stage VII was only just evident). A similar change occurred 4 days after testosterone withdrawal induced by immunoneutralization of luteinizing hormone. In the latter case and after EDS treatment, TE-supplementation of rats from day 0 maintained the normal control pattern of methionine incorporation into seminiferous tubule secreted proteins, although 6 days after EDS and TE treatment, incorporation into stages VI-VIII secreted proteins was 19% lower (P less than 0.05) than in the control group. In contrast, incorporation of methionine into proteins secreted by seminiferous tubules at stages II-V and IX-XII was unaffected by EDS and TE pretreatment, as was incorporation into intracellular proteins at all stages.(ABSTRACT TRUNCATED AT 400 WORDS)


Isolation and characterization of the nifUSVW-rpoN gene cluster from Rhodobacter sphaeroides. The rpoN gene from Rhodobacter sphaeroides was isolated from a genomic library via complementation of a Rhodobacter capsulatus rpoN mutant. The rpoN gene was located on a 7.5-kb HindIII-EcoRI fragment. A Tn5 insertion analysis of this DNA fragment showed that a minimal DNA fragment of 5.3 kb was required for complementation. Nucleotide sequencing of the complementing region revealed the presence of nifUSVW genes upstream from rpoN. The rpoN gene was mutagenized via insertion of a gene encoding kanamycin resistance. The resulting rpoN mutant was not impaired in diazotrophic growth and was in all respects indistinguishable from the wild-type strain. Southern hybridizations using the cloned rpoN gene as a probe indicated the presence of a second rpoN gene. Deletion of the nifUS genes resulted in strongly reduced diazotrophic growth. Two conserved regions were identified in a NifV LeuA amino acid sequence alignment. Similar regions were found in pyruvate carboxylase and oxaloacetate decarboxylase. It is proposed that these conserved regions represent keto acid-binding sites.


Sequence analysis and characterization of the Porphyromonas gingivalis prtC gene, which expresses a novel collagenase activity. In order to examine the potential role of bacterial collagenases in periodontal tissue destruction, we recently isolated a gene, prtC, from Porphyromonas gingivalis ATCC 53977, which expressed collagenase activity (N. Takahashi, T. Kato, and H. K. Kuramitsu, FEMS Microbiol. Lett. 84:135-138, 1991). The nucleotide sequence of the gene has been determined, and the deduced amino acid sequence corresponds to a basic protein of 37.8 kDa. In addition, Southern blot analysis indicated that the prtC gene is conserved among the three major serotypes of P. gingivalis. The enzyme has been purified to near homogeneity from Escherichia coli clone NTS1 following Mono Q anion exchange and sequential gel filtration chromatography. The molecular mass of the purified enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be ca. 35 kDa, and the active enzyme behaved as a dimer following gel filtration chromatography. The collagenase degraded soluble and reconstituted fibrillar type I collagen, heat-denatured type I collagen, and azocoll but not gelatin or the synthetic collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg. Enzyme activity was enhanced by Ca2+ and inhibited by EDTA, sulfhydryl-blocking agents, and the salivary peptide histatin. Preliminary evidence for the existence of a second collagenase expressed by strain 53977 was also obtained.


Two-stage induction of the soxRS (superoxide response) regulon of Escherichia coli. soxR and soxS are adjacent genes that govern a superoxide response regulon. Previous studies revealed that induction of the regulon is accompanied by increased transcription of soxS, which can activate the target genes. Therefore, induction may occur in two stages: the soxR-dependent activation of soxS, followed by the soxS-dependent induction of other genes. However, the requirement for soxR was unproven because the only existing soxR mutations either were of the regulon-constitutive type or also involved soxS. Therefore, we produced an insertion mutation that was shown by complementation to inactivate only soxR. In confirmation of the two-stage model, soxR was required for the induction by paraquat of the target genes studied (nfo, zwf, and sodA), for paraquat resistance, and for the 47- to 76-fold induction of soxS-lacZ gene fusions. Paraquat did not affect the expression of soxR-lacZ gene fusions. In a soxRS deletion mutant, the regulon was constitutively activated by a runaway soxS+ plasmid. However, a lower-copy-number plasmid failed to activate nfo, zwf, or sodA but did increase the paraquat resistance of a soxRS mutant. Therefore, there is a differential response of the regulon genes to soxS overproduction. A soxR regulon-constitutive mutation was suppressed by a soxR+ plasmid, suggesting a competition between native and activated forms of SoxR. It is proposed that to enhance the sensitivity of the response, the cell minimizes such potential competition by manufacturing only a small amount of this sensor protein, thereby necessitating signal amplification via induction of soxS.


Cloning and nucleotide sequence of the leucyl-tRNA synthetase gene of Bacillus subtilis. The leucyl-tRNA synthetase gene (leuS) of Bacillus subtilis was cloned and sequenced. A mutation in the gene, leuS1, increases the transcription and expression of the ilv-leu operion, permitting monitoring of leuS alleles. The leuS1 mutation was mapped to 270 degrees on the chromosome. Sequence analysis showed that the mutation is a single-base substitution, possibly in a monocistronic operon. The leader mRNA predicted by the sequence would contain a number of possible secondary structures and a T box, a sequence observed upstream of leader mRNA terminators of Bacillus tRNA synthetases and the B. subtilis ilv-leu operon. The DNA of the B. subtilis leuS open reading frame is 48% identical to the leuS gene of Escherichia coli and is predicted to encode a polypeptide with 46% identity to the leucyl-tRNA synthetase of E. coli.


Identification of novel loci affecting entry of Salmonella enteritidis into eukaryotic cells. There are an estimated 2 million cases of salmonellosis in the United States every year. Unlike the incidence of many infectious diseases, the incidence of salmonellosis in the United States and other developed countries has been rising steadily over the past 30 years, and the disease now accounts for 10 to 15% of all cases of acute gastroenteritis in the United States. The infecting organism is ingested and must traverse the intestinal epithelium to reach its preferred site for multiplication, the reticuloendothelial system. Despite several recent studies, the genetic basis of the invasion process is poorly understood. An emerging theme from these studies is that wild-type Salmonella organisms probably have several chromosomal loci that are required for the most efficient level of invasion. In this study, we have identified and characterized 13 TnphoA insertion mutants of Salmonella enteritidis CDC5 that exhibit altered invasion phenotypes. The mutants were identified by screening a bank of TnphoA insertions in S. enteritidis CDC5str for their invasion phenotype in three tissue culture cell lines (HEp-2, CHO, and MDCK). These 13 mutants were separated into six classes based on their invasive phenotypes in the tissue culture cell lines. Several mutants were defective for entry of some cell lines but not for others, while two mutants (SM6 and SM7) were defective for entry into all three tissue culture cell lines. This suggests that Salmonella spp. may express more than one invasion pathway. Southern analysis and chromosomal mapping indicated that as many as nine chromosomal loci may contribute to the invasion phenotype. It is becoming clear that the invasive phenotype of Salmonella spp. is multifactorial and more complex than that of some other invasive members of the family Enterobacteriaceae.


The genes required for heme synthesis in Salmonella typhimurium include those encoding alternative functions for aerobic and anaerobic coproporphyrinogen oxidation. Insertion mutagenesis has been used to isolate Salmonella typhimurium strains that are blocked in the conversion of 5-aminolevulinic acid (ALA) to heme. These mutants define the steps of the heme biosynthetic pathway after ALA. Insertions were recovered at five unlinked loci: hemB, hemCD, and hemE, which have been mapped previously in S. typhimurium, and hemG and hemH, which have been described only for Escherichia coli. No other simple hem mutants were found. However, double mutants are described that are auxotrophic for heme during aerobic growth and fail to convert coproporphyrinogen III to protoporphyrinogen IX. These mutant strains are defective in two genes, hemN and hemF. Single mutants defective only in hemN require heme for anaerobic growth on glycerol plus nitrate but not for aerobic growth on glycerol. Mutants defective only in hemF have no apparent growth defect. We suggest that these two genes encode alternative forms of coproporphyrinogen oxidase. Anaerobic heme synthesis requires hemN function, while either hemN or hemF is sufficient for aerobic heme synthesis. These phenotypes are consistent with the requirement of a well-characterized class of coproporphyrinogen oxidase for molecular oxygen.


Localization of a chromosomal mutation affecting expression of extracellular lipase in Staphylococcus aureus. We describe a Tn551 chromosomal insertion in Staphylococcus aureus S6C that results in sharply reduced expression of extracellular lipase. With Tn917 as a probe, the insertion in the original mutant (KSI905) was localized to a 12.6-kb EcoRI DNA fragment. The 12.6-kb fragment was cloned and used as a probe to identify a 26-kb EcoRI fragment containing the Tn551 insertion site in the S6C parent strain. Restriction endonuclease analysis of the 12.6- and 26-kb EcoRI fragments confirmed that the Tn551 insertion in KSI905 was accompanied by a deletion of 18.7 kb of chromosomal DNA. Tn551 was transduced from KSI905 back into the S6C parent strain. All transductants exhibited the same lipase-negative (Lip-) phenotype and contained the same mutation with respect to both the insertion and the 18.7-kb deletion. The inability to produce lipase was not caused by disruption of the lipase structural gene, since all Lip- mutants carried intact copies of geh. Moreover, the Tn551 insertion was localized to a region of the staphylococcal chromosome at least 650 kb from geh. Taken together, these results suggest that the Tn551 insertion occurred in a region of the chromosome encoding a trans-active element required for the expression of extracellular lipase. A 20-bp oligonucleotide corresponding to a sequence within the region encoding RNA II near the Tn551 insertion site in ISP546 (H.L. Peng, R.P. Novick, B. Kreiswirth, J. Kornblum, and P. Schlievert, J. Bacteriol. 170:4365-4372, 1988) and a 1.75-kb DNA fragment representing the region encoding RNA III were used as gene probes to show that the Tn551 insertion did not occur in the agr locus. We conclude that the genetic element functions independently of agr or as an unrecognized part of that regulatory system.


Conjugative transposition of Tn916: the transposon int gene is required only in the donor. Conjugative transposition of transposon Tn916 has been shown to proceed by excision of the transposon in the donor strain and insertion of this element in the recipient. This process requires the product of the transposon int gene. We report here the surprising finding that the int gene is required only in the donor during conjugative transposition. We find that Tn916 int-1, whose int gene has been inactivated by an insertion mutation, transposes when a complementing wild-type int gene is present only in the donor during mating. When the int+ gene is present in a plasmid and is expressed from the spac promoter, conjugative transposition is very inefficient. However, when the Int+ function is supplied from a coresident distantly linked Tn916 tra-641 mutant, which is defective in a function required for conjugation, efficient conjugative transposition of Tn916 int-1 occurs. This suggests either that Int is not required for integration of Tn916 in gram-positive bacteria or that the protein is transferred from the donor to the transconjugant during the mating event. When the nonconjugative plasmid pAT145 was present in the donor, it was rarely cotransferred with Tn916. This suggests that complete fusion of mating cells is not common during conjugative transposition.


An Escherichia coli DNA topoisomerase I mutant has a compensatory mutation that alters two residues between functional domains of the DNA gyrase A protein. Nucleotide sequence analysis revealed that the compensatory gyrA mutation in Escherichia coli DM750 affects DNA supercoiling by interchanging the identities of Ala-569 and Thr-586 in the DNA gyrase A subunit. These residues flank Arg-571, a site for trypsin cleavage that splits gyrase A protein between DNA breakage-reunion and DNA-binding domains. The putative interdomain locations of the DM750 mutation and that of E. coli DM800 (in gyrase B protein) suggests that these compensatory mutations may reduce DNA supercoiling activity by altering allosteric interactions in the gyrase complex.


Cloning and functional expression of a human neuropeptide Y/peptide YY receptor of the Y1 type. Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides that primarily function as neurotransmitter and gastrointestinal hormone, respectively. Previous functional and binding data have indicated the existence of at least three distinct receptor types, Y1, Y2, and Y3, for NPY and/or PYY in mammals. We describe here a human Y1 cDNA clone, hY1-5, isolated from a fetal brain library. The human Y1 receptor consists of 384 amino acids and has seven putative transmembrane domains like other members of the G-protein-coupled superfamily of receptors. In the region spanning the transmembrane domains, the Y1 receptor displays 29% sequence identity to human tachykinin receptors, but it only shows 21% and 23% homology with proposed bovine (LCR1) and Drosophila (PR4) NPY receptor clones, respectively. Northern blot analysis of a human neuroblastoma cell line, SK-N-MC, previously used by many investigators as a model system for studies on the Y1 receptor, revealed a single 3.5-kilobase mRNA species. Reverse transcriptase-polymerase chain reaction analysis indicated expression also in human cultured vascular smooth muscle cells, supporting the view that the Y1 receptor is associated with NPY/PYY-evoked vasoconstriction. When expressed in COS1 cells, hY1-5 conferred specific 125I-PYY binding sites with displacement patterns characteristic of the Y1 receptor, i.e. PYY greater than or equal to NPY greater than or equal to [Leu31,Pro34]NPY much greater than NPY2-36 greater than C2NPY greater than pancreatic polypeptide greater than NPY13-36 greater than NPY18-36. Moreover, in the Y1 receptor-transfected COS1 cells, but not in type 1 angiotensin II receptor-transfected control cells, NPY and PYY accelerated 45Ca2+ influx and inhibited forskolin-stimulated cAMP accumulation, both phenomena being characteristic of the mammalian Y1 receptor.


Repression of myogenin function by TGF-beta 1 is targeted at the basic helix-loop-helix motif and is independent of E2A products. The muscle-specific helix-loop-helix (HLH) proteins myogenin, MyoD, myf5, and MRF4 form hetero-oligomers with ubiquitous HLH proteins encoded by the E2A gene and activate muscle transcription by binding to a DNA sequence known as an E-box (CANNTG). Transforming growth factor-beta (TGF-beta) can inhibit muscle differentiation by silencing the transcription-activating potential of myogenic HLH proteins without affecting their ability to bind DNA. We show that repression by TGF-beta is directed at the basic-HLH motif of myogenin and is independent of E2A products. Using a series of reporter genes as targets for trans-activation by myogenin, transcriptional repression by TGF-beta is also shown to map to the E-box motif and to not require heterologous DNA sequence elements. These results demonstrate that TGF-beta represses muscle-specific transcription through a post-translational mechanism that renders the basic-HLH regions of the myogenic regulators nonfunctional. The selective repression of myogenic HLH proteins by TGF-beta indicates that the TGF-beta signaling system can discriminate between different classes of HLH proteins and implies that myogenic HLH proteins activate muscle-specific transcription through a unique mechanism.


The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity. The domain of thrombomodulin that binds to the anion-binding exosite of thrombin was identified by comparing the binding of fragments of thrombomodulin to thrombin with that of Hirugen, a 12-residue peptide of hirudin that is known to bind to the anion-binding exosite of thrombin. Three soluble fragments of thrombomodulin, containing (i) the six repeated growth factor-like domains of thrombomodulin (GF1-6), (ii) one-half of the second through the sixth growth factor-like repeats (GF2.5-6), or (iii) the fifth and sixth such domains (GF5-6), were examined. Hirugen was a competitive inhibitor for either GF1-6 or GF2.5-6 stimulation of thrombin activation of protein C. GF5-6, which binds to thrombin without altering its ability to activate protein C, competed with fluorescein-labeled Hirugen for binding to thrombin. Therefore, all three thrombomodulin fragments, each of which lacked the chondroitin sulfate moiety, competed with Hirugen for binding to thrombin. To determine whether GF5-6 and Hirugen were binding to overlapping sites on thrombin or were interfering allosterically with each other's binding to thrombin, the effects of each thrombomodulin fragment and of Hirugen on the active site conformation of thrombin were compared using two different approaches: fluorescence-detected changes in the structure of the active site and the hydrolysis of chromogenic substrates. The GF5-6 and Hirugen peptides affected these measures of active site conformation very similarly, and hence GF5-6 and Hirugen contact residues on the surface of thrombin that allosterically alter the active site structure to a similar extent. Full-length thrombomodulin and GF1-6 alter the active site structure to comparable extents, but the amidolytic activity of thrombin complexed to thrombomodulin or GF1-6 differs significantly from that of thrombin complexed to GF5-6 or Hirugen. Taken together, these results indicate that the GF5-6 domain of thrombomodulin binds to the anion-binding exosite of thrombin. Furthermore, the binding of GF5-6 to the anion-binding exosite alters thrombin specificity, as evidenced by GF5-6-dependent changes in both the kcat and Km of synthetic substrate hydrolysis by thrombin. The contact sites on thrombin for the GF4 domain and the chondroitin sulfate moiety of thrombomodulin are still unknown.


Cloning, sequencing, and expression of the nhaB gene, encoding a Na+/H+ antiporter in Escherichia coli. In Escherichia coli, expulsion of sodium ions is driven by proton flux via at least two distinct Na+/H+ antiporters, NhaA and NhaB. When the nhaA gene is deleted from the chromosome, the cell becomes sensitive to high salinity and alkaline pH (Padan, E., Maisler, N., Taglicht, D., Karpel, R., and Schuldiner, S. (1989) J. Biol. Chem. 264, 20297-20302). In the current work we cloned the nhaB gene by complementation of the delta nhaA strain. The gene codes for a membrane protein 504 amino acids long. Hydropathic analysis of the sequence indicates the presence of 12 putative transmembrane helices. NhaB has been specifically labeled with [35S]methionine; it is a membrane protein and displays an apparent M(r) of 47,000, slightly lower than that predicted from its amino acid sequence. Membranes from cells containing multiple dose of nhaB display enhanced Na+/H+ antiporter activity, as measured by the ability of Na+ to collapse a preformed pH gradient or by direct measurement of 22Na+ fluxes. In contrast to NhaA, whose activity increases with pH, NhaB is practically insensitive to pH. Limited homologies with Na+ transporters have been identified.


Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor. The signal for rapid internalization of the mannose 6-phosphate/insulin-like growth factor II receptor has been localized to the sequence Tyr-Lys-Tyr-Ser-Lys-Val in positions 24-29 of its 163-residue cytoplasmic tail. Most of the activity of this signal is mediated by the carboxyl 4 amino acids, especially Tyr26 and Val29 (Canfield, W. M., Johnson, K. F., Ye, R. D., Gregory, W. and Kornfeld, S. (1991) J. Biol. Chem. 266, 5682-5688). In this study, we have tested the effect of a series of mutations on the internalization rate of a mutant receptor that contains a 29-amino acid cytoplasmic tail terminating with the 4-amino acid internalization sequence Tyr-Ser-Lys-Val. Replacement of Tyr26 with Phe or Trp gave rise to mutant receptors that were internalized at 10% the wild-type rate, while receptors with Ala, Leu, Ile, Val, or Asn at this position were totally inactive. Val29 could be replaced by other large hydrophobic residues (Phe, Leu, Ile, or Met) with no loss of activity, but the presence of Ala, Gly, Arg, Gln, or Tyr in this position inactivated the signal. Ser27 could be effectively replaced by many different amino acids, but not by Pro or Gly. However, Gly27 could be tolerated if the residues at positions 28 and 29 were also changed. A change in the 2-residue spacing between Tyr26 and Val29 destroyed the signal. These data show that the essential elements of this signal are an aromatic residue, especially a Tyr in the first position, separated from a large hydrophobic residue in the last position by 2 amino acids. The residues in positions 2 and 3 of the signal may have a modulating effect on its activity. The Tyr-Ser-Lys-Val signal could be moved to a more proximal region of the cytoplasmic tail with only a modest loss of activity. In addition, the signal could be effectively replaced by the putative 4-residue signals of seven other receptors and membrane proteins known to undergo rapid endocytosis, including the Tyr-Thr-Arg-Phe sequence of the transferrin receptor, a Type II membrane protein. These results are compatible with the 4-residue signals of this type being interchangeable, even among Type I and Type II membrane proteins.


Prostacyclin is a specific effector of adipose cell differentiation. Its dual role as a cAMP- and Ca(2+)-elevating agent. The mitogenic-adipogenic activity of carbaprostacyclin (cPGI2), a stable analogue of prostacyclin (PGI2), has been proposed to be related to its ability to elicit cAMP production and to activate the protein kinase A cascade (Négrel, R., Gaillard, D., and Ailhaud, G. (1989) Biochem. J. 257, 399-405). In the present study, cPGI2 has been compared with other activators of the cAMP pathway, namely isoproterenol, forskolin and 8-bromo-cAMP, with respect to adipose cell differentiation. Carbaprostacyclin behaved as a much more potent and efficient effector of mouse Ob1771 preadipocyte differentiation than the latter agents. Moreover, cPGI2 also exerted a specific amplifying mitogenic-adipogenic role, as compared with isoproterenol in rat as well as human adipose precursor cells in primary culture, suggesting that the prostanoid was able to generate an additional second messenger. The fact that ionomycin was able to potentiate and amplify the differentiation induced by 8-bromo-cAMP led us to give evidence, using preadipocytes preloaded with the fluorescent calcium chelator Indo-1, that cPGI2, besides its ability to activate adenyl cyclase, was also able to induce a transient increase in intracellular free calcium. This phenomenon was independent of cAMP production or inositol phospholipid breakdown and appeared to be mediated after binding to a single class of PGI2 receptor. The potential to generate simultaneously two synergistic intracellular signals allows us to ascribe to PGI2 a key and specific role in the differentiation of adipose precursor cells in vitro that would likely lead in vivo to the recruitment of "dormant" preadipocytes to become adipocytes.


Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli. Escherichia coli contains a soluble, [2Fe-2S] ferredoxin of unknown function (Knoell, H.-E., and Knappe, J. (1974) Eur. J. Biochem. 50, 245-252). Using antiserum to the purified protein to screen E. coli genomic expression libraries, we have cloned a gene (designated fdx) encoding this protein. The DNA sequence of the gene predicts a polypeptide of 110 residues after removal of the initiator methionine (polypeptide M(r) = 12,186, holoprotein M(r) = 12,358). The deduced amino acid sequence is strikingly similar to those of the ferredoxins found in animal mitochondria which function with cytochrome P450 enzymes and to the ferredoxin from Pseudomonas putida which functions with P450cam. The overall sequence identity is approximately 36% when compared with human mitochondrial and P. putida ferredoxins, and the identities include 4 cysteine residues proposed to coordinate the iron cluster. The protein was overproduced approximately 500-fold using an expression plasmid, and the holoprotein was assembled and accumulated in amounts exceeding 30% of the total cell protein. The overexpressed ferredoxin exhibits absorption, circular dichroism, and electron paramagnetic resonance spectra closely resembling those of the animal ferredoxins and P. putida ferredoxin.


The stereoselective recognition of substrates by phosphoinositide kinases. Studies using synthetic stereoisomers of dipalmitoyl phosphatidylinositol. Soluble phosphatidylinositol (PtdIns) 4- and 3-kinase activities were partially purified and characterized from human placental extracts. The placental PtdIns 4-kinase (type 3) has a Km for ATP of 460 microM and is kinetically different to a partially purified human erythrocyte, membrane-bound, PtdIns 4-kinase (type 2). These three inositol lipid kinases were then used to compare their substrate specificities against the four synthetic stereoisomers of dipalmitoyl PtdIns. Only the placental 4-kinase was influenced by the chirality of the glycerol moiety of PtdIns. However, neither of the 4-kinases was able to phosphorylate L-PtdIns and, therefore, these kinases have an absolute requirement for the inositol ring to be linked to the glyceryl backbone of the lipid through the D-1 position. Phosphoinositide 3-kinase, on the other hand, was found to phosphorylate both D- and L-PtdIns. While the 3-kinase phosphorylated exclusively the D-3 position of D-PtdIns, further analyses demonstrated that the same enzyme phosphorylated two sites on L-PtdIns, namely the D-6 and D-5 positions of the inositol ring. Some implications of these findings are discussed.


Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver. Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.


Kinetic studies on the formation and decomposition of compounds II and III. Reactions of lignin peroxidase with H2O2. The present study characterizes the serial reactions of H2O2 with compounds I and II of lignin peroxidase isozyme H1. These two reactions constitute part of the pathway leading to formation of the oxy complex (compound III) from the ferric enzyme. Compounds II and III are the only complexes observed; no compound III* is observed. Compound III* is proposed to be an adduct of compound III with H2O2, formed from the complexation of compound III with H2O2 (Wariishi, H., and Gold, M. H. (1990) J. Biol. Chem. 265, 2070-2077). We provide evidence that demonstrates that the spectral data, on which the formation of compound III* is based, are merely an artifact caused by enzyme instability and, therefore, rule out the existence of compound III*. The reactions of compounds II and III with H2O2 are pH-dependent, similar to that observed for reactions of compounds I and II with the reducing substrate veratryl alcohol. The spontaneous decay of the compound III of lignin peroxidase results in the reduction of ferric cytochrome c. The reduction is inhibited by superoxide dismutase, indicating that superoxide is released during the decay. Therefore, the lignin peroxidase compound III decays to the ferric enzyme through the dissociation of superoxide. This mechanism is identical with that observed with oxymyoglobin and oxyhemoglobin but different from that for horseradish peroxidase. Compound III is capable of reacting with small molecules, such as tetranitromethane (a superoxide scavenger) and fluoride (a ligand for the ferric enzyme), resulting in ferric enzyme and fluoride complex formation, respectively.


DNA topoisomerase I phosphorylation in murine fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate and in vitro by protein kinase. The phosphorylation of DNA topoisomerase I in quiescent murine 3T3-L1 fibroblasts treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was characterized by in vivo labeling with [32P] orthophosphate and immunoprecipitation with a scleroderma anti-DNA topoisomerase I autoantibody. DNA topoisomerase I phosphorylation was stimulated 4-fold by 2 h of TPA treatment (TPA at 100 ng/ml maximally enhanced phosphorylation). Purified DNA topoisomerase I was phosphorylated in vitro in a Ca2+ and phospholipid-dependent fashion by types I, II, and III protein kinase C. The phosphorylation reaction was stimulated by TPA and had an apparent K(m) of 0.4 microM. DNA topoisomerase I was phosphorylated in vivo and in vitro predominantly at serine. The major tryptic phosphopeptides from DNA topoisomerase I in TPA-treated fibroblasts and phosphorylated by protein kinase C comigrated in thin-layer electrophoresis. The half-life of incorporated phosphate on DNA topoisomerase I was 40 min in both TPA-treated and control cells. These results suggest that phosphorylation is a mechanism for activating DNA topoisomerase I in fibroblasts treated with TPA and that protein kinase C functions in the phosphorylation.


Triglyceride metabolism in 3T3-L1 cells. An in vivo 13C NMR study. 13C nuclear magnetic resonance spectroscopy has been used to study triglyceride metabolism in 3T3-L1 cells incubated with [1-13/14C] acetate, myristate, palmitate, stearate, or oleate. Labeled cells embedded in agarose filaments were perfused in a specially fitted NMR tube within the spectrometer magnet. Incubation of 3T3-L1 cells with a specific fatty acid enriched the cellular triglycerides with that fatty acid; the NMR signal observed in the carbonyl region of the cell spectrum was due in large part to that fatty acid. NMR data demonstrated that cellular enzymes preferentially esterified saturated fatty acids at the glyceride sn-1,3 position and unsaturated fatty acids at the sn-2 position. cellular triglyceride hydrolysis by hormone-sensitive lipase was monitored by measuring the decrease in the integrated intensities of resonances arising from fatty acyl carbonyls esterified at glycerol carbons sn-1,3 and sn-2. Under basal conditions, the time courses were first-order, and the average rates were 0.14% of signal/min at both carbonyl positions. Under isoproterenol stimulated conditions, these rates were still first-order and increased 6.4-fold at the sn-1,3 position and 2.4-fold at the sn-2 position. The observation that the hydrolysis time courses were first-order suggested that only a small amount of cellular triglyceride was available to hormone-sensitive lipase, supporting the view that lipolytic enzymes operate at lipid surfaces where only small amounts of neutral lipid may be soluble. Attempts to correlate the measured rates with the rates of hydrolysis at the sn-1,3 and sn-2 positions were hindered by the fact that the chemical shifts of the carbonyl carbons of the diglyceride hydrolysis product did not overlie those of the triglyceride. Analysis of hydrolysis kinetics revealed that hormone-sensitive lipase exhibited little preference for a particular esterified fatty acid under basal conditions; however, under stimulated conditions, the enzyme exhibited a preference for certain triglyceride species.


Molecular cloning of the gene encoding Thiobacillus ferrooxidans Fe(II) oxidase. High homology of the gene product with HiPIP. The amino-terminal sequence of Thiobacillus ferrooxidans Fe(II) oxidase (linked to cytochrome c552) was determined, and the iro gene that encodes this enzyme was cloned using degenerate oligonucleotides as a probe. The DNA sequence of a region (856 base pairs) which encompasses the iro gene revealed that the enzyme was encoded by a 273-base pair open reading frame and consists of 90 amino acids, including a possible 37-residue signal sequence. The iro gene seems to be transcribed independently of any other gene because the transcriptional products are 0.45 and 0.6 kilobases in size. Current protein databases revealed that the iro gene product is a new member of the high redox potential iron sulfur proteins, which generally function in electron transport but do not show enzymatic activity.


Peptidoglycan composition in heterogeneous Tn551 mutants of a methicillin-resistant Staphylococcus aureus strain. It was shown that Tn551 inactivation of two chromosomal (so-called auxiliary) loci other than the mec gene result in a dramatic reduction of methicillin resistance and decreased cell wall turnover and autolytic capacity in a methicillin-resistant Staphylococcus aureus strain (de Jonge, B. L. M., de Lencastre, H., and Tomasz, A. (1990) J. Bacteriol. 173, 1105-1110). To understand the mechanistic basis of these phenomena we have examined the status of the autolytic enzymes and the muropeptide composition of peptidoglycan using reversed-phase high-performance liquid chromatography and mass spectral analyses. While no differences could be detected in the number of autolytic hydrolases, the mutants showed major changes in peptidoglycan composition. Nine prominent muropeptides of the parental strain each carrying a pentaglycyl substituent were missing from the cell wall of one group of mutants. The second mutant lacked four parental muropeptides which were composed of the unsubstituted disaccharide pentapeptide and its alanyl-tetraglycine derivative. The auxiliary genes are genetic determinants involved with the biosynthesis of peptidoglycan precursors, the presence of which in the cell wall may be needed for optimal cell wall turnover.


Assignment of interchain disulfide bonds in platelet-derived growth factor (PDGF) and evidence for agonist activity of monomeric PDGF. Platelet-derived growth factor (PDGF) is a dimeric factor stabilized by disulfide bonds. Using an approach involving partial reduction of PDGF, we have identified the 2nd and 4th cysteine residues in the PDGF chains as the cysteine residues forming interchain disulfide bonds. Analysis of PDGF mutants in which the 2nd and 4th cysteine residues were mutated to serine residues revealed that the disulfide bonds are arranged in a cross-wise manner, with the 2nd cysteine residue in one chain being linked to the 4th cysteine residue in the other. A PDGF B-chain mutant, in which both the 2nd and 4th cysteine residues were substituted with serine residues, migrated as a monomer in sodium dodecyl sulfate gel electrophoresis and retained receptor binding activity. When analyzed in receptor dimerization and autophosphorylation assays, this mutant showed agonistic activity. Thus, structural information has been obtained that will allow the large scale production of properly folded monomeric PDGF, as well as design of specific PDGF heterodimers.


Effects of the neuronal phosphoprotein synapsin I on actin polymerization. I. Evidence for a phosphorylation-dependent nucleating effect. Synapsin I is a synaptic vesicle-specific phosphoprotein which is able to bind and bundle actin filaments in a phosphorylation-dependent fashion. In the present paper we have analyzed the effects of synapsin I on the kinetics of actin polymerization and their modulation by site-specific phosphorylation of synapsin I. We found that dephosphorylated synapsin I accelerates the initial rate of actin polymerization and decreases the rate of filament elongation. The effect was observed at both low and high ionic strength, was specific for synapsin I, and was still present when polymerization was triggered by F-actin seeds. Dephosphorylated synapsin I was also able to induce actin polymerization and bundle formation in the absence of KCl and MgCl2. The effects of synapsin I were strongly decreased after its phosphorylation by Ca2+/calmodulin-dependent protein kinase II. These observations suggest that synapsin I has a phosphorylation-dependent nucleating effect on actin polymerization. The data are compatible with the view that changes in the phosphorylation state of synapsin I play a functional role in regulating the interactions between the nerve terminal cytoskeleton and synaptic vesicles in various stages of the exoendocytotic cycle.


Association of phosphorylated insulin-like growth factor-I receptor with the SH2 domains of phosphatidylinositol 3-kinase p85. Insulin-like growth factor-I (IGF-I) stimulates the production of 3-inositides and markedly increases the phosphatidylinositol 3-kinase activity that is immunoprecipitated by anti-phosphotyrosine antibodies, a portion of which is also associated with the IGF-I receptor. In this study, recombinant p85, the regulatory subunit of phosphatidylinositol 3-kinase, and fusion proteins containing various subdomains were used to investigate the association of p85 with the IGF-I receptor and to demonstrate that p85 is a direct in vitro substrate of the IGF-I receptor kinase. Solubilized IGF-I receptor was immobilized on antireceptor antibody-agarose beads. Following in vitro receptor phosphorylation and incubation with cell lysate, immobilized receptor became associated with phosphatidylinositol 3-kinase activity and with protein bands with molecular masses of 85 and 110 kDa, which correspond to the known molecular masses of the subunits of phosphatidylinositol 3-kinase. These associations were inhibited by the addition of recombinant intact p85 or SH2-containing fusion proteins, but not by fusion proteins containing its SH3 domain or breakpoint cluster homology region. A fusion protein containing the SH2 domains of Ras GTPase-activating protein also inhibited the association of phosphatidylinositol 3-kinase activity with immobilized IGF-I receptor, although less effectively than p85, whereas a similar construct containing the SH2 domain of pp60src was without effect. When immobilized phosphorylated IGF-I receptor was incubated with intact p85 or the SH2-containing fusion proteins, it became associated with and phosphorylated these proteins. These results demonstrate that at least in vitro, a tight association occurs between phosphorylated IGF-I receptor and phosphatidylinositol 3-kinase, that the region of phosphatidylinositol 3-kinase that contains its SH2 domains is directly involved in this association, and that this region is a direct substrate for IGF-I receptor tyrosine kinase. Furthermore, these results suggest that Ras GTPase-activating protein can also interact with the IGF-I receptor and that different SH2 domain-containing proteins interact with the IGF-I receptor with widely differing affinities.


Differential inhibition of the DNA translocation and DNA unwinding activities of DNA helicases by the Escherichia coli Tus protein. Binding of the Escherichia coli Tus protein to its cognate nonpalindromic binding site on duplex DNA (a Ter sequence) is sufficient to arrest the progression of replication forks in a Ter orientation-dependent manner in vivo and in vitro. In order to probe the molecular mechanism of this inhibition, we have used a strand displacement assay to investigate the effect of Tus on the DNA helicase activities of DnaB, PriA, UvrD (helicase II), and the phi X-type primosome. When the substrate was a short oligomer hybridized to a circular single-stranded DNA, strand displacement by DnaB, PriA, and the primosome (in both directions), but not UvrD, was blocked by Tus in a polar fashion. However, no inhibition of either DnaB or UvrD was observed when the substrate carried an elongated duplex region. With this elongated substrate, PriA helicase activity was only inhibited partially (by 50%). On the other hand, both the 5'----3' and 3'----5' helicase activities of the primosome were inhibited almost completely by Tus with the elongated substrate. These results suggest that while Tus can inhibit the translocation of some proteins along single-stranded DNA in a polar fashion, this generalized effect is insufficient for the inhibition of bona fide DNA helicase activity.


Induction of 103-kDa gelatinase/type IV collagenase by acidic culture conditions in mouse metastatic melanoma cell lines. Gelatinases/type IV collagenases have been shown to be involved in tumor invasion and metastasis. In this study, we examined the effect of culture medium pH on the secretion of the gelatinases from mouse B16 melanoma cell lines and human tumor cell lines using zymography analysis. The highly metastatic clone F10 of B16 melanoma did not secrete any gelatinase in neutral culture media (pH 7.1-7.3), whereas it secreted a high level of a 103-kDa gelatinase in an initial pH range of 5.4-6.1. The addition of an excess amount of glucose into a neutral culture medium also induced the gelatinase secretion from the cells by decreasing the medium pH during incubation. The extent of the acid-induced gelatinase secretion by the B16 melanoma cell lines was in the order of BL6 greater than F10 greater than F1 much greater than the parent B16 line, in good agreement with the order of their metastatic potentials. Two human cell lines (A549 and HT1080) secreted a higher level of a 90-kDa gelatinase at pH 6.8 compared with pH 7.3. The acid-induced gelatinase secretion from B16-F10 cells was blocked by cycloheximide, indicating that the enzyme induction was due to de novo synthesis. When in vitro tumor cell invasion was assayed in Boyden chambers, B16-F10 cells incubated in an acidic medium exerted a more active migration through type IV collagen gel than those in a neutral medium. These results suggest that the acidic environment formed around tumor tissues may be an important factor in invasion and metastasis of some types of tumors.


Site-directed mutagenesis of the rat m1 muscarinic acetylcholine receptor. Role of conserved cysteines in receptor function. There are 9 cysteine residues in the rat m1 muscarinic acetylcholine receptor (mAChR) that are conserved among all five mammalian mAChR subtypes sequenced to date. To study the role of these cysteines in rat m1 mAChR function, site-directed mutagenesis was used to convert each Cys residue to Ser, and the mutant receptor genes were transfected into mAChR-deficient Chinese hamster ovary (CHO) cells. Substitution of Cys391 (extracellular loop III) or Cys421 and Cys435 (carboxyl terminus) produces receptors with wild type phenotype. Cells transfected with Ser98 or Ser178 (extracellular loops I and II, respectively) receptor genes display no carbachol-mediated hydrolysis of phosphoinositides (PI), and membranes prepared from these cells do not bind the muscarinic antagonist [3H] quinuclidinyl benzilate, even though the cells express transcripts of the m1 mAChR as determined by RNA hybridization analysis. Since biochemical evidence suggests that these cysteines form a disulfide bridge (Curtis, C. A. M., Wheatley, M., Bansal, S., Birdsall, N. J. M., Eveleigh, P., Pedder, E. K., Poyner, D., and Hulme, E. C. (1989) 264, 489-495), our findings imply that this disulfide linkage may be critical for formation of the ligand binding domain or for proper protein folding. The Ser394 mAChR (extracellular loop III) exhibits a 44% decrease in efficacy for carbachol-mediated stimulation of PI hydrolysis relative to the wild type receptor, but displays normal ligand binding affinities. The Ser407 m1 mAChR (transmembrane helix VII) displays a decreased efficacy for eliciting carbachol-mediated PI hydrolysis (39% that of CHO cells transfected with the wild type receptor) and a 4-fold shift to the right in the carbachol dose-response curve, which is consistent with the 4-fold decrease in carbachol affinity at the Ser407 m1 mAChR. In contrast, the Ser417 m1 mAChR (transmembrane helix VII) displays an increase in carbachol affinity and a shift to the left in the carbachol dose-response curve for PI hydrolysis. These findings suggest that cysteine residues in the seventh transmembrane helix of the m1 mAChR may influence agonist binding and the efficiency of receptor activation.


Functional and immunological distinction between insulin-like growth factor I receptor subtypes in KB cells. Subtypes of insulin-growth factor I (IGF-I) receptors, including hybrid receptors containing insulin receptor alpha beta dimers associated with IGF-I receptor alpha beta dimers, have been described in a number of systems. The molecular basis of the multiple subtypes and their functional significance is not understood. Ligand-dependent phosphorylation of insulin and IGF-I receptors and immunoprecipitation with antipeptide and monoclonal antibodies have been used to characterize the subpopulations of these receptors in the human KB cell line. IGF-I receptors exhibit beta subunits of 95 and 102 kDa in these cells. IGF-I receptors containing 102-kDa beta subunits are immunoprecipitated by the IGF-I receptor-specific antibody alpha-IR3. Antibody alpha-IR3 does not appear to recognize a hybrid receptor in these cells. However, an antipeptide antibody against the carboxyl-terminal domain of the insulin receptor (AbP5) immunoprecipitates a population of receptors phosphorylated in response to IGF-I (1 nM) which contains both 95- and 102-kDa beta subunits. These receptors must be hybrid complexes because AbP5 does not recognize the 102-kDa beta subunit directly. The inability of antibody alpha-IR3 to recognize these complexes suggests that their IGF-I receptor alpha subunits must differ from typical IGF-I receptor alpha subunits either in primary sequence or conformation. Therefore, KB cells may contain more than one type of IGF-I receptor alpha subunit. Hybrid IGF-I receptors can also be distinguished from homotypic IGF-I receptors by their responsiveness to IGF-II. Stimulation of autophosphorylation in hybrid IGF-I receptors by IGF-I is 3-4-fold greater than that seen in response to IGF-II. In contrast, IGF-I and IGF-II are nearly equipotent in stimulating autophosphorylation in the alpha-IR3-reactive receptor population. This suggests the existence of functionally distinct receptor subtypes which may differ in their ability to mediate the biological effects of IGF-II.


Translocation of preproinsulin across the endoplasmic reticulum membrane. The relationship between nascent polypeptide size and extent of signal recognition particle-mediated inhibition of protein synthesis. Signal recognition particle (SRP) induces elongation arrest of nascent presecretory proteins as the signal peptide protrudes from the large ribosomal subunit. To examine the relationship between the size of the precursor and extent of SRP mediated inhibition of polypeptide chain elongation, we performed in vitro translation experiments in the presence of SRP using a series of truncated preproinsulin mRNA molecules. These precursors possessed the same NH2 terminus as native preproinsulin followed by progressively shorter COOH termini. SRP inhibited translation of precursors as short as 64 amino acids in length, however, the extent of inhibition diminished for shorter precursors. This correlated with a reduction in the time required for ribosomes to transit through the mRNA encoding the shortened precursors. By exploiting a chimeric protein comprising the first 71 residues of preproinsulin fused to the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase, we demonstrate that the largest size a nascent chain can reach and still be susceptible to SRP-mediated elongation arrest is approximately 17 kDa. Our data support the model that SRP binding to the signal peptide is a reversible process even in the absence of microsomal membranes, and that SRP can arrest polypeptide chain elongation at multiple stages during translation.


The calcium pump of the liver nuclear membrane is identical to that of endoplasmic reticulum. The envelope membrane of rat liver nuclei contains a P-type Ca(2+)-transporting pump, revealed by the presence of a Ca(2+)-stimulated phosphoenzyme. The level of the nuclear phosphoenzyme in autoradiographed polyacrylamide gels was decreased by lanthanum, as typically observed in the endoplasmic reticulum Ca2+ pump. It was also decreased by thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, two accepted inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. Comparative proteolysis of the phosphorylated enzyme of liver microsomes (endoplasmic reticulum) and nuclear membranes revealed an identical cleavage pattern. In addition, antibodies raised against the endoplasmic reticulum Ca2+ pump cross-reacted with the pump in the nuclear membranes. The findings show that nuclear membranes contain a Ca(2+)-transporting pump closely related to that of the endoplasmic reticulum, if not identical to it. The pump is likely to be involved in the control of nuclear free calcium.


Nonselective utilization of the endomannosidase pathway for processing glycoproteins by human hepatoma (HepG2) cells. Endo-alpha-D-mannosidase, a Golgi-situated processing enzyme, provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins (Moore, S. E. H., and Spiro, R. G. (1990) J. Biol. Chem. 265, 13104-13112). The present report demonstrates that at least five distinct glycoproteins secreted by HepG2 cells (alpha 1-antitrypsin, transferrin, alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, and alpha-fetoprotein) as well as cell surface components can effectively utilize this alternate processing route. During a castanospermine (CST)-imposed glucosidase blockade, these glycoproteins apparently were produced with their usual complement of complex carbohydrate units, and upon addition of the mannosidase I inhibitor, 1-deoxymannojirimycin (DMJ), to prevent further processing of deglucosylated N-linked oligosaccharides, Man6-8GlcNAc, but not Man9GlcNAc, were identified; the Man8GlcNAc component occurred as the characteristic isomer generated by endomannosidase cleavage. Although the endomannosidase-mediated deglucosylation pathway appeared to be nonselective, a differential inhibitory effect on the secretion of the various glycoproteins was noted in the presence of CST which was directly related to the number of their N-linked oligosaccharides, ranging from minimal in alpha-fetoprotein to substantial (approximately 65%) in alpha 1-acid glycoprotein. Addition of DMJ to CST-incubated cells did not further decrease secretion of the glycoproteins, although processing was now arrested at the polymannose stage, and a portion of the oligosaccharides were still in the glucosylated form. These latter findings indicate that complex carbohydrate units are not required for secretion of these glycoproteins and that any effect which glucose residues exert on their intracellular transit would be related to movement from the endoplasmic reticulum to the Golgi compartment.


Mutations in the Trp-Ser-X-Trp-Ser motif of the erythropoietin receptor abolish processing, ligand binding, and activation of the receptor. The erythropoietin receptor (EPOR) is a member of the newly identified cytokine receptor superfamily. A common sequence motif, Trp-Ser-X-Trp-Ser (WSXWS), near the transmembrane domain is highly conserved in this family. To determine the function of this motif, we constructed deletion and insertion mutations in this part of the EPOR and introduced them into an interleukin-3 (IL-3)-dependent hematopoietic Ba/F3 cell line. Cells expressing the wild-type EPOR displayed 1,500 erythropoietin (EPO)-binding sites/cell with a single affinity of about 300 pM and proliferate in the presence of IL-3 or EPO. Ba/F3 cells expressing receptors mutated in the WSXWS motif displayed little EPO binding on the cell surface and did not grow in the presence of EPO. The mutant receptors were retained in the endoplasmic reticulum (ER) and, as such, were unable to bind EPO. A single Gly insertion between the two WS sequences caused defects in receptor structure and function similar to mutations lacking all or part of the WSXWS motif. The EPOR can be activated, resulting in proliferation independent of EPO either by an Arg129 to Cys point mutation or by association with the Friend spleen focus-forming virus (SFFV) envelope glycoprotein gp55. Introduction of the point mutation (Arg129 to Cys) did not activate any of the receptors mutated in the WSXWS motif. Moreover, gp55 did not activate the mutant receptors in Ba/F3 cells. Our study indicates that the WSXWS motif is critical for protein folding, ligand-binding, and signal transduction.


Purification of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii and identification of a subunit of the enzyme. UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) from the soil amoeba Acanthamoeba castellanii has been purified over 100,000-fold by means of wheat germ agglutinin-Sepharose affinity chromatography, DEAE-cellulose chromatography, concanavalin A-Sepharose affinity chromatography, orange A-agarose dye chromatography, and gel filtration on Superose 6. The most purified enzyme has an estimated specific activity of at least 5 mumol of GlcNAc-phosphate transferred/min/mg of protein using alpha-methylmannoside as acceptor. The molecular weight of the native enzyme is approximately 250,000, as determined by gel filtration and glycerol gradients in H2O and D2O. A protein with an apparent M(r) of 97,000 in small scale preparations and its putative proteolytic fragment of 43,000 in large scale preparations co-purifies with the enzyme activity. This protein is covalently modified with GlcNAc-[32P]phosphate when the enzyme preparation is incubated with [beta-32P]UDP-GlcNAc in the absence of an acceptor substrate. The labeling of the 97(43)-kDa protein requires active enzyme and is completely inhibited by the addition of the acceptor substrate alpha-methylmannoside. The GlcNAc-[32P]phosphate transferred to the protein is not bound to serine, threonine, tyrosine, or mannose residues. The 97(43)-kDa protein with covalently bound GlcNAc-P does not serve as a kinetically competent enzyme-substrate intermediate. However, preincubation of GlcNAc-phosphotransferase with UDP-GlcNAc does result in a decrease in the Vmax of the enzyme in subsequent assays. Taken together, these data are consistent with the 97(43)-kDa protein being a subunit of GlcNAc-phosphotransferase.


Characterization of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii. The kinetic properties of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) partially purified from the soil amoeba Acanthamoeba castellanii have been studied. The transferase phosphorylated the lysosomal enzymes uteroferrin and cathepsin D 3-90-fold better than nonlysosomal glycoproteins and 16-83-fold better than a Man9GlcNAc oligosaccharide. Deglycosylated uteroferrin was a potent competitive inhibitor of the phosphorylation of intact uteroferrin (Ki of 48 microM) but did not inhibit the phosphorylation of RNase B or the simple sugar alpha-methylmannoside. Deglycosylated RNase (RNase A) did not inhibit the phosphorylation of RNase B or uteroferrin. These results indicate that purified amoeba GlcNAc-phosphotransferase recognizes a protein domain present on lysosomal enzymes but absent in most nonlysosomal glycoproteins. The transferase also exhibited a marked preference for oligosaccharides containing mannose alpha 1,2-mannose sequences, but this cannot account for the high affinity binding to lysosomal enzymes. A. castellanii extracts do not contain detectable levels of N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, the second enzyme in the biosynthetic pathway for the mannose 6-phosphate recognition marker. We conclude that A. castellanii does not utilize the phosphomannosyl sorting pathway despite expression of very high levels of GlcNAc-phosphotransferase.


An ELISA capture assay for the E7 transforming proteins of HPV16 and HPV18. ELISA capture assays were established for the E7 transforming proteins of HPV16 and HPV18, based on a range of previously characterised polyclonal and monoclonal antibodies. No cross-reactivity was observed in the ELISAs between HPV18 E7 and HPV16 E7. Immunoreactive E7 protein (iE7) was measured in a series of HPV-transformed cell lines, and ranged from 0.6 to 17.7 ng iE7/mg cell protein. iE7 was labile at 22 degrees C (t1/2 = 37 min) but relatively more stable at 4 degrees C (t1/2 = 210 min). HPV16 E7 protein at concentrations from 0.10 to 0.69 ng iE7/mg cell protein was detected in 5 of 13 smears from women with abnormal cervical cytology. Assay of E7 protein may play a role in the detection of HPV-induced cervical lesions with malignant potential.


Detection of hepatitis C viral RNA by the polymerase chain reaction in serum of patients with post-transfusion non-A, non-B hepatitis. Serial serum samples from cardiac patients with a history of chronic or resolved post-transfusion non-A, non-B hepatitis were analyzed by a combination of cDNA synthesis and the polymerase chain reaction (cDNA/PCR) to amplify HCV RNA. Analysis of sera drawn after the acute hepatitis episode from 8 of the patients who had an acute, resolving HCV infection showed no detectable levels of HCV RNA when primers from the NS3 region were used. Evaluation of these sera with primers from the 5'-untranslated (5'-UT) region revealed that one patient was positive for HCV RNA. Further analysis of serial serum samples available from two of these patients indicated that a resolved infection was associated with a disappearance of detectable HCV RNA after a peak level during the acute phase of the disease. In contrast, post-acute samples from 4 of 6 patients with symptomatic acute HCV infection evolving to chronicity were positive for HCV RNA using primers from the NS3 region, however, upon retesting with primers from the 5'-UT region, all 6 patients were found to be positive. Analysis of serial serum samples from 2 of these patients showed the persistence of HCV RNA in 70% of the samples. These two patients were subsequently treated with interferon alpha-2b. One patient resolved his disease and normalized his aminotransferase level during treatment and thereafter, while the other relapsed upon cessation of treatment. In these two patients, normalization of ALT levels was consistent with the absence of HCV RNA while relapse of disease was confirmed by the reappearance of detectable levels of HCV RNA. These results indicate the utility of HCV RNA as a marker for persisting HCV viremia and in differentiating patients with ongoing active HCV infection from those with an acute resolving disease.


Propagation of adenovirus types 40 and 41 in the PLC/PRF/5 primary liver carcinoma cell line. The sensitivity of cell cultures to adenovirus types 40 and 41 (Ad40/41) was compared by means of cell culture infectious dose (ID50) assays using monolayer cultures in microtitre plates. The PLC/PRF/5 cell line derived from a primary human hepatocellular carcinoma was 100 times more sensitive to a laboratory strain of Ad41, and 10 times more sensitive to a laboratory strain of Ad40 and two Ad41 stool isolates, than Graham 293 and Chang conjunctival cells commonly used for the propagation of these viruses. In microtitre plate titration assays PLC/PRF/5 cells retained an optimal condition for longer and displayed cytopathogenic effects earlier and more clearly than the other cell lines. In contrast to previously used cells, PLC/PRF/5 cells also proved successful for the quantitation of Ad41, but not Ad40, by conventional plaque assays. The reason for the exceptional susceptibility of PLC/PRF/5 cells has not been elucidated, but the findings open attractive new doors for research on Ad40/41.


Increased sensitivity for detection of human cytomegalovirus in urine by removal of inhibitors for the polymerase chain reaction. The presence of inhibitors in urine interferes with the enzymatic reaction of the polymerase chain reaction (PCR) for detection of human cytomegalovirus (HCMV). To remove inhibitors, HCMV virions in urine were precipitated with polyethylene glycol, or DNA was extracted from urine by the use of glass powder and subjected to PCR followed by Southern blot hybridization with alkaline phosphatase-linked oligonucleotide probes. These simple, rapid methods increased significantly the sensitivity of PCR for detection of HCMV in urine.


Effects of cAMP and forskolin on caffeine-induced contractures and myofilament Ca-sensitivity in saponin-treated rat ventricular trabeculae. Trabeculae from the right ventricle of rat were chemically 'skinned' with saponin (50 micrograms ml-1 for 30 min). Caffeine (10 mM) induced a transient contracture as a consequence of Ca(2+)-release from the sarcoplasmic reticulum and subsequent activation of the myofilaments. The amplitude of the caffeine contracture was used as an index of the Ca(2+)-content of the sarcoplasmic reticulum. cAMP (0.1-10 microM) markedly potentiated the caffeine-induced response under standardized conditions. This was interpreted as a cAMP-induced increase in Ca2+ accumulation by the sarcoplasmic reticulum during the Ca2+ loading period before the application of caffeine. After removal of cAMP, the amplitude of the regularly evoked contractures slowly declined towards standard levels. The characteristic effects of cAMP on the caffeine contracture were mimicked by addition of forskolin (10(-6) M), a substance known to stimulate cAMP production by adenylate cyclase. The results suggest that a significant quantity of functional adenylate cyclase persists after saponin treatment. In these preparations cAMP had no effect on the apparent Ca(2+)-sensitivity of the myofilaments. If the preparations were exposed briefly to saponin, cAMP caused a decrease in the apparent Ca2+ sensitivity of the contractile proteins. However, further exposure to saponin, or to Triton X-100, caused a marked increase in maximum Ca(2+)-activated force (Cmax). It was concluded that 'briefly' saponin-treated preparations, exhibiting a reduction in Ca2+ sensitivity in response to cAMP, are not uniformly permeable to the bathing solution.


Dispositions of junctional feet in muscles of invertebrates. The structure and disposition of the feet occupying the junctions between sarcoplasmic reticulum (SR) and surface membrane/transverse tubules were studied in muscles from a variety of invertebrates. Feet were imaged by rotary shadowing of isolated junctional SR vesicles and by filtering of micrographs from grazing views of the junction in thin sections. The overall size and shape of invertebrate feet is the same as that of feet in skeletal and cardiac muscle of vertebrates. However, the arrangement of feet in invertebrate muscles differs from that in vertebrates. These findings are discussed in terms of known variations in properties of excitation-contraction coupling of the two phyla.


Integration of human papillomavirus types 16 and 18 in cervical adenocarcinoma. To determine which type of human papillomavirus (HPV) is associated with cervical adenocarcinoma and whether the virus was integrated or episomal in two continents. Biopsy specimens from the UK (n = 16) and South Africa (n = 22) were analysed by non-isotopic in situ hybridisation (NISH) for HPV types 6, 11, 16, 18, 31, 33, and 35 on archival biopsy specimens using digoxigenin labelled probes. A total of 20 adenocarcinomas (53%) from both groups contained HPV DNA. In the UK group, seven and four cases contained HPV 18 (44%) and 16 (25%) respectively. In the South African group, nine cases contained HPV 18 (41%) while HPV DNA was not detectable in the other 13 cases. Hence HPV 18 was present in 80% of HPV positive adenocarcinomas. The HPV 16 or 18 genome was integrated in all viral positive cases. In two cases HPV 18 was also present in an episomal form. These data indicate that HPV integration is common to cervical adenocarcinoma in two continents by the same methodology. The lower prevalence of HPV 18 detection in the South African group may have been due to the presence of other or unsequenced HPV types.


Epstein-Barr virus DNA in nasopharyngeal carcinomas from Chinese patients in Hong Kong. To investigate the presence of Epstein-Barr virus (EBV) in cases of nasopharyngeal carcinoma (NPC) in Chinese patients living in Hong Kong. Nasopharyngeal biopsy specimens, formalin fixed and paraffin wax embedded, from 24 patients, eight with undifferentiated nasopharyngeal carcinoma, eight with well differentiated squamous carcinoma, and eight showing normal tissue histology, were analysed for the presence of Epstein-Barr virus (EBV) DNA by slot-blot hybridisation on extracted unamplified DNA, and also after amplification of EBV specific sequences by the polymerase chain reaction (PCR). DNA slot-blot analysis showed viral DNA in all the undifferentiated, five of the well differentiated tumours, and none of the normal biopsy specimens. PCR studies confirmed positivity in the eight undifferentiated tumours, but six of the well differentiated tumours and three of the normal biopsy specimens showed viral DNA by this method, illustrating its greater sensitivity. EBV genome is present in appreciable copy number in most cases of well differentiated NPC in Chinese patients in Hong Kong.


Detection of high risk human papillomavirus in routine cervical smears: strategy for screening. To develop a methodology for direct detection of high risk human papillomavirus (HPV) infection in routine cervical smears by non-isotopic in situ hybridisation (NISH) which can be compared with cytopathological assessment of the same cells. The methodology was established using cultured cells and routine cervical smears hybridised with digoxigenin labelled probes for HPV, 16, 18, 31, and 33. The technique was applied to the analysis of 53 patients from a sexually transmitted disease clinic. The optimal sensitivity achieved for single HPV detection in cultured cells was 1-2 copies of HPV 16 per cell and that for detection of a cocktail of HPV types in routine cervical smears was 2.5-12 copies per cell. Of parallel smears taken from patients with a normal Papinacolau-stained smear 33.3% (24) contained a HPV 16, 18, 31, and 33 signal indicating an occult HPV infection. The prevalence of these HPV types was similar in women in whom a cytopathological diagnosis of wart virus infection was made (64.7%, 17) and in patients with mild dyskaryosis (75%, 12). The methodology evolved localises HPV sequences directly to epithelial cell nuclei, which can be morphologically assessed by haematoxylin counterstaining. Sample contamination with exogenous viral sequences can be distinguished from true infection. In this study, a HPV signal was not found in morphologically normal epithelial cells. The methods described will permit the detection of HPV sequences in routinely collected cervical smears and the evaluation of the natural history and potential clinical relevance of HPV infection without changes in clinical practice.


Reactivation of polyomavirus in bone marrow transplant recipients. Polyomavirus was detected in the urine samples of 12 (48%) out of 25 patients within three months of receiving a bone marrow transplantation. The virus was first detected 11 to 46 days after the transplantation and excretion persisted for up to 42 days. Detection of the virus was not associated with symptoms and it seemed to be a marker of immunosuppression.


Myofibroblasts in hepatitis B related cirrhosis and hepatocellular carcinoma. Peritoneal liver biopsy specimens from eight patients with hepatitis B associated cirrhosis, complicated by hepatocellular carcinoma, were studied for identification and localisation of myofibroblasts. The avidin-biotin peroxidase complex technique was used on paraffin wax sections, using monoclonal antibodies for actin and desmin, and ultrastructural examination was performed. Myofibroblasts were found in seven of the eight cirrhotic specimens and in all eight tumour specimens. They were identified in the fibrotic areas by the immunohistochemical technique, but ultrastructural examination disclosed their presence in the perisinusoidal space and between tumour cells.


Signet-ring cell carcinoma of the prostate mimicking primary gastric carcinoma. A post mortem examination of a 70 year old man, who died three years after a poorly differentiated adenocarcinoma of prostate had been diagnosed, showed widespread signet-ring cell carcinoma, with an associated linitis plastica. The signet-ring cells stained positively with prostatic specific antigen and with prostatic specific acid phosphatase, but failed to react with mucopolysaccharide staining. The electron microscopic appearance of the signet-ring cell tumour was due to the presence of large cytoplasmic vacuoles. This case emphasises the possibility that cases of metastatic signet-ring cell carcinoma may be prostatic in origin. This can be confirmed by specific immunohistochemical studies.


Macrophage function in the acute phase of lactic dehydrogenase virus-infection of mice: suppression of superoxide anion production in normal mouse peritoneal macrophages by interferon-alpha in vitro. The effect of interferon (IFN)-alpha on the release of superoxide anions (O2-) by normal mouse macrophages (PEM) was examined. Sera from LDV-infected mice at 1 day, but not at 7 days post-infection, suppressed the O2- release by PEM. When PEM were exposed in vitro for 24 h to IFN-alpha, their capacity to release O2- was significantly suppressed. Progressive suppression of O2- release with increasing IFN-alpha concentration was observed. These results suggest that IFN-alpha in the circulation may be one of several suppressive factors on macrophage function in the early phase of infection and IFN-alpha may play a modulatory role in inflammation and immunity.


Basal cell carcinoma of the scrotum. Report of three cases and review of the literature. Although basal cell carcinoma (BCC) is the most common human malignancy, only 21 cases involving the scrotum have been previously reported. Our purpose is to describe three additional cases of scrotal BCC and review the literature summarizing the clinical features and identifying any predisposing factors. We retrospectively reviewed 21 cases of scrotal BCC and described three new cases. Polymerase chain reaction (PCR) was used to detect human papillomavirus (HPV) DNA in our biopsy specimens. Scrotal BCCs present as persistent ulcerations or plaques without identifiable predisposing factors. Lymphatic, pulmonary, or skin metastases were present in 3 of 24 cases (13%) resulting in death in one case. PCR did not detect HPV DNA in our three cases. Scrotal BCC rarely occurs and should be considered in the diagnosis of a persistent scrotal ulcer or plaque. Metastatic disease may be more common than with other BCCs and wide local excision or Mohs micrographic surgery may be the most appropriate initial therapeutic approach.


Detection of herpes simplex virus DNA in the peripheral blood during acute recurrent herpes labialis. Although herpes simplex virus (HSV) has been detected in the peripheral blood of immunocompromised patients and in neonates with disseminated disease, the extent to which this virus may be present in the blood during a localized infection in otherwise healthy adults is unknown. The purpose of this study was to determine whether HSV may be detected in the peripheral blood during acute recurrent herpes labialis. Peripheral blood mononuclear cells (PBMCs) were obtained from otherwise healthy adults with recurrent herpes labialis, both during an acute episode and several weeks after the lesions had healed. The PBMCs were examined for the presence of HSV with the polymerase chain reaction (PCR) and viral culture. By PCR, HSV DNA was detected in 7 of 34 specimens from an acute episode but in none of 24 specimens in the convalescent stage (p less than 0.004). PBMCs from seven donors, who were seronegative for HSV, were also negative for HSV by PCR. Viral cultures of 22 PBMC specimens were negative (including four specimens that were positive by PCR). The presence of HSV DNA in the blood is a transient phenomenon limited to the period of active infection in a minority of patients with herpes labialis, although it may be important in the development of disseminated disease as well as in the pathogenesis of herpes-associated cutaneous processes such as erythema multiforme.


Lithium chloride-sodium propionate agar for the enumeration of bifidobacteria in fermented dairy products. Lithium chloride-sodium propionate agar has been developed for the enumeration of bifidobacteria in fermented dairy products. The medium contains lithium chloride and sodium propionate to inhibit the growth of other lactic acid bacteria. Pure cultures of bifidobacteria, lactobacilli, and streptococci were tested for growth in this medium. With one exception, all bifidobacteria were able to grow in this medium and in a nonselective agar with a difference not exceeding .4 log units. However, none of the lactobacilli tested and only one strain each of Streptococcus salivarius ssp. thermophilus and Lactococcus lactis ssp. cremoris grew in lithium chloride-sodium propionate agar. In those cases, the numbers of colonies were lower in lithium chloride-sodium propionate agar by 1.26 and 2.51 log units, respectively, compared with a nonselective agar. Bifidobacteria were also selectively isolated from all fermented milks and cheeses analyzed.


Impact of in vitro fermentation techniques upon kinetics of fiber digestion. Three in vitro fermentation experiments were conducted to examine the impact on kinetics of fiber digestion of microminerals and tryptone addition, media reduction, fermentation vessel, CO2 gassing regimen, and buffer type. Alfalfa and bromegrass hays were incubated for 0, 4, 8, 12, 18, 24, 30, 36, 48, 72, and 96 h and analyzed for NDF. Kinetic measures of fiber digestion were estimated using nonlinear regression with iteratively reweighted least squares. In Experiment 1, continuous CO2 gassing increased rate and decreased lag time prior to NDF digestion compared with purging a non-CO2-saturated buffer at inoculation. Vessel type (50-ml polypropylene tube, 125-ml pyrex Erlenmeyer flask), use of additives (microminerals, tryptone), and media reduction had no effect on kinetics of NDF digestion. In Experiment 2, elimination of both media reduction and nutritive additives increased the lag time prior to NDF digestion. In Experiment 3, continuous CO2 gassing of buffer in 125-ml Erlenmeyer flasks resulted in faster rates of NDF digestion than CO2-saturated buffer in 50-ml screw-cap polypropylene tubes. The method that yielded the fastest rates and shortest lag times of NDF digestion consisted of continuous CO2 gassing, reduction, and use of additives to ensure that no nutrient limited fiber digestion.


In vitro buffering capacity changes of seven commodities under controlled moisture and heating conditions. Effects of adding 0 or .25 g of water/g of feed and heating for 0, 20, or 40 h at 100 degrees C on buffering capacity of beet pulp, almond hulls, alfalfa hay, oat hay, wheat bran, oat bran, and barley were determined in vitro. Feeds were examined for ash, N, NDF, ADF, acid detergent lignin, total buffering capacity (pH 4 to 9), and physiological buffering capacity (pH 5.5 to 7). Minerals were measured at 0 g of water/g of feed, 0 h of heat and at .25 g of water/g of feed, 40 h of heat; differences were negligible. Total buffering capacity was high for alfalfa, oat hay, and almond hulls; low for barley and oat bran; and intermediate for beet pulp and wheat bran. Physiological buffering capacity was consistently lower than total buffering capacity, but differences varied among and within feeds. Moisture had little effect on buffering capacity, NDF, ADF, or lignin. Time of heating had a significant effect on buffering capacity, NDF, ADF, and lignin but not on N or ash. Heating increased buffering capacity in most cases. Lignin was a poorer predictor of buffering capacity than total ash across feeds but a better predictor within feed across treatments. Large errors could occur when using total ash to predict buffering capacity for feeds subjected to heat.


A computer model of a cochlear-nucleus stellate cell: responses to amplitude-modulated and pure-tone stimuli. A computer model of a ventral-cochlear-nucleus (VCN) stellate cell with chop-S type response properties is presented and evaluated. The model is based on a simplified model of spike generation preceded by a stage that simulates dendritic low-pass filtering. Input to the model is in the form of simulated auditory-nerve spikes produced by a model of the auditory periphery [Meddis and Hewitt, J. Acoust. Soc. Am. 89, 2866-2882 (1991)]. Outputs from the stellate-cell model are shown to qualitatively replicate a wide range of typical in vivo responses. These include: (a) realistic onset and steady-state rate-level functions, (b) "chopper"-type post-stimulus time histogram responses; (c) typical "chop-S"-type neuron responses characterized by a low coefficient of variance (CV less than 0.3) of interspike intervals as a function of time; (d) level-dependent amplitude-modulation transfer functions; (e) intrinsic oscillations in responses to pure-tone stimuli; (f) amplitude-modulation encoding over a wide dynamic range; and (g) frequency-limited phase locking to pure tones. It is shown that these responses can be explained primarily by the membrane properties of the cells. More specifically, how the model encodes signal amplitude modulation was studied and an explanation was suggested for the generation of the bandpass modulation transfer functions. Such functions are observed neurally in response to amplitude-modulated stimuli presented at moderate to high signal levels.


Molecular biology of smooth muscle.

Molecular biology of smooth muscle. Several myosin isoforms are expressed in smooth muscle, based upon both heavy and light chain polymorphisms. These myosin isoenzymes reveal distinct detachment rate constants, thus regulating the responsiveness of the VSMC contractile apparatus to Ca2+, as well as its energetical and mechanical behavior.


The effect of a converting enzyme inhibitor upon renal damage in spontaneously hypertensive Fawn Hooded rats. To determine the effect of angiotensin converting enzyme inhibition (CEI) upon renal function and the incidence of glomerulosclerosis in spontaneously hypertensive Fawn Hooded rats (FHR). Male FHR were treated with captopril from the age of 5 months when mild hypertension, proteinuria and glomerulosclerosis are present, and sacrificed at 12 months of age. Renal function was determined in separate groups of FHR at 6 months of age. Proteinuria, body weight and systolic blood pressure were determined at regular intervals. Blood pressure was measured by the tail-cuff method. Kidneys were prepared for histological examination by standard methods. Renal function was determined by inulin clearance and urinary kallikrein by an amydolitic assay. In untreated FHR blood pressure, proteinuria and glomerulosclerosis increased with time. Captopril normalized blood pressure and stabilized proteinuria at pretreatment levels. At the end of the study, the incidence of glomerulosclerosis was significantly lower and comparable with the incidence at 5 months. Glomerular volume did not show a correlation with the incidence of glomerulosclerosis. Hemodynamic studies showed a significant increase of glomerular filtration rate in captopril-treated rats. No statistically significant effect was seen on renal plasma flow or filtration fraction. Urinary excretion of kallikrein was increased in captopril-treated rats. CEI is effective in protecting the kidney from structural damage in hypertensive FHR even when treatment is started under conditions of established glomerular injury. The protection given by captopril is probably related to intrarenal effects.