Feasibility and reliability of ankle/arm blood pressure index in preventive medicine.

Despite its potential usefulness for assessing preclinical atherosclerosis and cardiovascular risk, the ankle/arm blood pressure index (AAI) has not yet been the matter of study evaluating its feasibility and reliability by nonspecialist doctors in a general population. This study was planned for two steps. In step 1, the measurement of AAI, (ratio between Doppler systolic pressure at the ankle for each lower limb and the highest value of Doppler systolic pressure of the two upper limbs), should be performed by 50 general practitioners (GPs), 50 social security center physicians, and 50 occupational health physicians in 3,000 male smokers, 40 to 59 years, without clinical cardiovascular disease. In step 2, AAI measurement, coupled with echography-Doppler of iliofemoral arteries, should be repeated by a specialist in all subjects with decreased AAI (<0.90) and the first two subjects with normal AAI recruited in step 1 by each nonspecialist. The number of physicians and subjects participating in step 1 was lower than planned (80 physicians and 962 subjects) with the greatest defect for GPs (six physicians and 35 subjects) and the prevalence of decreased AAI was low (28 subjects). AAI measurement was repeated in step 2 in only 12 subjects with decreased AAI in step 1 and in 124 subjects with normal AAI in step 1. Five of the six subjects with decreased AAI in step 2 also had decreased AAI in step 1 and 123 of the 130 subjects with normal AAI in step 2 also had normal AAI in step 1. As regards echographic stenosis, decreased AAI had a sensitivity of 44% and a specificity of 98%. AAI seems more feasible for occupational health physicians and social security center physicians and AAI is also reliable for nonspecialists previously trained, but its predictive value as regards echographic stenosis is poor in asymptomatic subjects, which may limit its usefulness for detecting preclinical atherosclerosis.

Unusual activation of the integrated preS1 promoter of woodchuck hepatitis virus in a liver tumour.

We have analysed abnormal virus RNAs produced from integrated woodchuck hepatitis virus (WHV) sequences in two woodchuck liver tumours. Analysis of cDNA clones revealed that these transcripts consisted of rearranged, virus-specific RNAs encoding the WHV surface antigens. In one tumour, transcription was driven by the major preS2/S promoter and terminated at a cryptic poly(A) signal in the 5' end of the P gene, giving rise to a truncated version of the normal viral S message. In contrast, the integrated preS2/S promoter remained silent in the second tumour. The start sites of two abundant WHV transcripts encoding the large and middle surface proteins were localized about 100 bp upstream and 300 bp downstream of the preS1 translation initiation codon, corresponding to minor start sites of the normal surface protein mRNAs in chronically infected liver. Thus, the preS1 promoter, a weak promoter in episomal replicative forms of the virus, was activated in the integrated state in this tumour. Our results indicate that alternative usage of the preS1 or the preS2/S promoter in the integrated state may yield differential production of the three virus surface proteins in woodchuck liver tumours.

Hepadna virus integration generates virus-cell cotranscripts carrying 3' truncated X genes in human and woodchuck liver tumors.

Integration of the human and woodchuck hepatitis B viruses (HBV and WHV) in host chromosomes has been implicated in the development of hepatocellular carcinoma by different cis- and trans-acting mechanisms. The structure and coding capacity of abundant HBV and WHV transcripts of abnormal sizes produced from integrated viral sequences in one human and two woodchuck liver tumors were examined. Analysis of cDNA clones revealed in all cases hybrid virus-cell transcripts containing sequences of the viral surface gene, the viral enhancer, and different truncated versions of the viral X transactivator. Cotranscribed cellular sequences showing no significant coding function provided the signals for transcription termination. In two transcripts, the HBX and WHX genes truncated at carboxy terminal positions conserved transcriptional trans-acting capacity in transient transfection assays. These results lend support to the hypothesis that the integrated hepadnavirus X transactivator might participate in the development of woodchuck as well as human liver tumors by a common trans-acting mechanism.

Liver-specific expression and high oncogenic efficiency of a c-myc transgene activated by woodchuck hepatitis virus insertion.

The high oncogenic efficiency of woodchuck hepatitis virus (WHV) has been correlated with the ability of this virus to provoke insertional activation of myc family genes. To assess the impact of viral integration on liver cell transformation, we have generated transgenic mice carrying the mutated c-myc gene and adjacent viral DNA from a woodchuck tumor, in original configuration. Virtually all mice from two different strains developed hepatocellular carcinoma with a mean latency period of 8-12 months. The c-myc transgene was expressed transiently in neonatal livers, and re-expressed at preneoplastic and neoplastic stages in adult livers. Woodchuck c-myc mRNA driven by the normal P1 and P2 promoters and WHV-specific transcripts encoding viral surface antigens were produced in a strictly co-regulated fashion during development and tumorigenesis, indicating a predominant regulatory influence of the viral enhancer. Furthermore, the activity of the viral enhancer in response to various biological stimuli was apparently modulated by glucose uptake and glucagon/insulin balance in differentiated hepatocytes. In this model, a viral integration event selected from a naturally occurring tumor proved to be determinant for induction of hepatocarcinogenesis, although enforced, liver-specific expression of c-myc was limited to a particular developmental stage.

Evolutionary conservation of target sequences for cis-acting regulation in c-myc exon 1 and its upstream region.

Transcriptional control of the c-myc proto-oncogene, an important factor in cellular growth, differentiation and in the genesis of various neoplasms, is mediated by multiple positive and negative regulators in the 5' end region of the gene. Here, we report the nucleotide sequence of the first c-myc exon and its upstream region from woodchuck, a rodent which can develop liver tumors associated with c-myc activation [Möröy et al., Nature 324 (1986) 276-279]. Alignment of these sequences with the corresponding human and murine regions shows a surprisingly high homology between woodchuck and human, and suggests the absence of species-specificity in the fundamental regulatory elements which govern c-myc expression.

Integration of hepatitis virus DNA near c-myc in woodchuck hepatocellular carcinoma.

A total of 33 hepatocellular carcinomas, induced in woodchucks by chronic infection with woodchuck hepatitis virus (WHV), a virus closely related to the human hepatitis B virus, were analyzed for the state of viral DNA, the expression of viral genes and of different cellular proto-oncogenes. Low levels of viral replication and presence of integrated viral forms including sequences of the enhancer element, appeared as a general rule in these tumors. Enhanced expression of one or more of the nuclear protooncogenes: c-myc, N-myc, c-fos, c-jun and jun-B was frequently observed. In two hepatomas, elevated expression and allelic alterations of c-myc were subsequent to integration of WHV DNA near the c-myc coding domain. The viral strategy for insertional activation of c-myc in these tumors appeared basically identical to that of mammalian retroviruses in T-cell lymphomas of mice and rats. Whether insertional mutagenesis of different oncogenes may be more generally linked to liver oncogenesis induced by WHV and hepatitis B viruses remains to be determined.

Fused transcripts of c-myc and a new cellular locus, hcr in a primary liver tumor.

The proto-oncogene c-myc has been implicated in the formation of primary liver tumors in hepatitis virus-infected woodchucks. In one of these tumors, a DNA rearrangement placed the truncated c-myc gene downstream of a cellular sequence (hcr) in a head-to-tail configuration resulting in 50-fold enhanced levels of c-myc transcripts. Analysis of the tumor-specific c-myc RNA now demonstrates that transformed liver cells produce fused hcr/myc transcripts initiated from the hcr promoter and extending into c-myc coding sequences by differential splicing mechanisms. In phase fusion of the reading frames of both genes might result in the translation of the hcr/myc 2.0 kb RNA into a hybrid protein that would differ from the normal woodchuck c-myc gene product by 22 additional hcr amino acids at its amino-terminus. The production of inappropriate levels of modified or normal myc-encoded proteins is probably involved in the malignant process.

Structure and expression of hcr, a locus rearranged with c-myc in a woodchuck hepatocellular carcinoma.

We have previously described a rearrangement of the proto-oncogene c-myc with a new cellular sequence of unknown function in a woodchuck primary liver tumor. We have now cloned and further analysed the normal woodchuck locus (termed hcr) of the sequence involved in the rearrangement with c-myc. The hcr locus is highly expressed in hepatocytes but not in other cell types examined and is conserved in mammals. Two unspliced hcr transcripts 4.5 and 4.7 kb long accumulate in liver cell nuclei. These transcripts differ only in their 3' extremities, located 180 bases apart, and by additional poly(A) tailing of the longer RNA species. The genomic sequence flanking the transcription start site contains variant elements of a classical eukaryotic promoter. Nucleotide sequence analysis of cDNA clones for the hcr RNA reveals that the 5' end of the hcr transcripts contains a short open reading frame of only 3 gamma codons initiated by an ATG. The biological function of her RNA remains to be determined.

Activation of c-myc by woodchuck hepatitis virus insertion in hepatocellular carcinoma.

Two hepatocellular carcinomas, induced in woodchucks chronically infected with woodchuck hepatitis virus, were characterized for viral integration near c-myc and alterations of c-myc expression. In one tumor, viral integration within the untranslated region of c-myc exon 3 resulted in overexpression of a long c-myc viral cotranscript. In the second tumor, a single insertion of highly rearranged viral sequences 600 bp upstream of c-myc exon 1 was associated with increased levels of normal c-myc mRNA. In both cases, viral enhancer insertion and disruption of normal c-myc transcriptional or posttranscriptional control appear to be involved in c-myc activation. These results demonstrate that integration of woodchuck hepatitis virus near a cellular proto-oncogene, as in several retroviral models, can contribute to the genesis of liver tumors.

Rearrangement and enhanced expression of c-myc in hepatocellular carcinoma of hepatitis virus infected woodchucks.

Hepatocellularcarcinoma (HCC) that occur in woodchucks chronically infected with woodchuck hepatitis virus (WHV) were screened for activation of cellular oncogenes. Enhanced expression and allelic alterations of the c-myc oncogene were found in three HCC out of nine. Variations in the size of the c-myc transcripts, ranging from 2.0 kilobases (kb) to 5.6 kb, as well as in the level of c-myc gene expression, 5-50-fold higher than in adjacent liver tissues, were observed among the three HCC. Rearrangements of the c-myc locus were either upstream of the gene or within the first intron. Cloning and sequencing of the break-point region from one of the three tumours showed that the c-myc gene was truncated and joined to a unique cellular sequence of unknown function. WHV DNA was not integrated near the c-myc coding exons, excluding a direct role of the virus in c-myc activation. The novel type of rearrangement and activation of the c-myc gene, reported here in liver tumours of hepatitis virus infected animals, appears strikingly similar to those resulting from chromosomal translocations in human Burkitt's lymphomas, acute B- and T-cell leukaemias and mouse plasmacytomas.

Nucleotide sequence of the woodchuck hepatitis virus surface antigen mRNAs and the variability of three overlapping viral genes.

A cDNA library was constructed from the liver of a woodchuck chronically infected with woodchuck hepatitis virus (WHV). A clone, pWS23, encompassing the entire surface and X genes of WHV was isolated. Comparison of the complete nucleotide (nt) sequence of pWS23 with those of genomic DNAs from two different WHV isolates showed that it contained a nearly full-length copy of the major mRNA encoding the viral surface antigen (S mRNA). It was colinear with the WHV genome over 1858 nt and terminated 22 nt downstream from the variant polyadenylation signal within the core gene. Evidence for heterogeneity of the 5'-terminal region of the S mRNA came from direct sequencing of the 5' extremities of 20 cDNA inserts, similar to that of pWS23, isolated from a second cDNA library of the same woodchuck liver. In agreement with previous mapping studies of hepadnaviruses, two main initiation regions of S mRNA were localized 27-30 nt upstream and 22-49 nt downstream from the pre-S2 initiation codon. Further analysis of the amino acid sequences of the surface, polymerase and X genes of WHV showed a high conservation among three WHV isolates and a similar distribution of conserved and variable regions in woodchuck and human hepatitis B viruses.

Transcription of woodchuck hepatitis virus in the chronically infected liver.

The transcription of woodchuck hepatitis virus (WHV) genome was studied in the liver of chronically infected woodchucks by Northern blot, nuclease mapping and primer extension analysis. Two major transcripts, 2.1 and 3.7 kb in length, and several minor transcripts were found in samples which supported active WHV replication. The 2.1-kb RNA represents the major transcript of the S gene, encoding the viral surface antigen (WHsAg) as demonstrated by blot-hybridization experiments. Two transcription initiation sites were localized downstream of the second AUG of the pre-S region, 139 and 152 nucleotides upstream of the translation initiation codon of the S gene. The 3.7-kb transcript, present in an equal amount, is slightly larger than the WHV genome and could be involved in the expression of all viral proteins. The data derived from RNA mapping strongly suggest that this transcript is initiated approximately 70 nucleotides upstream of the C gene, encoding the viral core antigen (WHcAg), and represents the message for WHcAg. It might also serve in the viral replication cycle as a potential template for reverse transcription. All WHV-specific transcripts were found to be processed at a unique site, 20 nucleotides downstream of the polyadenylation signal situated within the core gene. A different set of WHV-specific mRNAs was observed in a woodchuck hepatocellular carcinoma when only integrated forms of WHV DNA could be detected. Two RNA species of 2.3 and 4.6 kb were characterized. The 3.7-kb RNA was absent, reinforcing the hypothesis that this transcript corresponds to the pre-genome.

Erythrocytic pyruvate kinase deficiency and hemolytic anemia inherited as a dominant trait.

A nonspherocytic hemolytic anemia, associated with a pyruvate kinase (PK) deficiency apparently inherited as a dominant trait has been identified in a family. In the affected members, the residual PK activities were 20% that of normal controls, an unusually low level for heterozygous subjects. The anemia was mild except in the proband, a 2-year-old boy who suffered from a severe anemia. The PKs of the proband and of his both parents have been characterized as kinetically and electrophoretically normal enzymes. Immunoprecipitation tests indicated a large amount of L-type inactive protein in the proband and his father. Moreover, an M2 type PK was present in the father's hemolysate, suggesting the existence of a compensatory process that derepressed the corresponding structural gene. We suggest that the presence of one or more mutated subunits in the tetrameric forms of L-type PK leads to the inactivation of these tetramers. This hypothesis accounts for the low residual activity in affected heterozygous members of this family. The severity of the hematological symptoms in the proband, in comparison with the mild hemolysis observed in the other heterozygous members of the family points to the existence of a large spectrum of pathologic expression for an identical PK defect present in a family.

Immunological studies of PK and PFK deficiencies induced by chemotherapy.

Pyruvate kinase (PK) and phosphofructokinase (PFK) erythrocyte deficiencies induced by chemotherapy were studied in 6 patients. From immunological tests it may be assumed that the PK and PFK deficiencies were due to different direct mechanisms: a disturbance of the synthesis of one of the PFK subunit; mutation(s) in the structural gene of PK which result in the synthesis of mutant proteins. Several molecular mechanisms are probably at the origin of all the disturbances induced by chemotherapy in the red blood cells. The study of these alterations which mimic those detected in preleukaemic and leukaemic states, provides information on the molecular events in the possible production of chemo-induced cancers.

A liver-type mutation in a case of pronounced erythrocyte phosphofructokinase deficiency without clinical expression.

A marked erythrocyte phosphofructokinase deficiency was detected in a healthy man. His enzymatic activity was only 25% that of normal controls. His father and his son had erythrocytic phosphofructokinase activities of 50-55% that of normal controls. The chromatographic separation of erythrocytic phosphofructokinase isozymes, as well as immunological studies revealed a decrease in L-type phosphofructokinase activity. The lowered erythrocytic L-type phosphofructokinase activity was not accompanied by a decreased level of L-type phosphofructokinase in proteins. The L/M subunit ratio was similar to that of normal subjects. The defect resulted from the synthesis of stable L-type mutant subunit with high electrophoretic mobility. White blood cells, which synthesize mostly the same isozyme as L-type phosphofructokinase also showed a decreased activity and a high electrophoretic mobility. In spite of this important deficiency, and of significant metabolic alterations (a slight decrease in ATP; 2,3-diphosphoglycerate; triose phosphate), hemolysis did not appear in the propositus.

A red cell pyruvate kinase mutant with normal L-type PK in the liver.

The erythrocytic and liver pyruvate kinases (PK) from a patient with congenital nonspherocytic hemolytic anemia have been studied. In red blood cells, the residual activity, 28% of the normal control, presented normal kinetic properties, instability to heat and urea, and slow electrophoretic mobility. The L-type PK from the patient's liver was characterized by normal activity, kinetic properties, stability to heat and urea, and electrophoretic mobility. The fact that erythrocyte mutant PK may, as in previous reports, or may not be associated, as in the present observation, with molecular abnormalities of the liver PK provides support for the hypothesis of a gene rearrangement compatible with two different tissue-specific mRNAs.

Influence of free Mg2+ on the kinetics of human erythrocyte phosphofructokinase.

The influence of Mg2+ on the reaction catalyzed by human erythrocyte phosphofructokinase has been investigated using kinetic methods. The catalytic activity of PFK is dependent upon the presence of Mg2+ which constitutes with ATP the true Mg-ATP2- substrate. Free Mg2+ has no influence on the affinity of the enzyme for Mg-ATP2- substrate. Erythrocyte PFK is more inhibited by ATP4- and uncomplexed citrate than it is by Mg-ATP2- and Mg-citrate. Free Mg2+ relieves the MgATP2- and Mg-citrate inhibition under conditions where free ATP4-is negligible. We can assume that uncomplexed Mg2+ acts as positive effector by direct binding to the enzyme. These results emphasize the role of Mg2+ in the regulation of PFK activity in the erythrocyte.

Studies on the mechanism of the erythrocyte enzyme abnormalities induced by chemotherapy.

With the aim of determining the possible mechanisms of the red cell enzyme deficiencies induced by chemotherapy, deficient red cell glucose-6-phosphate dehydrogenase (G-6-PD), pyruvate kinase (PK) and phosphofructokinase (PFK) from 17 patients were purified and characterized. In all cases G-6-PD showed normal kinetics, electrophoretic mobility and thermostability suggesting that a decreased enzyme synthesis was possible for the deficient enzyme activity. In each case studied, at least one of the PK properties was modified, either in affinity for phosphoenol pyruvate, thermal stability or electrophoretic mobility, indicating a primary or secondary molecular abnormality. In some patients PFK had significantly increased affinity for citrate inhibitor; however, neither the quantity nor quality of the M subunits seemed to be altered. Thus it appears that chemotherapy can induce qualitative as well as quantitative red cell enzyme abnormalities by different mechanisms. These are similar to those observed in spontaneous leukaemia and preleukaemic states. Such a similarity poses the question of whether or not the red cell enzyme abnormalities induced by chemotherapy could be considered as the first sign of secondary leukaemia due to treatment by oncostatic drugs.