Optimal number of multiple rapid swallows needed during high-resolution esophageal manometry for accurate prediction of contraction reserve.

BACKGROUND: Multiple rapid swallows (MRS) is a provocative test for assessment of contraction reserve, however reproducibility on repetitive MRS is incompletely understood. Our aim was to determine the optimal number of MRS sequences for consistent assessment of contraction reserve. METHODS: One hundred and fifty-nine consecutive patients (79 IEM and 80 normal motility) who underwent high-resolution manometers were enrolled. Ten single swallows (SS) and 10 MRS were performed. Gold standard for evaluation of the contraction reserve was the ratio between the mean DCI of 10 MRS and the mean DCI of 10 SS (MRS/SS DCI ratio). Rates of false negatives and false positives were calculated for increasing numbers of MRS sequences, using either mean DCI or the MRS with the highest DCI. KEY RESULTS: According to the gold standard, 50 IEM and 50 normal motility patients had contraction reserve. With progressively increasing numbers of MRS sequences, contraction reserve was detected using mean MRS DCI within three and four MRS sequences in IEM and normal motility respectively, whereas two and three MRS sequences were needed using the MRS sequence with the highest DCI. False positives were much higher with highest DCI method compared with mean DCI, (22% vs 9% respectively in IEM; 24% vs 9% in normal motility) when three MRS sequences were considered. CONCLUSIONS & INFERENCES: At least three MRS are needed to reliably assess contraction reserve. The mean DCI of the three MRS sequences is the best variable to utilize as evidence of contraction reserve.

Gastro-esophageal reflux and antisecretory drugs use among patients with chronic autoimmune atrophic gastritis: a study with pH-impedance monitoring.

BACKGROUND: Patients with chronic autoimmune atrophic gastritis (CAAG) often refer digestive symptoms and are prescribed antisecretory medications. Aims were to investigate: (i) gastro-esophageal reflux (GER), (ii) psychopathological profile, (iii) frequency of use and clinical benefit of antisecretory drugs. METHODS: Prospective observational study on 41 CAAG patients who underwent: 24 h multichannel intra-luminal impedance-pH (MII-pH) monitoring off-therapy, standardized medical interview and psychological questionnaire (i.e., SCL-90R). The medical interview was repeated at least 1 month after MII-pH in patients who were using antisecretory drugs. Statistical analysis was performed calculating median (10th-90th percentiles) and risk ratios (RR) with 95% confidence interval. KEY RESULTS: Median intra-gastric pH was 6.2 (4.6-7.0). One patient had acid reflux (AC) associated with symptoms, five had increased total reflux number and four had symptoms associated to non-acid reflux (NA) (patients referred as 'GER positive'). Using patients 'GER negative' with normal SCL-90R as reference, the RR of being symptomatic in patients GER positive was 2.1 (1.1-4.1) if SCL-90R was normal and 0.9 (0.5-1.7) if it was altered (difference in RR significant being p = 0.04). Seventeen/28 (61%) symptomatic patients were on antisecretory drugs, which were stopped in 16 of them according to results of MII-pH and clinical evaluation after 574 days (48-796) showed that symptoms were unchanged. CONCLUSIONS & INFERENCES: In patients with CAAG (i) AC reflux rarely occurred whereas increased NA reflux was not infrequent both being related to symptoms in some patients, (ii) psychopathological profile has a role in symptoms' occurrence, (iii) antisecretory drugs were generally inappropriately used and clinically ineffective.

The CD30 gene promoter microsatellite binds transcription factor Yin Yang 1 (YY1) and shows genetic instability in anaplastic large cell lymphoma.

CD30 is a member of the TNF receptor family. Our interest lies in understanding the control of CD30 expression, particularly as its over-expression provides a diagnostic marker for a subset of non-Hodgkin's lymphomas, particularly anaplastic large cell lymphoma (ALCL), and because anti-CD30 treatment has been shown to be efficacious. We have identified a number of regulatory regions, including an Sp1 element in the minimal promoter, and a downstream promoter element that is required for start site selection. The discovery of both an activating AP1 site and an upstream microsatellite that represses transcriptional activity of CD30 suggests that this region is involved in dysregulation of CD30 expression. We have now identified the major microsatellite binding activity as transcription factor Yin Yang 1 by both one-hybrid cDNA library screening and peptide mass fingerprinting. Due to the strong repressive effect of the microsatellite, we also investigated whether microsatellite instability may induce changes in CD30 expression and hence explain the over-expression of CD30 in ALCL. Laser capture microdissection of ALCL biopsies and CD30 microsatellite typing indicated that the neoplastic cells show a high degree of variation, but this does not correlate with high CD30 expression seen in ALCL.

Constitutive and stimulated production of VEGF by human megakaryoblastic cell lines: effect on proliferation and signaling pathway.

Release of vascular endothelial growth factor (VEGF) and other candidate angiogenic factors such as basic fibroblast growth factor and transforming growth factor beta, may play a role in sustaining neoplastic cell proliferation and tumor growth. We evaluated VEGF expression and synthesis in the two erythromegakaryocytic cell lines B1647, HEL and one megakaryocytic cell line MO7 expressing erythroid markers. In this study RT-PCR was performed to evaluate VEGF expression and that of its receptor KDR; VEGF production was assayed by Elisa test and western blot analysis; sensitivity to VEGF was tested by thymidine incorporation. VEGF and its receptor KDR were expressed in B1647 and HEL, both as mRNAs and as proteins, while only KDR transcript was found in MO7 cells. Only B1647 and HEL cells showed a strong spontaneous proliferating activity. In fact, measurable amounts of VEGF were present in the unstimulated cell medium, thus suggesting an autocrine production of VEGF by B1647 and HEL cells, but not by MO7, which was inhibited in mRNA-silencing conditions. This production could not be further boosted by other growth factors, whereas it was inhibited by TGF-beta1. Finally, analysis of Shc signal transduction proteins following stimulation with VEGF indicated that only p46 was tyrosine phosphorylated. These data indicate that leukemic cells may be capable of autocrine production of VEGF which, in turn, maintains cell proliferation, possibly mediated by Shc p46 phosphorylation.

Distribution of human CD30 gene promoter microsatellite alleles in healthy and human immunodeficiency virus-1 infected populations.

The human CD30 gene, found on chromosome 1; 1p36, contains a microsatellite of the type [(CCAT)2-12CCACTTATGCAT]n within the promoter. As the microsatellite has been shown to be both polymorphic and involved in the transcriptional regulation of this gene, it is of potential interest with respect to interindividual differences in CD30 expression. Here we describe a method for determining length variation of this repeat region and determine the frequency and distribution of alleles of the CD30 microsatellite within the Western Australian population. As soluble CD30 levels are predictive for disease associated with human immunodeficiency virus (HIV)-1 infection, we also determined allele frequencies in an HIV+ cohort.

Novel nucleotide substitutions within the coding region of DNMT2 are in strong linkage disequilibrium in Caucasians and Japanese.

Investigations into mechanims by which cytosine methylation may be genetically controlled have led to the identification of single nucleotide polymorphisms within the coding region of DNMT2 that are conserved in different ethnic groups. The DNMT2 I allele includes a G at nucleotide position 104 of exon 2 and a C at position 50 of exon 4. The alternative allele, DNMT2 II, includes an A and T, respectively, at these positions. G was never found in the absence of C and vice versa and A was never found in the absence of T and vice versa. The gene products of DNMT2 I and DNMT2 II differ by the inclusion of a histidine or tyrosine residue at the position specified by codon 101. This amino acid substitution alters the amino acid composition of a conserved methylating enzyme motif shown to be involved in S-adenosylmethionine binding in M.HhaI, a bacterial methyltransferase that is almost identical to DNMT2 in size and structure. Demonstration of strong linkage disequilibrium between the nucleotide substitutions associated with each DNMT2 allele provides valuable tools for the investigation of molecular genetic mechanisms of evolution and speciation.

Five novel alternatively spliced transcripts of DNA (cytosine-5) methyltransferase 2 in human peripheral blood leukocytes.

Alternative splicing of RNA molecules transcribed from DNA (cytosine-5) methyltransferases has been proposed as a mechanism by which methylation is able to effect diverse biological processes in higher eukaryotes. This study has investigated transcriptional versatility of DNA (cytosine-5) methyltransferase 2, which may methylate cytosine residues within 5'-CCTGG-3' pentanucleotides in regions of the human genome devoid of 5'-CG-3' methylation. Five novel splice variants of DNA (cytosine-5) methyltransferase 2 were identified in the peripheral blood leukocytes of healthy subjects following cloning and sequencing of RT-PCR products amplified using gene specific oligodeoxyribonucleotide primers. The generation of some of these splice variants may be influenced by the formation of secondary structures within pre-mRNA due to the repetition of sequences flanking alternatively spliced exons in a reverse and complementary orientation on the same strand. These findings enable novel approaches to investigate the role of RNA secondary structures in alternative splicing. The DNA (cytosine-5) methyltransferase 2 splice variants are generated in all the major cell types of peripheral blood, as well as in neoplastic lymphoid cells indicating that they are unlikely to generate proteins involved in control of the cell cycle or cellular differentiation. Interestingly, the gene products generated by some splice variants completely or partially lack highly conserved amino acid motifs shown to be important for the catalysis of cytosine methylation. The possibility cannot be excluded, therefore, that alternative splicing of DNA (cytosine-5) methyltransferase 2 pre-mRNA may generate protein isoforms which have different methylating capabilities or which are involved in biological processes other than the catalysis of cytosine methylation.

Evidence that cytosine residues within 5'-CCTGG-3' pentanucleotides can be methylated in human DNA independently of the methylating system that modifies 5'-CG-3' dinucleotides.

In contrast to the complex sequence specificities of the prokaryotic DNA methylating systems, the mammalian machinery identified thus far methylates cytosine residues within the context of a 5'-CG-3' dinucleotide. To explore the possibility that cytosine residues that do not precede guanine may be independently methylated in mammalian DNA, we have examined a region of the human myogenic gene, Myf-3, which is not targeted by the methylating system that methylates 5'-CG-3' dinucleotides. Our investigations have revealed cytosine methylation within the 5'-CCTGG-3' pentanucleotides specified by the 0.8-kb Myf-3 probe. We have also found that in DNA from neoplastic cells, in which 5'-CG-3' dinucleotides within Myf-3 become abnormally hypermethylated, cytosine residues within 5'-CCTGG-3' pentanucleotides are not methylated. Moreover, methylation of 5'-CCTGG-3' pentanucleotides was not detected within the closely related Myf-4 gene, which is normally 5'-CG-3' hypermethylated. These findings indicate the existence of a system that methylates 5'-CCTGG-3' pentanucleotides independently of the system that methylates cytosine residues within 5'-CG-3' dinucleotides. It is possible that the 5'-CCTGG-3' methylating system influences the fate of foreign integrated DNA.

Allele-specific variation in the gene copy number of human cytosine 5-methyltransferase.

Previously, we have identified two alternate allelic forms of cytosine 5-methyltransferase, 5-MT I and 5-MT II, specified by polymorphic fragments of 1.5 and 1.1 kb, respectively. In the presence study, a 0.8-kb genomic probe was prepared which was confirmed to be included within the polymorphic fragments. The 0. 8-kb probe hybridised with greater intensity to the 1.1-kb fragment than the 1.5-kb fragment. Densitometric analysis indicated that there is 1 copy of 5-MT associated with 5-MT I, whereas there may be 1-4 copies of the gene associated with the 5-MT II allele. Segregation studies demonstrated that the multiple copies of 5-MT II are inherited in a Mendelian fashion. These results allow novel approaches to investigating the underlying mechanisms of cytosine methylation and gene duplication.

HPLC-fluorescence determination of equilin and equilenin in postmenopausal women's urine.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection (lambda(ex) = 280 nm; lambda(em) = 410 and 312 nm) in combination with a post-column on-line photochemical derivatization is described for the determination of equilin and equilenin in urine from normal postmenopausal women after therapy with conjugated oestrogens. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo-induced modifications were useful for the identification of the analytes. The conjugated (sulphate and glucuronide) forms were analysed after enzymatic or chemical hydrolysis and extracted with chloroform. Solid-phase extraction using strong anion-exchange sorbent was applied to the analysis of unconjugated oestrogen fraction to obtain a practical and reliable sample clean-up. The HPLC separations were achieved using ODS columns with a mobile phase consisting of 0.05 M triethylamine phosphate buffer (pH 4.0)-acetonitrile (64:36, v/v) at a flow rate of 1.0 mL/min. The method was accurate and reproducible; for the equilin and equilenin separation isocratic conditions were satisfactory, allowing a sensitive detection in urine samples with a detection limit of about 50 fmol for equilin (lambda(ex) = 280 nm; lambda(em) = 312 nm, after photoderivatization) and 10 fmol for equilenin (lambda(ex) = 280 nm; lambda(em) = 410 nm).

A novel MspI PCR-RFLP in the human cytosine 5-methyltransferase gene: lack of relevance for malignant lymphoproliferative disease and breast cancer.

Two alternate allelic forms of human cytosine 5-methyltransferase, 5-MT I and 5-MT II, which differ by the absence or presence of an intronic MspI recognition sequence, have been recognised. The polymorphic region was localised using a series of subprobes prepared upon MspI digestion of the 2.5-kb cDNA probe (hmt-2.5). A PCR-based method was then developed for rapid 5-MT genotyping. The gene and phenotype frequencies of 5-MT I and 5-MT II were not significantly different in genomic DNA samples from a series of non-Hodgkin's lymphomas and breast cancer cases compared with DNA from normal subjects. Allelism of 5-MT allows new approaches to the assessment of variation in gene copy number of 5-MT in different types of neoplasia.

Glycosaminoglycan analysis in amniotic fluid and in cultured fibroblasts from normal and holoprosencephalic human embryonic organs.

Glycosaminoglycans are polysaccharides involved in epithelial-mesenchymal interaction and cell differentiation and provide a meshwork which is essential to maintain a proper intercellular milieu. The development of embryonic organs can be accompanied by alterations in the glycosaminoglycan pattern. In pregnancies with malformed fetuses, there are alterations in total glycosaminoglycans and their components (chondroitin 4-6 sulphate, dermatan sulphate, and hyaluronic acid) in amniotic fluid. We examined total glycosaminoglycans and the percentage variations of the single classes in both amniotic fluid and culture medium of fibroblasts from heart, lung, and skin obtained from five normal human fetuses and one with holoprosencephaly. In the amniotic fluid total glycosaminoglycans and their sulphate classes were increased, whereas hyaluronic acid was decreased, compared with controls. The extracellular glycosaminoglycans showed hyaluronic acid reduction in skin, while chondroitin 4-6 sulphate plus dermatan sulphate and heparan sulphate were higher in skin and heart. Our data demonstrate that variations in the glycosaminoglycan pattern are associated with alterations of the cellular environment, which can prevent normal organogenesis.

Serous tumors of the ovary: ultrastructural observations.

One borderline primary serous ovarian tumor and six carcinomas were studied by means of electron microscopy. Borderline malignant tumor evidenced concomitant presence of both benign and malign serous epithelium. Ultrastructural observations revealed differentiation characteristics which involved complex architecture of cell arrangement and polarity in the distribution of the organelle and intercellular junctions. The cilia believed to be more frequent in benign tumors were also reported in poorly differentiated carcinomas and so may not be considered as reliable prognostic markers.