Modulation of the hyperdynamic circulation of cirrhotic rats by nitric oxide inhibition. The effects of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) biosynthesis on the splanchnic and systemic circulation, were investigated in rats with cirrhosis induced by carbon tetrachloride. Portal hypertension in these rats was accompanied by decreased arterial blood pressure and peripheral vascular resistance as well as by splanchnic vasodilation with increased portal venous inflow and decreased splanchnic resistance. Intravenous bolus administration of L-NMMA (25 mg/kg) significantly increased systemic blood pressure and decreased cardiac output. L-NMMA also significantly increased systemic and splanchnic vascular resistance; whereas blood flow to the stomach, small intestine, colon, pancreas, mesentery, spleen, and kidney was decreased significantly. L-NMMA did not alter the portal pressure or portosystemic shunting in these cirrhotic rats, yet portal vascular resistance increased, suggesting effects on the intrahepatic and collateral circulation. Pretreatment with L-arginine (300 mg/kg) prevented the hemodynamic changes induced by L-NMMA. These findings support the concept that local excess formation of NO contributes to changes in splanchnic circulation associated with portal hypertension in cirrhosis.


Comparison of different treatment modalities in experimental pancreatitis in rats. Lipolytic enzymes may play a role in the pathogenesis of acute pancreatitis. Therefore, the effects of a lipase inhibitor, THL (tetrahydrolipstatin), a protease inhibitor, FUT (nafamostat mesilate), and albumin under different conditions in rats were investigated. (a) Isolated pancreatic acini were incubated with pancreatic homogenates and triglycerides or lecithin with or without albumin and the degree of cellular destruction quantitated. (b) Taurocholate was injected into the pancreatic duct of isolated pancreas and the organ continuously perfused with either FUT, THL, or albumin. Organ damage was evaluated by measurement of pancreatic enzymes in the portal effluence. (c) Necrotizing pancreatitis was induced in vivo via retrograde taurocholate injection. FUT, THL, or albumin was applied either intravenously or injected into the pancreatic parenchyma. (a) Albumin prevented the cellular damage caused by both fatty acids and lysolecithin. (b) THL was ineffective, FUT lowered the release of pancreatic enzymes into the portal effluence, and albumin was most effective. (c) Albumin prevented the development of panlobular necrosis and lowered the degree of extrapancreatic fat necrosis. Albumin, via its ability to bind detergents, may have therapeutic implications.


Possible role of transforming growth factor alpha in the pathogenesis of Ménétrier's disease: supportive evidence form humans and transgenic mice. Ménétrier's disease is an uncommon disorder of unknown etiology characterized by enlarged gastric folds with foveolar hyperplasia and cystic dilatation of gastric glands. Biochemical features that are seen frequently include hypoproteinemia, hypochlorhydria, and increased gastric mucus. Because transforming growth factor alpha (TGF alpha) is an epithelial cell mitogen that inhibits gastric acid secretion and increases gastric mucin content, we hypothesized that its altered expression might be involved in the pathogenesis of this disease. Therefore, we characterized TGF alpha immunoreactivity in the gastric mucosa of 4 patients with Ménétrier's disease. In contrast to the normal pattern of TGF alpha immunostaining in which TGF alpha appears most concentrated in parietal cells, there was intense staining in the majority of mucous cells in the gastric mucosa of patients with Ménétrier's disease. In one patient from whom sufficient fresh tissue was obtained to isolate RNA, expression of TGF alpha and the epidermal growth factor receptor was higher in the gastric mucosa relative to a normal control. In addition, metallothionein-TGF alpha transgenic mice, which overexpress TGF alpha in gastric mucosa, show a number of features characteristic of Ménétrier's disease. These include foveolar hyperplasia and glandular cystic dilatation, increased gastric neutral mucin staining, and reduced basal and histamine-stimulated rates of acid production. Taken together, observations derived from the human material and correlation with data from a transgenic mouse model support an important role for TGF alpha in the pathogenesis of Ménétrier's disease.


[Hormonal contraception and substitution therapy: the importance of progestogen for cardiovascular diseases]. Epidemiological data have demonstrated, that the progestogen component of oral contraceptives is involved in the development of hypertension, ischaemic heart diseases and stroke. It had been suggested, that atherosclerotic lesions due to the unfavourable effect on lipid metabolism of progestogens with androgenic properties, play a causal role. It has, however, been shown, that there is no development of atherosclerosis despite reduced HDL and elevated LDL, presumably because of the induction of hepatic LDL- and remnant-receptors by the strong effect of ethinyl-oestradiol upon the liver. A series of experimental and clinical findings indicates that vasospasms caused by the vasoconstrictory effect of progestogens are involved in the development of arterial thromboses. In postmenopausal women, the additional administration of progestogens to the oestrogen treatment may trigger ischaemic diseases, particularly in the presence of vascular lesions. Oestrogens exercise a pronounced vasodilatory effect and stabilize the vascular tonus--through changes in the responsiveness of endothelium and smooth muscle cells to vasoactive compounds, through modulation of neurotransmitter release from nerve endings, and through direct blocking of calcium channels. The effects depend essentially on an intact endothelium. By a direct action on the vascular wall, progestogens increase the sensitivity of arteries to vasoconstrictory compounds and reduce blood flow. As aldosterone increases the number of beta-adrenergic receptors in the arterial smooth muscle cells and thus act vasodilatorily, it cannot be excluded, that progestogens with high affinity to the aldosterone receptor and antimineralocorticoid properties, may exert a strong vasoconstrictory effect.(ABSTRACT TRUNCATED AT 400 WORDS)


[Diagnosis of tubal function using intraluminal ultrasound--initial results]. The extremely small ultrasound transducers of the intraluminal ultrasound instruments, introduced via catheters, enable diagnosis to be made inside hollow organs. In order to test the possible uses and indications for this new method in gynaecology, we conducted preliminary examinations in the diagnosis of the uterus and tubes. We employed an intraluminal instrument supplied by Dornier. The intraluminal transducers of this instrument have a diameter of 3.5 and 5 F. Following in-vitro examinations, hysteroscopy and laparoscopy/laparotomy were performed in 15 patients during which the transducer was pushed up via the cervix uteri to the tubes with full vision. This was successful in all 15 patients; in 9 cases the transducer could be pushed as far as the distal end. The tubal walls were examined in detail by this method and for the first time it became possible to achieve functional diagnosis of the motility of the tubes. Strictures can be visualised. The endometrium of the uterus, however, cannot as yet be diagnosed exactly by the present-day state of examination technique. If image quality can be further improved, this method will be the first to enable a functional diagnosis of the tubes and the uterus.


[Fetal plexus cysts. An indication for prenatal karyotyping]. From March 1988 to September 1990, 1844 patients (pregnant women) were sonographically examined following a pregnancy monitoring programme and for further diagnostic clarification of unclear sonographic patterns. The ultrasound examinations were carried out with a 3.5 Mhz convex transducer, and by pregnancies before the 16th week with a 5 Mhz vaginal transducer (Toshiba SSA 250 a), respectively. In 13 cases (0.7%), foetal chorioidal plexus cysts were found; in 6 of them they occurred unilaterally and in the other 7 bilaterally. In general, most of the cases were diagnosed between the 13th and 22nd weeks of pregnancy. All unilateral cysts appeared transitorily, were of small size and receded by the 24th week of pregnancy. From the 7 patients with bilateral plexus cysts, 4 foetuses showed no signs of anatomical abnormalities. In the other 3 foetuses, it was possible, to recognise further anatomical abnormalities, when performing ultrasound i.e. 2 of them proved to be a trisomy 18 and the third case a trisomy 21.


[Pelviscopic hysterectomy--a prospective comparative study of 40 cases]. We review to date 20 pelviscopic extirpations of the uterus. This new method of hysterectomy shows some advantages over the common vaginal hysterectomy. Anatomy of the supporting ligaments of the uterus remains the same, recovery from the operation goes quicker. Morbidity rate seems to be less than in the case of vaginal hysterectomy.


[Systematic aspects of depot peritoneal lavage in gynecology]. After 9 years of experience in gynaecology, the systematics of depot peritoneal lavage is now open for discussion. Intraoperatively, a liquid residual depot of 1.5-2.0 l Ringer's solution remains, while the abdominal cavity is continuously rinsed with varying amounts of liquid according to the indication. By maintaining the depot in all lavages, the formation of adhesions is therefore prevented. Forms and indications of the lavages: 1. "Postoperative Clear Lavage": a) Removal of blood and tissue residues must be removed b) Control/early diagnosis of secondary haemorrhage 2. "Adhesion Lavage": Prophylaxis 3. "Anti-septic Massive Lavage": In all cases of inflammatory diseases of the abdominal cavity, to prevent abscess and adhesion formation, and in peritonitis, to remove bacteria, endotoxins and detritus. In severe ascending infections, complete organ preservation was possible in 94% of 330 patients by combining early laparoscopy with an antiseptic massive lavage. Results of the first two indications will be published shortly.


[The magnesium and calcium content of pre- and postmenopausal myometrium]. Magnesium and calcium, as well as sodium and potassium, have an important influence on the physiology of contraction of the uterus. The ionic concentrations in the pre- and postmenopausal myometrium were examined. We assumed, that there are differences in the electrolytic contents because of the different functions of the corpus, isthmic part and cervix uteri during pregnancy and throughout labour. Small tissue samples from 37 premenopausal and 20 postmenopausal patients, who underwent an operation with different indications, were dissected after hysterectomy. Magnesium and calcium levels were determined by atomic absorption spectrophotometry and potassium and sodium by emission spectrometry in the wet tissue. In both groups, we found significant differences of the electrolytic contents of the myometrium. In the premenopausal uteri, the magnesium levels decrease significantly (p < 0.001) from the corpus (mean = 4.14 mmol/kg) to the cervix (mean = 2.14 mmol/kg). The same scenario can be observed for the potassium levels (Corpus mean = 56.2 mmol/kg and cervix mean = 25.4 mmol/kg). In contrary, the calcium levels (Corpus mean = 1.60 mmol/kg and cervix mean = 2.26 mmol/kg) and sodium levels (Corpus mean = 69.3 mmol/kg and cervix mean = 93.0 mmol/kg) increase significantly (p < 0.001). In the postmenopausal group, we found slightly elevated concentrations of magnesium, sodium and potassium. Only the calcium content of the corpus uteri rises up to three times about 20 years after the menopause (from mean = 1.60 mmol/kg) (p < 0.001). The electrolytic contents in the myometrium were shown to be different in the corpus, isthmus and cervix uteri.(ABSTRACT TRUNCATED AT 250 WORDS)


[Subjective stress of adjuvant chemotherapy in breast cancer patients]. 50 patients, suffering from carcinoma of the breast pT1-3 with concomitant metastatic affection of the lymph nodes with no evidence of distant metastases, and who received adjuvant chemotherapy (either CMF od AC/EC) were examined. Somatic and psychic interferences, the feeling of well-being and general condition, as well as a critical estimation of the clinical situation were assessed. Besides nausea and vomiting, and still prior to hair fall out, chemotherapy activated the memory of the carcinoma. 92% of the patients agreeing to chemotherapy described their decision as having been based exclusively on the medical information. Nevertheless, 30% feel insufficiently informed, so the fear of side effects, the waiting period prior to application, and the confrontation with the seriously ill, were described as a negative experience. An optimistic view with respect to improving the prognosis of the disease were correlated with chemotherapy: 74% would agree to a further chemotherapy. Psychic abnormalities, e.g. depressions, could not be shown following chemotherapy when compared to a reference population.


[Acute non-immunologic hydrops fetalis with bilateral chylothorax in the 36th week of pregnancy]. Since the introduction of anti-D gamma-globulin prophylaxis non-immune hydrops fetalis (NIHF) has become the relevant and more common form of fetal hydrops. A case of an NIHF with chylothorax, diagnosed in the 36th week of pregnancy, is discussed with regard to obstetrical and neonatological management.


[Intraplacental hematoma following chorionic villi biopsy]. One of the most typical complications following chorionic villae sampling (CVS) is vaginal bleeding shortly or some days after the intervention, resulting in abortion in some cases. We report on a case of intraplacental haematoma (pathology: massive subtotal acute haemorrhagic placental infarct), followed by acute placental insufficiency in the 29th week of pregnancy. Therefore, we decided on an emergency caesarean section.


[In the light of a petroleum lamp the operation could begin. Richard Frommel (1854-1912): pioneer in treatment of ruptured extrauterine pregnancy]. Richard Frommel (1854-1912) can be called an outstanding German gynaecologist during the late 19th century. This article is intended to give a picture of his life, work, and personality. Frommel was director of the department of obstetrics and gynaecology at the university of Erlangen from 1887 to 1901. Science owes to him, amongst other contributions, an important impulse for changing the therapy of ruptured ectopic pregnancy. Up to the present day Frommel's name together with that of Johann Chiari (1817-1854), serve as eponyms for a special form of the amenorrhoea-galactorrhoea syndrome, namely, the persistent one-post partum. It remains puzzling why Frommel resigned office, when he was just 46 years old, giving up gynaecology completely only a short time later.


Fine structure mapping and complementation studies of the metD methionine transport system in Salmonella typhimurium. A fine structure deletion map of the metD region of the chromosome of Salmonella typhimurium responsible for a high-affinity methionine transport system has been constructed. Complementation tests involving the introduction of metD+DNA contained in a pUC8 vector into metD strains indicated the presence of four complementation groups in the metD region. This suggested that the methionine system belongs to the osmotic shock-sensitive class of transport system, and therefore should possess a periplasmic methionine-binding protein and several membrane proteins. But a deletion mutation covering all known metD point mutations did not affect the level of a methionine binding activity in osmotic shock fluids, suggesting either that the deletion did not extend into the gene encoding the binding protein, or that the binding activity is not associated with the metD system. Possible reasons for the failure to isolate mutations in the gene for the binding protein are discussed.


Genetics of sexual isolation in females of the Drosophila simulans species complex. Genetic analysis of hybrids between Drosophila simulans and D. sechellia shows that sexual isolation in females is caused by at least two genes, one on each major autosome, while the X chromosome has no effect. These results are similar to those of a previous study of hybrids between D. simulans and another sibling species, D. mauritiana. In this latter hybridization, each arm of the second chromosome carries genes causing sexual isolation in females, implying a total divergence of at least three loci. The genetic similarity between the D. simulans/D. mauritiana and D. simulans/D. sechellia hybridizations probably results from independent evolution and not phylogenetic artifacts, because the dominance relationships and behavioural interactions differ between the two hybridizations. The lack of an X-chromosome effect on sexual isolation contrasts with genetic studies of post-zygotic reproductive isolation, which invariably show strong effects of this chromosome.


Polymorphisms distinguishing different mouse species and t haplotypes. Three anonymous chromosome 17 DNA markers, D17Tu36, D17Tu43, and D17Le66B, differentiate between house mouse species and/or between t chromosomes. The D17Tu36 probe, which maps near the Fu locus and to the In(17)4 on t chromosomes, identifies at least 15 haplotypes, each haplotype characterized by a particular combination of DNA fragments obtained after digestion with the Taq I restriction endonuclease. Ten of these haplotypes occur in Mus domesticus, while the remaining five occur in M. musculus. In each of these two species, one haplotype is borne by t chromosomes while the other haplotypes are present on non-t chromosomes. The D17Tu43 probe, which maps near the D17Leh122 locus and to the In(17)3 on t chromosomes, also identifies at least 15 haplotypes in Taq I DNA digests, of which nine occur in M. domesticus and six in M. musculus. One of the nine M. domesticus haplotypes is borne by t chromosomes, the other haplotypes are borne by non-t chromosomes; two of the six M. musculus haplotypes are borne by t chromosomes and the remaining four by non-t chromosomes. Some of the D17Tu43 haplotypes are widely distributed in a given species, while others appear to be population-specific. Exceptions to species-specificity are found only in a few mice captured near the M. domesticus-M. musculus hybrid zone or in t chromosomes that appear to be of hybrid origin. The D17Leh66B probe, which maps to the In(17)2, distinguishes three haplotypes of M. domesticus-derived t chromosomes and one haplotype of M. musculus-derived t chromosomes. Because of these characteristics, the three markers are well suited for the study of mouse population genetics in general and of t chromosome population genetics in particular. A preliminary survey of wild M. domesticus and M. musculus populations has not uncovered any evidence of widespread introgression of genes from one species to the other; possible minor introgressions were found only in the vicinity of the hybrid zone. Typing of inbred strains has revealed the contribution of only M. domesticus DNA to the chromosome 17 of the laboratory mouse.


The role of DNA replication and isochores in generating mutation and silent substitution rate variance in mammals. It has been suggested that isochores are maintained by mutation biases, and that this leads to variation in the rate of mutation across the genome. A model of DNA replication is presented in which the probabilities of misincorporation and proofreading are affected by the composition and concentration of the free nucleotide pools. The relationship between sequence G+C content and the mutation rate is investigated. It is found that there is very little variation in the mutation rate between sequences of different G+C contents if the total concentration of the free nucleotides remains constant. However, variation in the mutation rate can be arbitrarily large if some mismatches are proofread and the total concentration of free nucleotides varies. Hence the model suggests that the maintenance of isochores by the replication of DNA in free nucleotide pools of biased composition does not lead per se to mutation rate variance. However, it is possible that changes in composition could be accompanied by changes in concentration, thus generating mutation rate variance. Furthermore, there is the possibility that germ-line selection could lead to alterations in the overall free nucleotide concentration through the cell cycle. These findings are discussed with reference to the variance in mammalian silent substitution rates.


Evidence for random distribution of sequence variants in Tenebrio molitor satellite DNA. Tenebrio molitor satellite DNA has been analysed in order to study sequential organization of tandemly repeated monomers, i.e. to see whether different monomer variants are distributed randomly over the whole satellite, or clustered locally. Analysed sequence variants are products of single base substitutions in a consensus satellite sequence, producing additional restriction sites. The ladder of satellite multimers obtained after digestion with restriction enzymes was compared with theoretical calculations and revealed the distribution pattern of particular monomer variants within the satellite. A defined higher order repeating structure, indicating the existence of satellite subfamilies, could not be observed. Our results show that some sequence variants are very abundant, being present in nearly 50% of the monomers, while others are very rare (0-1% of monomers). However, the distribution of either very frequent, or very rare sequence variants in T. molitor satellite DNA is always random. Monomer variants are randomly distributed in the total satellite DNA and thus spread across all chromosomes, indicating a relatively high rate of sequence homogenization among different chromosomes. Such a distribution of monomer variants represents a transient stage in the process of sequence homogenization, indicating the high rate of spreading in comparison with the rate of sequence variant amplification.


Involvement of the Escherichia coli RNA polymerase alpha subunit in transcriptional activation by the bacteriophage lambda CI and CII proteins. Escherichia coli cells harbouring the rpoA341 mutation produce an RNA polymerase which transcribes inefficiently certain operons subject to positive control. Here, we demonstrate that the rpoA341 allele also prevents lysogenization of the host strain by bacteriophage lambda, a process dependent upon the action of two phage-encoded activators. This phenomenon was shown to arise from an inability to establish an integrated prophage rather than a failure to maintain the lysogenic state. The inability of the rpoA341 host to support lysogenization could be completely reversed by CII-independent expression of int and cI in trans. These results led us to propose that the inhibition of lysogenization arises from a defective interaction between the phage lambda transcriptional activator CII and the mutant RNA polymerase at the phage promoters pI and pE. Finally, we also provide genetic evidence for impaired transcription of the cI gene from the CI-activated promoter, pM in the rpoA341 background.


Multiple protein tyrosine phosphatase-encoding genes in the yeast Saccharomyces cerevisiae. In higher eukaryotic organisms, the regulation of tyrosine phosphorylation is known to play a major role in the control of cell division. Recently, a wide variety of protein tyrosine phosphatase (PTPase)-encoding genes (PTPs) have been identified to accompany the many tyrosine kinases previously studied. However, in the yeasts, where the cell cycle has been most extensively studied, identification of the genes involved in the direct regulation of tyrosine phosphorylation has been difficult. We have identified a pair of genes in the yeast Saccharomyces cerevisiae, which we call PTP1 and PTP2, whose products are highly homologous to PTPases identified in other systems. Both genes are poorly expressed, and contain sequence elements consistent with low-abundance proteins. We have carried out an extensive genetic analysis of PTP1 and PTP2, and found that they are not essential either singly or in combination. Neither deletion nor overexpression results in any strong phenotypes in a number of assays. Deletions also do not affect the mitotic blockage caused by deletion of the MIH1 gene (encoding a positive regulator of mitosis) and induction of the heterologous Schizosaccharomyces pombe wee1+ gene (encoding a negative regulator of mitosis). Molecular analysis has shown that PTP1 and PTP2 are quite different structurally and are not especially well conserved at the amino acid sequence level. Low-stringency Southern blots indicate that yeast may contain a family of PTPase-encoding genes. These results suggest that yeast may contain other PTPase-encoding genes that overlap functionally with PTP1 and PTP2.


Imported sequences in the mitochondrial yeast genome identified by nucleotide linguistics. In addition to universally appearing mitochondrial (mt) genes, origins of replication and transcription start regions typical of all mt genome variants of the yeast Saccharomyces cerevisiae, the mt genomes of some of the strains contain variable sequences. These sequences are apparently largely dispensable. They are mainly composed of group-I and -II introns and intergenic open reading frames (ORFs). Many of the introns contain ORFs, some of which were shown by genetic and biochemical means to be involved in splicing and transposition of the mt introns. Some of the optional sequences are hypothesized to be mobile genetic elements. Nucleotide (nt) sequences of the mt genome of S. cerevisiae were examined by analyzing occurrences of oligodeoxyribonucleotide (oligo) 'words'. This linguistic technique had been found to be sensitive to both function and origin of the sequence [Pietrokovski et al., J. Biomol. Struct. Dyn. 7 (1990) 1251-1268]. A clear difference is found between the oligo vocabularies of the optional and basic yeast mt sequences. The difference is mainly located in protein coding segments of the optional sequences which contain conserved amino acid motifs, characteristic of intronic and intergenic ORFs. The use of nt linguistics to detect the sequence dissimilarity and its causes in yeast mitochondria provides fast and straightforward results, identifying the intronic and intergenic ORFs as DNA sequences of foreign, non-mt origin.


High-level production of a peroxisomal enzyme: Aspergillus flavus uricase accumulates intracellularly and is active in Saccharomyces cerevisiae. Strains of Saccharomyces cerevisiae producing Aspergillus flavus uricase (Uox) have been constructed. An artificial promoter which combined the upstream and downstream sequences of the GAL7 and ADH2 promoters, respectively, was found to be efficient in directing the synthesis of uaZ mRNAs encoding Uox. A good proportionality between the copy number of the uaZ expression cassette and the level of Uox production was found in the range of 1-10 copies. Transformants accumulated active and soluble Uox to a level exceeding 13% of total protein, as deduced from enzymatic assays. This relative level could be improved two- to threefold by using a recipient strain in which the wild-type GAL4 gene had been deleted and which expressed a GAL4 construct placed under the control of the ADH2 promoter.


Structural and functional analysis of the amdR regulatory gene of Aspergillus oryzae. We have isolated the Aspergillus oryzae homologue of the amdR regulatory gene of Aspergillus nidulans by cross hybridization. Sequence analysis and functional studies have shown that the amdR genes are highly conserved and functionally interchangeable between the two species. The homology between the two genes extends throughout most of the coding sequences, including sequences encoding the DNA-binding domain and putative activation domains. Two regions of nonconserved sequence were also identified. Studies using various amdS::lacZ fusion constructs indicate that the A. oryzae gene product binds similar sequences and responds to inducer in a similar manner to the A. nidulans protein. Inactivation of the A. oryzae gene results in the inability to grow on gamma-amino-butyric acid (GABA) as a carbon and/or nitrogen source indicating that GABA utilization is amdR-dependent in A. oryzae as it is in A. nidulans.


Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme. The cDNA gene encoding porcine pancreatic prophospholipase A2 (proPLA2) was cloned into an Aspergillus niger expression vector downstream of the glucoamylase (glaA) gene promoter region. When this construct was transformed into A. niger, no detectable PLA2 was produced. Evidence was obtained showing that the PLA2 gene was transcribed and that PLA2 is extremely susceptible to both intracellular and extracellular proteases of A. niger, thus indicating that translation products would be rapidly degraded. By fusing the proPLA2-encoding sequence to the entire glaA gene, secreted yields of PLA2 up to 10 micrograms/ml were obtained from a transformed protease-deficient strain of A. niger. PLA2 was secreted in young cultures as a fusion protein, but in older cultures, it was processed from the glucoamylase carrier protein. Secreted PLA2 was shown to be enzymatically active and to have the correct N-terminal amino acid (aa) sequence, although another form of processed PLA2 was also produced. This form included two aa of the proregion from PLA2. The potential for improving yields of secreted heterologous proteins from A. niger still further is discussed.


Cloning and differential expression during the sexual cycle of a meiotic endonuclease-encoding gene from the basidiomycete Coprinus cinereus. The naturally synchronous meiosis of the fungus, Coprinus cinereus, provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. We have cloned a cDNA from the fruiting body of C. cinereus encoding the 12-kDa subunit of a meiotic endonuclease (mENase). The identification of the 12-kDa subunit cDNA clone was achieved by the mENase antiserum against a lambda gt11 cDNA expression library. It was confirmed by a direct match of the amino acid (aa) sequence obtained from purified 12-kDa polypeptide with the nucleotide sequence. Northern blot analysis using the cDNA clone as a probe showed that the mENase-encoding gene (MenA) for the 12-kDa subunit was expressed mainly in fruiting bodies and at a very low level in the asexual vegetative mycelium. In addition, it was differentially expressed in the early meiotic stages. The MenA transcript was most abundant in fruiting body primordia prior to the premeiotic S-phase; it remained high from karyogamy to early pachytene, declined drastically by late pachytene and diplotene, and was undetectable by sterigma stage. Western blot analysis showed that the mENase protein was produced at a very low level in mycelium; it was produced in great quantity during the early meiotic stages and decreased to a low level at the end of meiosis.


Mutagenesis and regulation of the cysJ promoter of Escherichia coli K-12. The cysJ promoter of Escherichia coli K-12, which is positively controlled by the CysB regulatory protein, was localized through the formation of a fusion of cysJ, the gene encoding NADPH-cytochrome c reductase with lacZ. The position of the transcription start point was determined and the orientation of transcription was shown to be counterclockwise on the E. coli K-12 map. Oligodeoxyribonucleotide-directed mutagenesis of the inferred -10 and -35 regions indicated that both sites could be altered to produce promoter 'down' mutations. When the -10 region was made to agree with the -10 consensus sequence, there was increased function under conditions of repression (that is, in the presence of cysteine). Upstream deletions, as well as mutations in a region proposed to be involved in binding of the CysB regulatory protein, identified sequences important for promoter activity from -90 to -78 and from -71 to -66. By comparison of the sequences of four cys promoters, a possible CysB-binding site was found which included the region shown to be required for the positive regulation of the cysJ promoter.


A small plasmid for recombination-based screening. We reported recently the construction of the 4.4-kb R6K-derived pMAD1 plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with ColE1 and therefore permits recombination-based screening of lambda libraries that contain ColE1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the pi-encoding pir gene required for R6K replication. To supply pi [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans, we employed pPR1 delta 22pir116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz, J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger lambda inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1 (1991) 120-123].


Cloning and expression in Escherichia coli of the gene encoding an extracellular deoxyribonuclease (DNase) from Aeromonas hydrophila. The gene encoding an extracellular DNase from Aeromonas hydrophila CHC-1 has been cloned and sequenced. Following expression of the dns in Escherichia coli, it was revealed that some of the cloned enzyme was present in the cell-free extracellular supernatant fluid, and there was no cell lysis and concurrent release of cytoplasmic or periplasmic proteins. Therefore, results suggest that E. coli cells were capable of secreting the DNase extracellularly, albeit very inefficiently. The dns is transcribed from its own promoter in E. coli, and expressed as a 25-kDa product, as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of the culture supernatant preparations followed by a DNA-hydrolysis assay. Nucleotide sequence analysis predicted a single open reading frame of 690 bp encoding a 230-amino acid (aa) polypeptide, with a potential 20-aa signal peptide located at the N terminus of the predicted protein. The deduced aa sequence of the entire protein is highly homologous with that of the DNase of Vibrio cholerae.


Cloning of a gene from Bacillus cereus with homology to the mreB gene from Escherichia coli. We have cloned and sequenced a gene coding for a putative shape-determining protein (MreB) highly homologous to the mreB gene product of Escherichia coli. The amino acid (aa) identity was 53% and the similarity 72%. The gene is expressed early in the logarithmic phase. The aa sequence comparison showed that the protein, like the E. coli MreB, has structural similarity to actin and heat-shock protein Hsc70 encoded by a new super-gene family.


A series of shuttle vectors for Bacillus subtilis and Escherichia coli. A series of shuttle vectors for Bacillus subtilis and Escherichia coli was developed. These are derived from one basic construct composed of parts of the Gram+ plasmid pUB110 and the Gram- plasmid pBR322. They contain multiple cloning sites flanked by transcriptional terminators. In one plasmid, a vegetative B. subtilis promoter drives transcription of inserted genes. For the construction of operon and gene fusions, the cat of pUB112 and the lacZ gene of E. coli were employed as reporter genes. With these vectors, cloning and expression of genes as well as probing of regulatory signals can be performed in B. subtilis and E. coli.


Cloning and sequence analysis of the gene encoding L-lactate dehydrogenase from Lactococcus lactis: evolutionary relationships between 21 different LDH enzymes. Lactate dehydrogenase (LDH; EC1.1.1.27) is a key enzyme in the fermentation of milk by lactic acid bacteria used in the dairy industry. An 800-bp DNA fragment containing part of the gene (ldh) encoding LDH was amplified from Lactococcus lactis in a polymerase chain reaction using primers designed from the partial amino acid sequence of a lactococcal LDH. This fragment was radioactively labelled and used to probe a phage lambda library of Lc. lactis genomic DNA. Fragments containing ldh were subcloned from lambda to pUC13 and pUC18 and a 1.2-kb region was sequenced. The deduced aa sequence reveals that the lactococcal LDH is highly homologous to the LDHs of other organisms. The active site and several other domains of unknown function are highly conserved between all LDH enzymes (prokaryotic and eukaryotic). An evolutionary study of LDH sequences clearly divides the prokaryotic from the eukaryotic enzymes except for the Bifidobacterium longum LDH which anomalously groups with the eukaryotic enzymes. The LDHs from Gram-positive bacteria form a separate group from the enzymes from the Gram-negative organisms. The lactococcal LDH is phylogenetically closest to the streptococcal LDH.


Sequence of a Rhodococcus gene encoding a protein with extensive homology to the mammalian propionyl-CoA carboxylase beta chain. Sequence analysis of a 2-kilobase DNA fragment from Rhodococcus sp. NI86/21 revealed an open reading frame encoding a 476-amino-acid protein with striking homology to the rat and human propionyl-CoA carboxylase beta subunits. The nucleotide sequence of a corresponding prokaryotic gene has not yet been reported. Upstream, the C-terminal part of a putative beta-ketoacyl synthase was identified.


A homolog of the proteasome-related RING10 gene is essential for yeast cell growth. Proteasomes are intracellular protein complexes displaying multiproteolytic activities. These complexes have been implicated in the antigen degradation process that generates peptides associated with the major histocompatibility complex (MHC) class-I molecule. RING10 and RING12 are genes encoded by the class-II region of the human MHC that have sequence homology to proteasome-encoding genes. We have identified a yeast gene, called PRG1, that encodes a protein predicted to contain 55.6% sequence identity to 80% of the RING10 gene product. Genomic disruption of PRG1 revealed that it is essential for yeast cell growth. These data strongly indicate that the antigen-processing system present in vertebrates evolved from a basic cellular process present in all organisms.


Cloning the Cryptococcus neoformans TRP1 gene by complementation in Saccharomyces cerevisiae. We have cloned the phosphoribosyl anthranilate isomerase (PRAI)-encoding gene (TRP1) of Cryptococcus neoformans by genetic complementation in Saccharomyces cerevisiae. Sequence analysis of this gene revealed it to be 939 bp in length, and without known promoter or termination sequences. Unlike some of the filamentous fungi, where PRAI enzymatic activity is controlled by a trifunctional gene product, the C. neoformans PRAI appears to be unifunctional. PRAI of C. neoformans exhibits 39% amino acid (aa) sequence identity compared to the S. cerevisiae counterpart. The TRP1 gene of C. neoformans maps to different size chromosomes in strains with different serotypes. The cloning of this gene for vector constructions, and the demonstration that S. cerevisiae can be used as a surrogate for C. neoformans gene expression, should help with the molecular studies of this significant fungal pathogen in our increasing immunocompromised population.


An inducible expression system for the production of human lactoferrin in Aspergillus nidulans. The production and secretion of human lactoferrin (hLF) in Aspergillus nidulans is described. The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase (alcA) promoter. Recombinant hLF (re-hLF) is produced at levels up to 5 micrograms/ml. Approximately 30% of the re-hLF produced in this system is secreted into the growth medium. The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity. Furthermore, re-hLF is functional by the criterion of iron-binding capacity. The A. nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins.


Cloning and molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata. The glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) has been identified from a genomic DNA library prepared from the plant pathogenic fungus Glomerella cingulata. Nucleotide sequence data revealed that this gene codes for a putative 338-amino-acid protein encoded by two exons of 129 and 885 bp, separated by an intron 216 bp long. The 5' leader sequence is also spliced by an intron of 156 bp. A cDNA clone was prepared using the polymerase chain reaction, the sequence of which was used to confirm the presence of the intron in the coding sequence and the splicing of the 5' leader sequence. The transcriptional start point (tsp) was mapped at -253 nt from the site of the initiation of translation by primer extension and is adjacent to a 42-bp pyrimidine-rich region. The general structure of the 5' flanking region shows similarities to gpdA from Aspergillus nidulans. The putative protein product is 71-86% identical at the aa level to GPDs from Aspergillus nidulans, Cryphonectria parasitica, Curvularia lunata, Podospora anserina and Ustilago maydis.


Sequence of the URA1 gene encoding dihydroorotate dehydrogenase from the basidiomycete fungus Agrocybe aegerita. The URA1 gene encoding dihydroorotate dehydrogenase (DHOdehase) from the edible basidiomycete, Agrocybe aegerita, has been cloned by complementation of the Escherichia coli pyrD mutation. The nucleotide sequence of a 1531-bp genomic fragment carrying URA1 revealed two uninterrupted open reading frames (ORFs) separated by 61 bp. The larger ORF can encode a 328-amino acid (aa) DHOdehase that has 53% homology with the corresponding protein from E. coli. Comparison with other DHOdehase aa sequences showed essentially conservation of the cofactor-binding site of flavoproteins.


The Rhizobium meliloti pmi gene encodes a new type of phosphomannose isomerase. Interspecific complementation of a Xanthomonas campestris pv. campestris phosphomannose isomerase (PMI) mutant was used to isolate a cosmid from a genomic library of Rhizobium meliloti 2011 carrying the pmi gene of this strain. Subcloning experiments localized the coding region to a 2.0-kb SalI-ClaI fragment. Nucleotide sequence analysis of this fragment indicated the presence of two open reading frames (ORFs), coding for 18- and 43-kDa polypeptides. The analysis of the gene function by gene disruption experiments showed that ORF2 codes for pmi. A comparison of the deduced amino acid sequence with the corresponding sequences of the Pseudomonas aeruginosa and Escherichia coli PMIs revealed no significant homology, indicating that the isolated gene encodes a new type of PMI. The construction of a pmi-deficient mutant of R. meliloti using the sacB-sacR cassette technique showed that the loss of PMI activity does not affect the symbiotic properties of this strain.


Cloning and sequencing of the genes encoding glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase (gap operon) from mesophilic Bacillus megaterium: comparison with corresponding sequences from thermophilic Bacillus stearothermophilus. The structural genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and the N-terminal part of triosephosphate isomerase (TIM) from mesophilic Bacillus megaterium DSM319 have been cloned as a gene cluster (gap operon) by complementation of an Escherichia coli gap amber mutant. Subsequently, the entire tpi gene, encoding TIM, was isolated by colony hybridization using a homologous probe. Nucleotide (nt) sequence analysis revealed an unidentified open reading frame (urf1) of 1029 bp located 50 nt upstream from the start codon of the gap gene. Gene expression from subclones containing different coding regions was studied by enzyme assay and SDS-PAGE. Both GAPDH and TIM are synthesized in transformed E. coli cells, whereas PGK is not. There is no unequivocal evidence for urf1 expression. Two putative promoter sites are present: one 100 nt upstream from urf1 and one 200 nt upstream from the pgk gene. An inverted repeat following the second promoter site is postulated to be involved in the transcriptional regulation of the operon. Each coding region shows a G+C content of 40% attained by the adaptation of the G+C content of the third base in the codon to compensate the G+C content of the first and second bases. The deduced amino acid (aa) sequences of B. megaterium GAPDH, PGK and TIM were compared with those from the thermophilic Bacillus stearothermophilus by antisymmetrical matrices. The detected characteristic thermophilic-mesophilic exchange pattern concerning aa substitutions between hydrophobic-polar and charged-charged residues corresponds to data obtained for thermophilic and mesophilic lactate dehydrogenases (LDH). The determination of the thermostability of these enzymes revealed two regions of stability for B. megaterium TIM at high enzyme concentrations. Heat treatment seems to be responsible for the conversion of two differently active conformations or the induction of a new quaternary structure.


Four genes in Streptomyces aureofaciens containing a domain characteristic of principal sigma factors. Four genes encoding sigma-factor-like proteins, hrdA, hrdB, hrdD, and hrdE, were identified in a Streptomyces aureofaciens genomic library using an oligodeoxyribonucleotide probe encoding a peptide motif homologous to the core-binding domain in sigma factors. The deduced proteins have M(r) values of 43,363, 57,172, 36,591, and 57,565, respectively, and strongly resemble all known principal sigma factors, including possession of the characteristic 'rpoD box'. Transcription analysis of the hrd genes by Northern blot hybridization indicated only the expression of hrdB and hrdD and weak transcription of hrdA. A repetitive region of a pentapeptide tandemly repeated 6 and 4 times was identified in the N-terminal part of HrdB and HrdE, respectively. No such domain was found in any principal sigma factors.


tDNA(ser) sequences are involved in the excision of Streptomyces griseus plasmid pSG1. Plasmid pSG1 is maintained in some derivatives of Streptomyces griseus NRRL3851 mainly in the chromosomally integrated form (pSG1int). In others, the integrated plasmid is co-maintained with free pSG1 [Cohen et al., Plasmid 13 (1985) 41-50]. The pSG1 plasmid integration site (attP) and the pSG1int-chromosome boundaries (attL and attR) were cloned and sequenced. The results indicate that pSG1int is flanked at attL by a functional tRNA(ser) gene and at attR by a 60-nt sequence of the 3' end of the same tRNA(ser) gene. A single mismatch distinguishes the 60-nt sequence at attR from its direct repeat at attL. The attP site contains a single copy of the 60-nt repeat, identical to the one at attL. This observation indicates that pSG1, like integrating plasmids of other Actinomycetes, is integrated in a tRNA gene and suggests that the exact excision of pSG1int occurs by a recombinational crossing-over event at the first 43 nt of the 60-nt repeat.


Transcription map of the early region of the Streptomyces bacteriophage phi C31. Streptomyces coelicolor A3(2), lysogenised by the temperature-sensitive cts1 mutant of phi C31, can be synchronously induced into the lytic cycle by heat treatment. A transcription map of 10 kb of the phi C31 early gene cluster was deduced using low-resolution S1 nuclease mapping of RNA prepared 10 min after induction. At least nine early transcripts, early (e)RNAs 1-9, were localised reading exclusively rightwards with respect to the standard physical map of phi C31. The mRNAs were extensively overlapping, frequently initiating at the same place but terminating at different sites, and vice versa. Gene expression during the lytic cycle was tightly regulated; no transcription was observed before induction. Transcription was maximal at 10 min post-induction, and at 20 min, eRNAs 5 and 6 persisted whilst eRNAs 7-9 were severely reduced or absent. The pattern of transcription of the early region is consistent with the simultaneous activation of a large number of promoters and differential termination efficiency.


A method for generating transcripts with defined 5' and 3' termini by autolytic processing. Plasmids containing both the hammerhead and hairpin ribozyme autocatalytic cassettes were constructed for the purpose of generating RNA transcripts with specific termini at both the 5' and 3' ends. Following transcription, the RNA encoded by these cassettes was capable of intramolecular cleavage. This resulted in the generation of a processed RNA, which was located between the two cassettes, with specifically engineered 5' and 3' ends. The two different ribozymes were selected for their efficient intramolecular cleavage ability and to reduce the possibility of DNA recombination that could occur if identical cassettes were used. An application of this technique was the generation of a processed RNA which was itself a ribozyme, with specific 5' and 3' termini. The ribozyme generated was a hairpin ribozyme specific for a sequence in the gene encoding hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA reductase). The processed ribozyme was fully catalytically active against an RNA substrate sequence of HMGCoA reductase.


Histone-like proteins are required for cell growth and constraint of supercoils in DNA. The gene hns of Escherichia coli K-12, which encodes the histone-like protein H-NS, was inactivated by insertion of a DNA (gene hph) encoding hygromycin phosphotransferase. The growth rates of two mutants lacking either one or the other of the histone-like proteins, HU and IHF, were not affected by introduction of the hns mutation. However, cells depleted of HU, IHF and H-NS simultaneously, could not be constructed by P1 phage-mediated transduction. These results, together with our previous finding that cells deficient in both HU and IHF are viable at 30-37 degrees C [Kano and Imamoto, Gene 89 (1990) 133-137] showed that E. coli cells deficient in any two of these three histone-like proteins are viable at 30-37 degrees C, and suggested that simultaneous deficiency of all three of the proteins is lethal. There were no detectable differences in the levels of superhelicity of the reporter plasmids isolated from cells deficient in either IHF or H-NS, or from wild-type cells, but about 15% decrease in negative superhelicity was detected for the reporter plasmid isolated from cells lacking HU and lacking both HU and H-NS. However, the cryptic bgl operon, whose expression was reported to be regulated through topological changes of cellular DNA, could not be activated in cells depleted of HU or IHF. The bgl operon was expressed in cells depleted of both HU and H-NS as well as in cells depleted of H-NS.(ABSTRACT TRUNCATED AT 250 WORDS)


The mosaic organization of the mitochondrial introns of Saccharomyces cerevisiae: features and evolutionary origins. The introns of three genes (oxi3, cob and 21S) from the mitochondrial (mt) genome of Saccharomyces cerevisiae contain closed reading frames (CRFs). In the present work, we have analyzed these sequences in their oligodeoxyribonucleotide (oligo; isostich) patterns. We have shown that the relative amounts of di- to hexanucleotides, when compared to random sequences having the same sizes and compositions, exhibit the same deviations as the intergenic noncoding sequences of the mt genome (except for the CRFs from 21S intron). In contrast, intronic open reading frames (ORFs) showed oligo patterns which were generally quite distinct from those of CRFs, although some similarities could be detected in some cases (especially for aI5 alpha). The mt introns of yeast, therefore, are endowed with a mosaic structure, in which CRFs derive from mt intergenic sequences, whereas ORFs have a different origin (indicated as exogenous by other evidences) yet show, in some cases, the effects of 'sequence assimilation' with CRFs.


A comparison of Twin Block, Andresen and removable appliances in the treatment of Class II Division 1 malocclusion. Cephalometric radiographs were taken before and after treatment in three groups of patients. Ten were treated with removable appliance therapy involving extraction of upper first premolars and retraction of anterior teeth. Ten were treated on a non extraction basis coupled with wearing an upper expansion appliance followed by an Andresen appliance. Another group of ten patients were treated in an identical manner except that they wore a Twin Block instead of the Andresen appliance. When comparing the patient groups before and after treatment, statistically significant differences were found for upper incisor angulation in the Removable (t = 9.49 p < 0.001) Andresen (t = 4.48 p < 0.001) Twin Block (t = 2.81 p < 0.001) groups and for angle ANB in the Andresen (t = 2.88 p < 0.01) and Twin Block (t = 2.65 p < 0.01) groups. Thus the overjet was reduced purely by incisor tipping with the removable appliance as would be expected, but both functional appliances produced some bodily correction with an improvement in the dental base relationship. Although the Twin Block appliance group showed a greater reduction in angle ANB this was not significantly different from that in the Andresen appliance group. The treatment time from insertion of the first appliance to reduction of the overjet to 3mm or less was also recorded. The treatment time for the Twin Block group (8.1 months) was significantly less (t = 2.79 p < 0.05) than the Andresen group (12.8 months), but neither functional group differed significantly from the removable appliance group (10.1 months).


Epidermal growth factor in the oesophagus. Epidermal growth factor (EGF) has been implicated in mitogenesis and oncogenesis in the gastrointestinal tract. To determine the role of EGF in oesophageal disease, its quantity and distribution in the oesophageal mucosa of control subjects and patients with oesophageal disease were studied. Oesophageal biopsy specimens, taken 20-40 cm from the incisors in 72 patients, were graded histologically and adjacent specimens were taken for immunohistochemical analysis of the distribution of EGF. In patients with Barrett's columnar lined oesophagus, specimens were also taken from the gastric cardia for comparison. Twenty two biopsy specimens showed oesophagitis, 20 Barrett's mucosa, and 30 were histologically normal. EGF was found in the capillary endothelium of the normal oesophageal papillae and basal mucosa. Significantly more EGF positive papillae were found in the normal mucosa (81%) than in the inflamed mucosa (42%) (p < 0.001). The 20 patients with Barrett's mucosa showed abnormal expression of EGF in 25% of the isthmus and superficial epithelial cells. This study has shown that EGF is found only in the endothelial cells of the capillaries of the normal oesophageal mucosa and that the peptide is detectable significantly less frequently than normal in the inflamed oesophageal mucosa. EGF is also abnormally present, in large quantities, in the cytoplasm of the epithelial cells of Barrett's mucosa compared with gastric mucosa.


Outcome of surgical treatment of adenocarcinoma in Barrett's oesophagus. A retrospective study was performed of an 11 year period (1978-88) to analyse the survival of 112 patients (85 men and 27 women, mean age 63 years) with adenocarcinoma in a columnar lined (Barrett's) oesophagus in respect of surgical treatment, tumour staging, and histological grading. Presenting symptoms were dysphagia (60%) and pain (25%). Only six patients were previously known to have a columnar lined oesophagus. Eighty five patients (76%) underwent partial resection of the oesophagus and cardia. Postoperative mortality was 6%. After resection (n = 85), the 5 year survival was 24%. Survival was significantly better for patients without regional lymph node metastases (stage 0, I, IIA (n = 61): 5 year survival 30%) and even better if the tumour was restricted to the submucosa (stage 0, I (n = 12): 5 year survival 63%). Survival was not influenced by the histological grade of the tumour. Staging based on infiltration of the oesophageal wall and lymph node spread is valuable in determining the prognosis for patients with adenocarcinoma in Barrett's oesophagus.


Endosonography can detect residual tumour infiltration after medical treatment of oesophageal cancer in the absence of endoscopic lesions. Endoscopic ultrasound (endosonography) is useful in the preoperative staging of oesophageal tumours. It may also have a role in evaluation and surveillance of patients with inoperable carcinomas. Thirty four patients with inoperable oesophageal cancer were investigated by endosonography and computed tomography before medical treatment. In 10 patients receiving combined chemotherapy and radiotherapy, the endoscopic lesions resolved and biopsy specimens were negative. When endosonography suggested the persistence of tumour infiltration in these patients, a local recurrence or distant metastases appeared within a few months. In contrast, when no infiltration was detected, no tumoral recurrence or progression was observed within eight months. These results suggest that endosonography is better than endoscopic biopsy specimens and computed tomography in assessing the response of oesophageal carcinoma to non-surgical treatment.


Dyspepsia, Helicobacter pylori, and peptic ulcer in a randomly selected population in India. There seems to be a worldwide geographic variation in the prevalence of peptic ulcer disease, although there are few reliable population based studies. This study aimed to determine the prevalence of peptic ulcer disease in a community in southern India and to evaluate the relationship between dyspeptic symptoms, Helicobacter pylori infection, gastritis, and peptic ulcer disease. A sample population was selected randomly from a rural monastic settlement in southern India. Subjects were interviewed using a standardised symptom and demography questionnaire then underwent upper endoscopy and antral biopsy for histology and CLO rapid urease test. Altogether 197 subjects from a population of 1499 (13.1%) were studied. All were male monks and ethnically Tibetan. The median age was 28 years (range: 21-81). None smoked or took NSAIDs. The six month period prevalence of dyspeptic symptoms was 68.5%. Current symptoms were present in 58.9% of subjects. Dyspepsia was more common in subjects aged 40 years or younger (p < 0.0001). H pylori was detected in 77.2% subjects. There was no association between dyspepsia and the presence of H pylori or histological gastritis, although there was a strong correlation between symptoms and ulcer (p < 0.003). The point prevalence of active peptic ulcer was 6.6% (13/197). All ulcers detected were either prepyloric or pyloroduodenal in location. A further 6.6% of subjects had definite evidence of scarring or deformity indicative of ulceration in the past. Subjects with past or present ulcers comprised 17.8% of dyspeptic subjects. H pylori was present in all subjects with active ulcers and in 12/13 of those with scarring. Dyspepsia, H pylori infection, gastritis, and peptic ulcer are all more common in this population than in those from developed countries. Ulcer disease, however, accounts for only a small proportion of subjects with symptoms and neither H pylori infection nor gastritis are significantly associated with the presence of dyspepsia.


Mucosal reactive oxygen metabolite production in duodenal ulcer disease. To investigate the hypothesis that reactive oxygen metabolites are important in the pathophysiology of duodenal ulcer disease, their production by duodenal mucosal biopsy specimens was measured using luminol and lucigenin amplified chemiluminescence. Luminol chemiluminescence, expressed as background corrected median photon emission/mg/min x 10(3) (95% confidence intervals), was increased in duodenal inflammation as assessed macroscopically: ulcers 20.3 (4.8 to 51.3), n = 29; severe duodenitis 13.9 (6.6 to 75.3), n = 16; mild duodenitis 0.0 (-0.5 to 0.8), n = 56; controls -0.8 (-1.3 to -0.1), n = 41; p = 0.0001, Kruskal-Wallis) and microscopically: severe 17.0 (9.3 to 51.3), n = 12; moderate 0.3 (-2.8 to 5.8), n = 17; mild -0.1 (-1.8 to 1.0), n = 17; controls -0.8 (-1.6 to 0.0), n = 15; (p = 0.0001). Luminol chemiluminescence was directly related to both the macroscopic and microscopic severity of duodenal damage (Spearman's R = + 0.53, + 0.55 respectively, both p = 0.0001), to histochemical assessment (myeloperoxidase activity) of neutrophil infiltration (R = + 0.63; p = 0.04), and to lucigenin chemiluminescence (R = + 0.56, p = 0.0002). Luminol chemiluminescence was inhibited by sodium azide (-80%), catalase (-73%), and dimethyl sulphoxide (-24%). Superoxide dismutase inhibited lucigenin more than luminol dependent chemiluminescence (-61% and -7% respectively, p < 0.05). Within disease groups, Helicobacter pylori antral infection was associated with increased duodenal chemiluminescence, whereas smoking, alcohol, and use of NSAIDs or H2 blockers had no influence. Their disease related generation in duodenal mucosa supports a role for reactive oxygen metabolites in the pathogenesis of duodenitis and duodenal ulcer. These metabolites might include superoxide, hydrogen peroxide, hydroxyl, and products of myeloperoxidase activity.


Effect of immunisation against vasoactive intestinal polypeptide on gastric corpus tone and motility in the ferret. The role of vasoactive intestinal polypeptide in the control of gastric corpus tone and motility was investigated using auto-antibodies to neutralise endogenous vasoactive intestinal polypeptide. Six ferrets were immunised with vasoactive intestinal polypeptide thyroglobulin conjugate in Freund's complete adjuvant which resulted in a significant increase in plasma vasoactive intestinal polypeptide binding activity compared with unimmunised control animals. In acute experiments the level of spontaneous motility in the period immediately after completion of the surgical preparation was 15 times higher in immunised v control animals (p < 0.02). Surprisingly, however, there was no deficit in the ability of the corpus to accommodate fluid. Peak pressure at the end of a 20 ml ramp distension was not different in immunised animals (5.7 (0.6) cm H2O) compared with controls (4.8 (0.3) cm H2O). It is concluded that the non-adrenergic non-cholinergic inhibitory mechanisms regulating corpus tone and motility are different and that vasoactive intestinal polypeptide acts primarily to regulate phasic contractile activity. Alternatively, because of plasticity in the mechanisms controlling corpus tone, the effect of vasoactive intestinal polypeptide may have been superceded during the timecourse of the immunisation procedure.


Gastric antral dysrhythmias in children with chronic idiopathic intestinal pseudoobstruction. Chronic idiopathic intestinal pseudoobstruction is a serious disorder of intestinal neuromuscular function resulting in recurrent episodes of intestinal obstruction, and is caused by primary disease of the enteric nerves or enteric smooth muscle. Gastric electrical control activity detected by the non-invasive technique of surface electrogastrography was investigated in 11 children (0.1-16 years) with proven chronic idiopathic intestinal pseudoobstruction (four with known disease of the enteric nerves, three with disease of smooth muscle cells, and four without defined pathology), to determine whether abnormalities were present and whether these were useful in detecting the underlying pathology. Abnormalities were present in eight of 11 patients. Persistent tachygastria (electrical control activity frequency > 5 cycles/minute) was found in three patients, all with a proven neuropathy. A continuously irregular frequency was found in five patients, three with a proven myopathy and two with undefined pathology. A normal electrical control activity frequency was present in three patients, one with a proven neuropathy and two with undefined pathology. It is suggested that this non-invasive technique may provide a useful screening test of the pathophysiological basis of the functional obstruction in children with chronic idiopathic intestinal pseudoobstruction.


Immunohistochemical analysis of mucosal gamma-interferon production in coeliac disease. The role of gamma-interferon in the pathogenesis of enteropathies with an immunological basis such as coeliac disease, is unclear. Gamma-interferon immunoreactive lymphocytes were quantified in jejunal biopsies from patients with coeliac disease and from normal controls. In coeliac disease, there was an apparent decrease in the percentage of both intraepithelial (3.5% v 13.5%) and lamina propria (10.3% v 47.2%) lymphocytes expressing gamma-interferon compared with controls. In patients successfully treated with a gluten free diet, the percentage of gamma-interferon immunoreactive intra-epithelial lymphocytes was 10.3%. Intraepithelial lymphocytes were immunonegative for class II major histocompatibility complex, while epithelial cells showed increased expression of this product in coeliac disease. The results show that a relatively large proportion of lymphocytes in normal small bowel express gamma-interferon. They also indicate that in coeliac disease the major increase in the numbers of mucosal lymphocytes is the result of infiltration by lymphocytes not expressing gamma-interferon.


Gliadin uptake in human enterocytes. Differences between coeliac patients in remission and control individuals. The pepsin trypsin digest of the wheat prolamin gliadin (PT-gliadin) is deleterious to the small intestinal mucosa of coeliac patients. The handling of PT-gliadin by the intestinal epithelium in coeliac patients in remission and control individuals was investigated by in vivo instillation of PT-gliadin. The uptake of PT-gliadin was monitored by immunofluorescence microscopy of intestinal biopsy specimens, using affinity purified PT-gliadin antibodies. Control individuals show weak staining in the apical region of the enterocytes thereby showing an uptake of PT-gliadin. Coeliac patients have a conspicuous fluorescence in relation to the lateral membrane/intercellular space of enterocytes and intense staining intracellularly in the apical region. There is only weak staining in the enterocytes after the instillation was terminated, indicating an intracellular clearance. The study shows that normal enterocytes are able to take up PT-gliadin. The increased uptake in coeliac patients might be of importance for the pathogenesis either by direct toxicity or by presentation to immunocompetent cells. Furthermore, the results are in agreement with the suggestion of a functional alteration in the zonula occludens in the intestinal epithelium of coeliac patients.


Colonic preservation reduces need for parenteral therapy, increases incidence of renal stones, but does not change high prevalence of gall stones in patients with a short bowel. Forty six patients with less than 200 cm of normal jejunum and no functioning colon were compared with 38 patients with similar jejunal lengths in continuity with a functioning colon. Women predominated (67%), and the most common diagnosis in each group was Crohn's disease (33 of 46 no colon, 16 of 38 with colon). All patients without a colon and less than 85 cm of jejunum and all those with a colon and less than 45 cm jejunum needed long term parenteral nutrition. Six months after the last resection 12 of 17 patients with less than 100 cm jejunum and no colon needed intravenous supplements compared with 7 of 21 with a colon. Between 6 months and 2 years, little change occurred in the nutritional/fluid requirements in either group, though there was weight gain. Of 71 patients assessed clinically at a median of 5 years, none with more than 50 cm of jejunum and a colon needed parenteral supplements. Most (25 of 27) of those without a colon who did not need parenteral supplements required oral electrolyte replacement compared with few (4 of 27) with a colon. None of the patients without a colon developed symptomatic renal stones compared with 9 of 38 (24%) with a colon (p < 0.001). Stone analysis in three patients showed calcium oxalate. Gall stone prevalence was high but equal in the two groups--43% of those without and 44% of those with a colon.


Ketotifen effectively prevents mucosal damage in experimental colitis. The effects of ketotifen, a 'mast cell stabiliser,' on two models of experimental colitis were examined. The inflammatory response elicited by either trinitrobenzene sulphonic acid or acetic acid resulted in increased colonic synthesis of platelet activating factor, prostaglandin E2, thromboxane B2, leukotrienes B4 and C4, and myeloperoxidase activity. Intragastric administration of ketotifen 100 micrograms/100 grams twice daily significantly decreased mucosal damage when given prophylactically 48 hours before the induction of colitis and then throughout the experiment. This effect was consistent in both models and was accompanied by a significant reduction in mucosal generation of platelet activating factor, prostaglandin E2, thromboxane B2, and leukotrienes C4 and B4. Myeloperoxidase activity was reduced as well, reaching significance only in the acetic acid model. This study shows that both trinitrobenzene sulphonic acid and acetic acid colitis can be pharmacologically manipulated by ketotifen. The mechanism of action of ketotifen has not yet been determined. Ketotifen's potential in the treatment of active inflammatory bowel disease or in the prevention of exacertations, or both, remains to be elucidated.


Predictors of presence, multiplicity, size and dysplasia of colorectal adenomas. A necropsy study in New Zealand. Three hundred and thirty six forensic necropsy specimens of large bowel were examined in order to identify subject related variables that independently predicted the following adenoma characteristics: presence, size (largest), multiplicity and high grade dysplasia. The variables were age, gender, body mass index, race (European origin versus Maori/Polynesian) and presence of hyperplastic (metaplastic) polyp(s). Subjects included 303 New Zealanders of European origin (M = 185, F = 118) yielding 149 adenomas and 251 hyperplastic polyps and 33 Maori/Polynesians (M = 25, F = 8) yielding five adenomas and one hyperplastic polyp. Independent predictors of adenoma presence as determined by regression analysis were age (p = 0.0001), presence of hyperplastic polyps (p = 0.0001) and male gender (p = 0.05). Models were poor at explaining variation in size, multiplicity, and dysplasia. Larger adenomas occurred more frequently in subjects with multiple adenomas (p = 0.03) and multiple adenomas were probably associated with hyperplastic polyps (p = 0.09) and male gender (p = 0.09) in Europeans. High grade dysplasia was more frequent in women (p = 0.05) and possibly in subjects with hyperplastic polyps (p = 0.2). Body mass index and ethnicity did not predict any adenoma characteristics, but hyperplastic polyp prevalence was influenced by European origin (p = 0.04) and to a lesser extent by body mass index (p = 0.08) as well as presence of adenoma (p = 0.0002) and age ( = 0.005). The association of hyperplastic polyp with presence, multiplicity but not size of adenoma and with a high risk group for colorectal cancer (New Zealanders of European origin) suggests that the hyperplastic polyp serves as a marker for a factor which influences neoplastic evolution at the stages of initiation/transformation but not promotion. Fifty nine per cent of individuals with adenoma(s) did not have hyperplastic polyp(s) emphasising that the last would serve only as a marker of populations and not individuals at high risk of bowel cancer. Low intracolonic butyrate may be the factor linking the expression of the two types of polyp.


Dietary calcium does not reduce experimental colorectal carcinogenesis after small bowel resection despite reducing cellular proliferation. It has been proposed that colorectal carcinogenesis is accompanied by increased mucosal cell proliferation and that the converse may also apply. To examine this thesis, the crypt cell production rate (CCPR) was measured in eight groups of rats (n = 187) that had received 1,2 dimethylhydrazine, 70% small bowel resection, supplemental dietary calcium, or a combination of these. Analysis of variance showed the following: (1) the CCPR decreased between the ileum and distal colon; (2) the CCPR decreased between 16 and 32 weeks; (3) 1,2 dimethylhydrazine and small bowel resection increased the CCPR and calcium decreased the CCPR independently of one another; (4) the CCPR interacted with 1,2 dimethylhydrazine x small bowel resection, calcium x 1,2 dimethylhydrazine and interacted between the site of bowel and calcium, 1,2 dimethylhydrazine, small bowel resection, and 1,2 dimethylhydrazine x small bowel resection (p = 0.014 to p < 0.001). The tumour yield was reduced by calcium in 1,2 dimethylhydrazine treated animals (chi 2 = 14.1, df = 3, p < 0.01) but was unaffected by calcium in 1,2 dimethylhydrazine and small bowel resection treated animals despite significant differences in the CCPR. An increase of the CCPR both preceded and accompanied colorectal carcinogenesis but reduction of the CCPR was not invariably accompanied by reduced carcinogenes.


Crypt cell production rates at various sites around the colon in Wistar rats and humans. The crypt cell production rate (CCPR) is considered the most robust estimate of cell turnover. The CCPR was determined at various sites around the colon in the Wistar rat (using an in vivo technique) and in six healthy humans (using in vitro organ culture). In both the rat and human colon, the CCPR increased proximally from the rectum to the caecum. The caecum had a significantly higher cell turnover than any other site in the colon (p < 0.05 in the rat, p < 0.01 in humans, Student's t test). These findings are of interest when considering cellular proliferation studies in both inflammatory bowel disease and colorectal cancer, and draw attention to the importance of choosing a constant reference site for comparative studies. This is the first time the CCPR had been measured along the length of the human colon.


Anorectal function in patients with complete supraconal spinal cord lesions. Anorectal manometry and sphincter electromyography were performed in 23 patients with complete supraconal traumatic spinal injuries and 30 age and sex matched control subjects. Basal pressures in the spinal group were similar to those in normal subjects but conscious control of sphincter activity was abolished in all spinal patients. Discriminant rectal sensation was also abolished during rectal distension, but 40% of patients experienced a dull pelvic ache at maximum levels of distension. Phasic rectal contraction and anal relaxation were present but exaggerated and induced at lower distending volumes than in normal subjects. The configuration of the rectal pressure/volume relationship was linear in patients compared with a reversed 'S' shape in normal subjects. The external anal sphincter response to rectal distension was noticeably attenuated, reinforcing the view that this spinal reflex is heavily modulated by supraspinal centres under normal circumstances. The external anal sphincter response to increases in abdominal pressure was also attenuated, and the anal pressures were strongly correlated with the level of the lesion and the abdominal pressure the patient could generate. No spinal patient showed a decrease in external anal sphincter activity during straining 'as if to defecate.' The exaggerated anorectal smooth muscle responses to rectal distension and the attenuated external sphincter response explain why patients with complete supraconal spinal lesions experience uncontrollable reflex defecation, while the persistance of external and sphincter contraction and the absence of any external anal sphincter relaxation during straining 'as if to defecate' might explain the difficulty that these patients have in consciously expelling rectal contents.


Detection of subclinical autonomic neuropathy in constipated patients using a sweat test. Chronic idiopathic constipation may be the result of an autonomic neuropathy. This hypothesis was tested in 23 constipated patients and 17 age matched controls, using the acetylcholine sweat spot test devised to test autonomic integrity in diabetes. Acetylcholine (0.01%) was injected in the dorsum of the foot painted with a mixture of starch and iodine. Active sweat glands appeared on the surface of the skin as small black dots which were photographed and counted, using a grid with 60 subareas. Two measurements were made: the number of dots per unit subarea (sweat spot test score) and the % number of abnormal subareas (with less than six spots). These two parameters were correlated. The median sweat spot test score was 9.53 in patients and 13.92 in controls (p = 0.0001), the receiver operating characteristic curve showing that a score of 12 delimited normal and abnormal subjects. Increasing age was correlated with a low score in patients, probably because of prolonged symptoms. Seventy per cent of patients and one control had a borderline or abnormal number of subareas. These results suggest that idiopathic constipation is associated with a degree of autonomic denervation. The sweat spot test is an easy, inexpensive method to test this hypothesis and deserves a place in the clinical assessment of slow transit constipated patients.


Intrahepatic expression of pre-S1 and pre-S2 antigens in chronic hepatitis B virus infection in relation to hepatitis B virus replication and hepatitis delta virus superinfection. Hepatocyte expression of pre-S1 and pre-S2 in relation to hepatitis B virus replication (hepatitis B virus-DNA in serum and HBcAg in the liver), histological activity and hepatitis delta virus superinfection was studied by indirect immunofluorescence on frozen sections of liver specimens from 68 patients with chronic hepatitis B virus infection. All 44 patients with chronic type B hepatitis had pre-S1 and pre-S2 display in the liver. The distribution of pre-S1 in the liver was membranous in one, mixed membranous and cytoplasmic in 12, and cytoplasmic in 31. The distribution of pre-S2 was membranous in one, mixed membranous and cytoplasmic in 26, and cytoplasmic in 17. Membranous expression of pre-S1 was significantly more prevalent in patients with active hepatitis B virus replication than in those without (13/28 v 0/16, p < 0.001), regardless of the histological activity, as was membranous expression of pre-S2 (27/28 v 0/16, p < 0.001). In contrast, a significantly higher extent of cytoplasmic expression of pre-S1 and pre-S2 was noted in patients without active hepatitis B virus replication than in those with. Of 24 patients with chronic type D hepatitis virus, eight had active hepatitis B virus replication, and the other 16 did not. The distribution and quantitative expression of pre-S1 and pre-S2 in the liver in these patients also correlated significantly with the status of hepatitis B virus replication and, moreover, showed little or no difference from those without hepatitis delta virus infection. In conclusion, all patients with chronic type B hepatitis had synthesis and display of pre-S1 and pre-S2 in the liver. The distribution and quantitative expression of pre-S1 and pre-S2, however, were closely related to the status of hepatitis B virus replication, but not to the histological activity. Hepatocyte expression of pre-S1 and pre-S2 in chronic type D hepatitis also correlated significantly with status of hepatitis B virus replication, and was not modulated by concurrent hepatitis delta virus infection.


Plasma concentrations of endogenous heparinoids in portal hypertension. Bleeding as a complication of liver disease can occur in the absence of recognised haemostatic defects. It is now possible to measure the concentration of endogenous heparinoid substances in the blood using a competitive binding assay. One such substance, heparan sulphate (normal range < 600 ng/ml) was assayed in the plasma of 49 patients admitted because of oesophageal varices. In 27 patients with recent upper gastrointestinal bleeding the median plasma heparan sulphate value was 1700 ng/ml (interquartile (IQ) range 900-3900) compared with 390 ng/ml (IQ range 256-800) in 22 patients with no recent bleed (p < 0.01). As heparan sulphate is metabolised by the same route as exogenous heparin, an attempt to establish a cause for the raised heparan concentrations was made by measuring the clearance of exogenous heparin in 10 portal hypertensive patients and 10 controls. The median half life of heparin in plasma in the portal hypertensive patients (25.5 minutes; IQ range 22-34) was significantly longer (p < 0.007) than the median half life in the controls (18.7 minutes; IQ range 17-21.5). Thus, there is evidence of raised concentrations of endogenous heparin like substances in portal hypertensive patients after gastrointestinal bleeding. These high concentrations may result from reduced hepatic clearance.


A prospective trial of endoscopic sclerotherapy v oesophageal transection and gastric devascularisation in the long term management of bleeding oesophageal varices. In a prospective three centre study oesophageal transection and gastric devascularisation have been compared with endoscopic sclerotherapy in the long term management of bleeding oesophageal varices. Cirrhotic patients (Child's A or B grade) with documented bleeding oesophageal varices were treated initially with emergency sclerotherapy, and after five days stability, were allocated to one of the two treatment regimes. The endoscopic sclerotherapy group underwent regular sclerotherapy until variceal obliteration while those undergoing surgery were not endoscoped unless bleeding recurred, when they were treated by sclerotherapy if appropriate. Ninety two patients were eligible for analysis (68% alcoholic cirrhosis; mean age 50.1 years) and follow up was achieved for a mean of 52.5 months (range 17-83). Mortality in the first three months was greater in the oesophageal transection and gastric devascularisation group (20% v 1%) but by two years the survival curves were the same and thereafter there was no difference in mortality. Rebleeding occurred in 13/41 (31%) patients, undergoing oesophageal transection and gastric devascularisation. The costs incurred during the first year of oesophageal transection and gastric devascularisation treatment were significantly greater than with endoscopic sclerotherapy (4369 pounds v 1067 pounds, p < 0.0001) and the high rate of rebleeding in the surgical group meant that no cost savings occurred in subsequent years. It is concluded that oesophageal transection and gastric devascularisation confers no benefit over endoscopic sclerotherapy in terms of long term survival and that it is not cost effective as judged by the current health care costs in the United Kingdom.


Value of ultrasound and liver function tests in determining the need for endoscopic retrograde cholangiopancreatography in unexplained abdominal pain. The value of serum liver function tests and abdominal ultrasound as screening tests of the need for endoscopic retrograde cholangiopancreatography (ERCP) was determined in patients with unexplained abdominal pain without associated jaundice. In 1989 and 1990 1005 ERCPs were undertaken, of which 138 (14%) were for this indication. The duct or ducts of interest were delineated by ERCP in 95% of patients. The lesions found were bile duct stones in 10 patients, chronic pancreatitis in five, pancreatic carcinoma in one, peptic ulcer or duodenitis in four. A satisfactory ultrasound examination had been performed in 94% of patients. For chronic pancreatitis, its sensitivity was 60% and specificity 95%. For choledocholithiasis, the ultrasonic detection of duct dilatation or stones had a sensitivity of 90% and specificity of 86%. Of the liver function tests, the alkaline phosphatase was more sensitive (67%) than the transaminases (44%) in indicating the presence of bile duct stones and had a high specificity (95%). None of the 10 patients with duct stones had normal ultrasound and normal alkaline phosphatase. Thus it was found that demonstration of a normal common bile duct by abdominal ultrasound and normal serum alkaline phosphatase together have 100% specificity in excluding bile duct stones. Using such knowledge over the two year period of this study would have spared 36 patients the need for ERCP.


Intracerebroventricular neuropeptide Y stimulates bile secretion via a vagal mechanism. The effect of intracerebroventricular injection of neuropeptide Y on biliary secretion was studied in conscious dogs, prepared with gastric and duodenal fistulas and cerebroventricular guides. Bile secretion was increased in a dose-dependent fashion by intracerebroventricular neuropeptide Y. The peak increase was seen after 500 pM/kg of neuropeptide Y which resulted in a 30 x 2% increase in bile flow over the period 30-150 minutes after injection. ( 23 x 2 (1 x 2) ml/2 hours; neuropeptide Y 500 pM/kg: 30 x 5 (1 x 1) ml/2 hours). Biliary lipid composition was not altered significantly but bicarbonate output was increased at all doses tested. Intravenous infusion of neuropeptide Y (1000 pM) for 1 hour had no significant effect. Intracerebroventricular neuropeptide Y (1000 pM/250-300 mg body weight) also increased bile flow in urethane-anaesthetised rats. This effect was abolished by cervical vagotomy. The demonstration of a central stimulation of alkaline bile flow suggests that bile secretion may be subject to central modulation.


Minimal change chronic pancreatitis.

Minimal change chronic pancreatitis. In patients with severe abdominal pain, of pancreatic origin, there are a few with minimal or equivocal findings on pancreatic investigation and in whom the aetiology of their pancreatic disease is elusive. The findings and outcome in 16 of these patients (four men and 12 women) who underwent resection are reported. Pancreatic imaging showed minimal or equivocal findings in all 16; pancreas divisum was present in five. All were managed conservatively at first but resection was required for progression of symptoms. A drainage procedure was performed initially in five patients but relief of pain was at best transitory before further surgery was required. Partial resection was needed in 12, of whom eight required subsequent completion pancreatectomy and four had a one stage total resection. Nine patients are currently pain free after resection or are very much improved, while six are no better and one patient has died from an unrelated cause. Histology of resected specimens showed chronic inflammatory changes accompanied by subtle non-inflammatory changes in all but one. These changes include duct proliferation, duct complex formation, adenomatous nodules, and acinar cell atrophy, the significance of which is unclear. These findings suggest a syndrome of minimal macroscopic and radiological change chronic pancreatitis with pain as its chief clinical feature and a distinct histology, the aetiology of which is unclear. It seems that there is a distinct syndrome of minimal change pancreatitis, among the group of patients which presents with the clinical features of chronic pancreatitis.


Effective treatment of diabetic diarrhoea with somatostatin analogue, octreotide. A 22 year old insulin dependent diabetic with high volume, secretory chronic diarrhoea refractory to standard andiarrhoeal drugs was treated with the somatostatin analogue octreotide, 50 micrograms twice daily by subcutaneous injection. She improved markedly with a decrease in mean stool weight from 1170 g/24 h range 440-2900 g) to 440 g/24 h (range 180-800 g) (p < 0.05). Stool frequency also decreased from six (range two to 12) to one (range one to three) bowel movements per day (p < 0.01). Mouth to caecum transit time increased from 45 minutes to > 210 minutes, although total gut transit time was unchanged and remained rapid at nine hours. Thus octreotide can reduce stool volume and frequency in high volume diabetic diarrhoea when conventional antidiarrhoeal agents have failed. Its therapeutic benefit appeared to be predominantly related to a marked increase in mouth to caecum transit time.


The possibilities of changing the production of exocellular polysaccharides of a mutant Mycobacterium strain. By second-step mutagenesis and treatment with N-methyl-N'-nitro-N-nitrosoguanidine a mutant strain of Mycobacterium sp. V-649 producing a glucan extracellular polymer and another new streptomycin-resistant mutant were prepared. This mutant strain formed more than 100% first-rate (1.0-1.2%) exocellular polysaccharide. Treatment with 1% dimethyl sulfoxide during submerged cultivation of the mutant strain did not increase the production of the extracellular polysaccharide.


Virulence of silver-resistant mutant of Klebsiella pneumoniae in burn wound model. A silver-resistant mutant of Klebsiella pneumoniae B-5 was produced by passaging in nutrient broth containing graded concentrations of silver nitrate up to 150 ppm. The development of silver resistance in the strain resulted in rough colonies, decrease in cell size, carbohydrate content and change in klebocin pattern. The virulence of the AgR strain as checked by the burn wound model decreased as the mutant could not establish itself in the skin and spleen of the animals and the organism was cleared more efficiently by human lymphocytes than the parent AgS strain.


Immobilized beta-glucosidase from Curvularia lunata. beta-Glucosidase from Curvularia lunata was immobilized in pellets of polyacrylamide, sodium alginate and agar. The activity of the enzyme was estimated at different times by measuring the absorbance of a solution into which 2-nitrophenol was released by the enzyme. The effect of pH and temperature was studied to select the optimum conditions. Thermostability of the beta-glucosidase in each of the carriers was assessed over a period of 12-26 d. The immobilized enzyme on all the three carriers retained its activity longer than free enzyme did. Polyacrylamide was the best carrier both in terms of thermostability and of reusability of the immobilized enzyme preparations. The Michaelis constant (Km) for each of the immobilized enzyme preparation was calculated.


UV-induced mutability in repair-deficient rad6-1 strains of Saccharomyces cerevisiae is caused by a suppressor gene. The RAD6 gene is a multifunctional gene required for DNA repair, induced mutagenesis and sporulation. The survival and revertibility of two loci in four rad6-1 mutant strains of different origin after UV irradiation were followed. As expected, all the rad6-1 strains tested were more sensitive to UV radiation in comparison with RAD6 strains. The reversion frequency per survivor in trp1-289 and arg4-17 alleles was significantly higher in all four rad6-1 mutant strains than in wild-type strains after equal doses of UV radiation. On the basis of genetic analysis we suggest that the phenomenon of increased frequency of induced mutagenesis is caused by a suppressor gene.


Expression of Japanese quail ovalbumin in Saccharomyces cerevisiae. A cDNA sequence coding for Japanese quail ovalbumin was used for the construction of expression plasmid under the ADH1 promoter of the yeast shuttle vector pVT101-U. The resulting recombinant expression vector pJK2 was used for the transformation of Saccharomyces cerevisiae. Expression of quail ovalbumin in yeast cells was demonstrated by Western blotting followed by immunochemical detection.


Intrapopulation variability of antibiotic biosynthesis in streptomycetes. Spontaneous variants making up parallel series of hereditary variability inside the populations of antibiotic-producing actinomycetes differ in the level of their antibiotic activity. As a rule, spontaneous variants of the basic type possess the highest antibiotic activity. Other variants representing parallel series have a lower activity level. This raises the possibility to carry out a directed selection of previously known active colonies from populations on the basis of their easily discernible morphological properties. It enhances the efficiency of selection work both in the case of stabilization of the level of antibiotic activity and in the case of obtaining more productive commercial strains.


Ethanol-induced death and lipid composition of Saccharomyces cerevisiae: a comparative study of the role of sterols. Ethanol tolerance of four Saccharomyces cerevisiae strains characterized by different amounts of delta 5,7-sterols was tested. The individual tolerances did not correlate with the strains sterol levels. The highly and medium-accumulating strains exhibited the highest and lowest ethanol tolerances, respectively.


The K3 type killer strains of genus Saccharomyces for wine production. Using simultaneous fusion of protoplasts of three strains the auxotrophic K3 killer designated MH1 was prepared at a very low frequency and further employed for the transfer of the K3 killer character into a commercial wine yeast. A novel Saccharomyces Bratislava 1K3 strain with desirable technological and anti-yeast killer abilities was thus constructed. Attempts to prepare double K1/K3 killers were made. The obtained fusion products contained M1 dsRNA and were able to produce only the K1 type killer toxin.


Enhanced biodegradation of a hard bis-quaternary ammonium salt. Bacterial strains with a high biodegradation potential were isolated from activated sludge. Their ability to decompose the hard bis-quaternary ammonium salt FB was determined by the method of chemical oxygen demand (COD) in a mineral medium, where the compound FB was the only source of carbon. The COD values were very low after 21 d and in the course of this period they reached zero level twice. The contribution of adsorption to decrease the COD value was small. The maximum COD decrease was accompanied by an increase of cell respiration. It is suggested that FB is effectively decomposed in spite of the fact that according to its structure it is a typical hard detergent.


Consumption of a large dose of alcohol in a short time span. Blood alcohol concentrations (BAC) and time to peak BAC were determined in 16 subjects after ingestion of a large quantity of alcoholic beverages within a short drinking time span not exceeding 30 min. The first group (7 subjects) consumed alcohol after a 3- to 4-h fast. In the second group (9 subjects) the consumption of alcohol took place after eating a large meal. Venous blood samples taken 30 min after drinking finished were compared to the near-simultaneous Breathalyzer results. In addition, the minimum duration of a BAC plateau for these drinking circumstances was assessed.


Enzyme-linked immunosorbent assay (ELISA) for the determination of the toxic glycoside cleistanthin B. Cleistanthus collinus is a highly toxic plant frequently implicated in suicidal and homicidal poisoning cases referred to our laboratory. An enzyme-linked immunosorbent assay (ELISA) is reported in this paper for the quantitation of the active principles of C. collinus. This method is highly reproducible and sensitive to detect as low as 2 ng/ml of cleistanthin B, a toxic constituent of C. collinus. The ELISA can be successfully applied for the specific identification of C. collinus poisoning in clinical and forensic toxicology.


Precision of estimating time of death by vitreous potassium--comparison of various equations. This paper is a study of the precision of estimating the time since death comparing the equations developed by different authors. Our aim is to determine with the maximum degree of accuracy the exact time of death of the individual. We consider that the study has been fully justified by the observed differences in the results obtained from the different equations under study when the concentration of potassium in the vitreous humour was identical.


Grading rigor mortis with myotonometry--a new possibility to estimate time of death. Myotonometry has been applied to grade rigor mortis. The myotonometer is a hand-held instrument which gives a mechanical impact against the muscle. The muscle responds with a damped vibration which is registered and treated mathematically. The period and the logarithmic decrement of decay of the vibrations were found to change with the development of rigor mortis. The method gives quantitative information on the muscular stiffness and it can be used when estimating the time of death.


Bupropion and alcohol fatal intoxication: case report. A fatality due to the ingestion of bupropion and ethanol is presented. Bupropion and its metabolites were extracted from several tissues and identified using gas chromatography with nitrogenphosphorus and mass spectrometry detection. The concentrations of bupropion, hydroxybupropion and the erythroamino and threoamino alcohol metabolites in heart blood were 4.2, 5.0, 0.6 and 4.6 mg/l, respectively. The heart blood ethanol concentration was 0.27 g/dl. In addition, bupropion was distributed as follows: subclavian blood, 6.2 mg/l; bile, 1.4 mg/l; kidney, 2.4 mg/l; liver, 1.0 mg/kg; stomach contents, 16 mg and urine, 37 mg/l.


Suicidal hanging in Cardiff--a 15-year retrospective study. A retrospective study of 84 cases of suicidal hanging was undertaken in which petechial haemorrhages or congestion were seen in 27%, soft tissue haemorrhage in 29% and fracture of the hyoid or laryngeal cartilages in 36%. Fractures were found to be related to increasing age and were not found when a soft ligature had been employed. Petechial haemorrhages and congestion were related to the completeness of suspension. Apart from the lack of fractures in the soft ligature group any of these findings, either singly or in combination, was found to occur without regard to age, suspension type or ligature type. No evidence was found of clustering or seasonality. The findings are compared to other series and discussed with regard to the mechanisms of causation.