Laparoscopic Heller's cardiomyotomy achieved lesser recurrent dysphagia with better quality of life when compared with endoscopic balloon dilatation for treatment of achalasia.

Achalasia is a rare primary motility disorder of esophagus; treatments include endoscopic balloon dilatation (EBD) and laparoscopic Heller's cardiomyotomy (LC). This study compared EBD versus LC for treatment of achalasia with focus on quality of life (QoL) and prevalence of post-treatment gastroesophageal reflux disease. This was a retrospective cohort study of all patients diagnosed with achalasia older than 16 treated with either EBD or LC from January 1998 to April 2008. Patients' demographic data, comorbidities, postintervention GERD symptoms, QoL, recurrence of dysphagia, reintervention rate, hospital stay, and time to resumption of diet were collected. Sixty-eight patients were recruited into the study (EBD n= 50; LC n= 18). A significant improvement in QoL was found in patients undergoing LC (0.917 vs. 0.807, P= 0.006). A higher proportion of patients treated with EBD developed post-treatment gastroesophageal reflux symptoms (60.5% vs. 43.8%) when compared with LC, although statistically insignificant (P= 0.34). Patients treated with balloon dilatation had a greater percentage of recurrence of dysphagia (55.1% vs. 26.7%; P= 0.235) and need of reintervention (42.1% vs. 9.1%; P= 0.045). However, these patients had a shorter median hospital stay (1d [range 0-4]) and earlier resumption of diet (0d [range 0-3]). Although EBD is associated with a quicker perioperative recovery, LC accomplished a better QoL, lower incidence of recurrence of dysphagia, and need of reintervention after treatment for achalasia.

A precursor form of vascular endothelial growth factor arises by initiation from an upstream in-frame CUG codon.

Vascular endothelial growth factor (VEGF) is a mitogen in physiological and pathological angiogenesis. Understanding the expression of different VEGF isoforms might be important for distinguishing angiogenesis in tissue development, vascular remodelling and tumour formation. We examined its expression and noted the presence of the isoforms VEGF(121) and VEGF(165) (121 and 165 residues long respectively) in fetal heart, lung, ovary, spleen, placenta and ovarian tumours. Unexpectedly, a 47 kDa species predominated in fetal intestine and muscle. The presumed initiation site in VEGF is an AUG codon (AUG(1039)), 1039 nt from its main transcriptional start site. AUG(1039) is preceded in the 5' untranslated region by an in-frame CUG at nt 499 (CUG(499)), which could produce the 47 kDa form with a 180-residue N-terminal extension. We therefore assessed whether CUG(499) functions as an initiator. CUG(499) initiation produced the 47 kDa VEGF(165) precursor, which was processed at two sites to yield VEGF and three N-terminal fragments. When CTG(499) was mutated to CGC, the precursor and N-terminal fragments were barely detectable. Although the precursor form was predominant in VEGF(165), both CUG(499) and AUG(1039) forms were found in VEGF(121). VEGF precursor induced neither the proliferation of human umbilical vein endothelial cells nor the expression of angiopoietin 2, which can be induced by, and act with, VEGF to induce tumour angiogenesis. The precursor also adheres to the extracellular matrix (ECM), suggesting that it might be a storage form for generating active VEGF in the cell or ECM. Alternate CUG(499) and AUG(1039) initiation and processing of the inactive precursor and its products might be important in regulating angiogenesis.

Lysophosphatidic acid induction of vascular endothelial growth factor expression in human ovarian cancer cells.

BACKGROUND: Lysophosphatidic acid (LPA) stimulates ovarian tumor growth at concentrations present in ascitic fluid. Vascular endothelial growth factor (VEGF) stimulates angiogenesis and plays a pivotal role in the formation of ovarian cancer-associated ascites. We examined whether LPA promotes ovarian tumor growth by increasing angiogenesis via VEGF. METHODS: VEGF expression was examined in a simian virus 40 T-antigen-immortalized ovarian surface epithelial cell line (IOSE-29) and in ovarian cancer cell lines (OVCAR-3, SKOV-3, and CAOV-3) treated with LPA. VEGF promoter activity was measured in OVCAR-3 cells after transfection or cotransfection with c-Fos and c-Jun, components of AP1 transcription factor, potential binding sites for which are present in the VEGF promoter. The expression of the LPA receptors Edg2 and Edg4 was also assessed. All statistical tests were two-sided. RESULTS: LPA treatment increased steady-state VEGF messenger RNA (mRNA) levels in OVCAR-3 cells in a time- and dose-dependent fashion and stimulated VEGF promoter activity without prolonging mRNA half-life in these cells, but LPA had little effect on IOSE-29 cells. Forced overexpression of c-Jun and c-Fos in OVCAR-3 cells stimulated VEGF promoter activity fourfold. LPA also elevated VEGF protein levels by 1.5-fold in SKOV-3 cells (P =.0148), 1.9-fold in CAOV-3 cells (P<.001), and threefold in OVCAR-3 cells (P<.0001). Both Edg2 and Edg4 were detected in ovarian cancer cells; however, only Edg2 was present in normal ovarian surface epithelial cells and IOSE-29 cells. CONCLUSIONS: LPA stimulates ovarian tumor growth, at least in part, via induction of VEGF expression through transcriptional activation. However, this LPA response is not evident in normal ovarian surface epithelial cells. Our data suggest that Edg4, but not Edg2, plays a role in LPA stimulation of ovarian tumor growth.

Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal endometrial and endometriosis-derived stromal cells.

Several investigators have noted that hormone-dependent development of endometriosis implants lags behind that of simultaneously analysed eutopic endometrium. With the recent discovery of the oestrogen receptor-beta (ER-beta) isoform, the aim of this study was to investigate whether differences in the expression of ER-alpha and ER-beta might explain this observation. mRNA transcripts from endometrial stromal cells isolated from normal endometrium (NE) and from endometriomas (EI) were analysed using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. RT-PCR and Southern blot analyses of the two ER isoforms indicated that NE and EI stromal cells predominantly express ER-alpha mRNA, however the relative concentrations of ER isoform mRNA transcripts differed between the two cell types. Steady-state ER-alpha:ER-beta mRNA ratios were 15.5 +/- 2.8 and 5.2 +/- 0.9 respectively for NE and EI cells (P = 0.02). NE and EI stromal cells expressed ER proteins with similar Kd ( approximately 0.9 nM) and densities ( approximately 24 500 binding sites/cell) respectively. Functional ER expression was indicated by an increase in progesterone receptor concentrations of approximately 60% (P = 0.03) after incubation with 10 nM oestradiol. We postulate that differential transcript processing, ligand specificity and biological actions of the ER-alpha and -beta isoforms may influence differential growth responses in normal and ectopic endometrium.

Estrogen receptor alpha (ER-alpha) and beta (ER-beta) mRNAs in normal ovary, ovarian serous cystadenocarcinoma and ovarian cancer cell lines: down-regulation of ER-beta in neoplastic tissues.

The prognosis in ovarian carcinoma, the most lethal of the gynecologic neoplasms, is poor and has changed little in the last three decades. Only a small number respond to antiestrogen therapy, although the classic estrogen receptor, ER-alpha, has been identified in ovarian surface epithelium, from which approximately 90% of ovarian cancers originate. We have previously shown that ER-beta mRNA is most abundant in human fetal ovaries, suggesting that it might play an important role in ovarian development. Therefore, we investigated the mRNA levels of both ERs in normal ovaries, ovarian serous cystadenocarcinomas, granulosa cells from patients undergoing in vitro fertilization (IVF), the ovarian surface epithelium cell line IOSE-Van, and the ovarian cancer cell lines SKOV3, HEY and OCC1. Northern blots of normal and neoplastic ovaries were hybridized with an ER-beta riboprobe that spans the A/B domain. We detected two major hybridizing bands at approximately 8 and 10 kb. An RNase protection assay using the same probe revealed a single band of the expected size. Hybridizing the same blot with an ER-alpha riboprobe showed a strong hybridizing band at approximately 6.5 kb. In ovarian cancer samples, ER-beta mRNA level was decreased when compared to normal ovaries. Using 25 cycles of RT-PCR followed by Southern blotting, we found equal amounts of ER-alpha and -beta mRNAs in normal ovaries in all age groups from 33 to 75 years; however, in ovarian cancer tissue, the level of ER-alpha mRNA was similar or slightly higher, comparable to 10(3) to 10(4) copies of plasmid DNA, but ER-beta mRNA levels were markedly decreased. Granulosa cells from IVF patients expressed high levels of ER-beta mRNA. The OSE cell line expressed a low level of ER-alpha, detectable after 40 cycles of RT-PCR and no ER-beta mRNA. SKOV3 showed a low level of ER-alpha and -beta mRNAs, whereas OCC1 showed a low level of ER-beta and a relatively high level of ER-alpha. HEY did not contain detectable amounts of either ER after 40 cycles of RT-PCR. We found no evidence of differential splicing or major deletions in almost the entire coding region of ER-beta in either normal ovaries or tumor samples.

Tissue distribution of estrogen receptors alpha (ER-alpha) and beta (ER-beta) mRNA in the midgestational human fetus.

We compared the expression profiles of the mRNAs of both estrogen receptors, ER-alpha and the recently cloned ER-beta, in the midgestational human fetus by semiquantitative RT-PCR. ER-alpha was most abundant in the uterus, and smaller quantities were detected in the ovary, testis, skin and gut. High amounts of ER-beta mRNA were present in fetal ovaries, testes, adrenals and spleen. In these tissues, the levels of ER-beta mRNA were higher than ER-alpha. In the uterus, however, ER-alpha mRNA was more abundant, and ER-beta mRNA was expressed only moderately. ER-beta mRNA was present at moderate to low levels in the thymus, pituitary gland, skin, lung, kidney and brain cortex. In the course of our work, using the ER-beta primers on genomic DNA, an intron of 2468 bp in length, located between nt 222 and 223 in the A/B domain of ER-beta cDNA, was detected, cloned and sequenced. The study shows that the expression profile of the two ERs is different, and ER-beta is expressed in a variety of tissues during human fetal development, suggesting different, organ-specific roles for the two receptors.

T-->A transversion 11 bp from a splice acceptor site in the human gene for steroidogenic acute regulatory protein causes congenital lipoid adrenal hyperplasia.

Congenial lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH. Affected individuals can make no adrenal or gonadal steroids. All affected individuals are phenotypic females irrespective of gonadal sex, and frequently die in infancy if mineralocorticoid and glucocorticoid replacements are not instituted. Recent data implicate the steroidogenic acute regulatory (StAR) protein in this disorder. We now describe a 46,XY patient of Vietnamese ancestry with lipoid CAH who had a somewhat milder form of the disease. Diagnosis was at 10 weeks of age, and low levels of plasma progesterone, corticosterone, 180H-corticosterone and androstenedione were detectable. Testicular RNA for StAR was reverse transcribed, amplified, cloned and sequenced, revealing a 185 bp deletion corresponding to all of exon 5. The corresponding mRNA did not encode active protein in transfected cells. Cloned genomic DNA from the patient revealed only a T-->A transversion in intron 4,11 bp from the splice acceptor site of exon 5. This transversion destroys an NcoI site; digestion of PCR-amplified genomic DNA from the patient and both parents confirmed that the patient was homozygous and the parents were heterozygous. Expression vectors for StAR minigenes were constructed containing all StAR exons plus introns 4, 5 and 6 either with or without the T-->A mutation in intron 4. RNase protection assays showed that expression of the vector with normal intron 4 yielded correctly spliced StAR mRNA in transfected COS-1 cells, while most, but not all StAR mRNA from the vector with the T-->A transversion in intron 4 was abnormally spliced. RNase protection of the patient's testicular RNA confirmed that most, but not all StAR mRNA was similarly spliced abnormally. Splicing errors appear to be a rare cause of genetic diseases, but subtle intronic mutations may be missed when genomic DNA is the only material available for study. The low level of normal StAR mRNA produced may account for the later clinical presentation and low levels of steroid hormones detected in this patient.

A promoter within intron 35 of the human C4A gene initiates abundant adrenal-specific transcription of a 1 kb RNA: location of a cryptic CYP21 promoter element?

Complement component C4 is encoded by two nearly identical genes, C4A and C4B, that encode a C4 precursor that is proteolytically cleaved into the alpha, beta and gamma subunits of the mature protein. C4 is expressed primarily in liver and to a much lesser extent in immune cells. We have identified a unique 1 kb RNA transcript, termed Z, that arises from a cryptic promoter lying in the intron between exons 35 and 36 of the C4 gene. Primer extension, RNase protection, and 5' RACE experiments locate the cap site in intron 35, 55 bases upstream from exon 36. Northern blotting and RNase protection assays show that expression of this 1 kb Z RNA transcript is confined to the adrenal gland. Z RNA contains the same open reading frame as C4 which predicts a protein of 131 amino acids, but antisera to C4 do not interact with epitopes on this protein when it is synthesized by cell-free translation, hence the presence or absence of a Z protein in vivo could not be determined. Transfection of Z promoter/reporter constructs into human adrenal NCI-H295 cells shows that most if not all of the sequences required for high-level adrenal expression lie within 235 bases upstream from the cap site, but that this region is inactive when transfected into COS-1, JEG-3 and Hep-G2 cells, suggesting it contains an adrenal-specific element. The 222 bases upstream from the cap site are 75% identical in the human C4A and mouse Slp genes, and contain a potential binding site for steroidogenic factor 1 (SF-1), an orphan zinc-finger nuclear receptor. We propose that this region, like a nearby region in the mouse genome, functions as an upstream element of the P450c21 promoter, and may be a component of an adrenal-specific locus-control region.

Sequences promoting the transcription of the human XA gene overlapping P450c21A correctly predict the presence of a novel, adrenal-specific, truncated form of tenascin-X.

A compact region in the human class III major histocompatibility locus contains the human genes for the fourth component of human complement (C4) and steroid 21-hydroxylase (P450c21) in one transcriptional orientation, while the gene for the extracellular matrix protein tenascin-X (TN-X) overlaps the last exon of P450c21 on the opposite strand of DNA in the opposite transcriptional orientation. This complex locus is duplicated into A and B loci, so that the organization is 5'-C4A-21A-XA-C4B-21B-XB-3'. Although this duplication event truncated the 65-kb X(B) gene to a 4.5-kb XA gene, the XA gene is transcriptionally active in the adrenal cortex. To examine the basis of the tissue-specific expression of XA and C4B, we cloned the 1763-bp region that lies between the cap sites for XA and C4B and analyzed its promoter activity in both the XA and the C4 orientations. Powerful, liver-specific sequences lie within the first 75 to 138 bp from the C4B cap site, and weaker elements lie within 128 bp of the XA cap site that function in both liver and adrenal cells. Because these 128 bp upstream from the XA cap site are perfectly preserved in the XB gene encoding TN-X, we sought to determine whether a transcript similar to XA arises within the XB gene. RNase protection assays, cDNA cloning, and RT/PCR show that adrenal cells contain a novel transcript, termed short XB (XB-S), which has the same open reading frame as TN-X.(ABSTRACT TRUNCATED AT 250 WORDS)

Tenascin-X: a novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B.

A human gene termed XB overlaps the P450c21B gene encoding steroid 21-hydroxylase and encodes a protein that closely resembles extracellular matrix proteins. Sequencing of phage and cosmid clones and of cDNA fragments shows that the XB gene spans 65 kb of DNA, consisting of 39 exons that encode a 12-kb mRNA. The predicted protein of over 400 kD consists of five distinct domains: a signal peptide, a hydrophobic domain containing three heptad repeats, a series of 18.5 EGF-like repeats, 29 fibronectin type III repeats, and a carboxy-terminal fibrinogen-like domain. Because the structure of the protein encoded by the XB gene closely resembles tenascin, we term this protein tenascin-X (TN-X), and propose a simplified nomenclature system for the family of tenascins. RNase protection experiments show that the TN-X transcript is expressed ubiquitously in human fetal tissues, with the greatest expression in the fetal testis and in fetal skeletal, cardiac, and smooth muscle. Two adrenal-specific transcripts, P450c21B (steroid 21-hydroxylase) and Y (an untranslated transcript) overlap the XB gene on the complementary strand of DNA, yielding a unique array of overlapping transcripts: a "polygene." In situ hybridization histochemistry experiments show that the TN-X transcript and the P450c21 and Y transcripts encoded on the complementary DNA strand are all expressed in the same cells of the human adrenal cortex. Genetic data suggest that TN-X may be essential for life.

Abundant adrenal-specific transcription of the human P450c21A "pseudogene".

Human adrenal steroid 21-hydroxylase (P450c21) is encoded by the CYP21A1 (21B) gene located in the class III region of the HLA locus. A tandemly duplicated gene designated CYP21A1P (21A), which lies 30 kilobases upstream, contains several point mutations and an 8-base pair deletion so that it cannot encode P450c21 protein; as a result, it is generally considered to be a pseudogene. We previously showed that two additional genes, XA and XB, lie on the opposite strand of DNA overlapping the 3'-ends of the 21A and 21B genes. We have now identified a third pair of duplicated overlapping genes in this locus, termed YA and YB, whose transcriptional orientation is the same as 21A and 21B and opposite to XA and XB. YA transcripts use the 21A promoter, have 5'-ends that are similar to 21B mRNA, and have approximately 10-20% of the abundance of 21B transcripts, but have unique 3'-ends. The YA gene encodes a 7.5-kilobase RNA that overlaps XA completely and a 3.0-kilobase RNA that excludes most of XA. The YB gene appears to be similar in size and organization to YA. The YA and YB genes extend beyond the limit of the duplication in this locus; hence, their cDNAs are distinguishable by differences in their 3'-sequences. YA and YB transcripts are expressed only in the fetal and adult adrenal glands, but their cDNAs do not contain a long open reading frame. Although the function of these genes is not yet clear, the complex genetic organization of three overlapping genes (21/X/Y) appears to be unique among higher eukaryotes. As YA transcription is initiated by the 21A 5'-flanking DNA and includes 21A sequences, the designation of 21A as a "pseudogene" merits reconsideration.

Metastatic spread of osteosarcoma to the temporal bone in a patient with Paget's disease: a case report.

Secondary tumors involving the temporal bone are comparatively rare. We report a case of secondary osteosarcoma presenting in the temporal bone which arose from a primary lesion in the tibia in a patient with Paget's disease. To our knowledge secondary osteosarcoma of the temporal bone has never previously been described.

Temperature gradient gel electrophoresis: detection of a single base substitution in the cattle beta-lactoglobulin gene.

An A in equilibrium with G transition in exon III is known to differentiate alleles A and B of the cattle beta-lactoglobulin (BLG) gene. A BLG exon III fragment containing the transition site was amplified by the polymerase chain reaction. Temperature gradient gel electrophoresis (TGGE) was then used to detect this transition and hence to genotype cattle: the AT base-pair in allele A was readily distinguished from the GC base-pair of allele B. TGGE can be used to detect any single base-pair substitution, and thus is a powerful method of detecting genetic variability.