Three-dimensional ultrasonographic evaluation of ovarian tumours.
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Female↗
Biomedical subjects
M Rehn
Publications and source records attributed to M Rehn.
The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro.
Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.
Basement Membrane↗
Type XIII collagen is identified as a plasma membrane protein.
The complete primary structure of the mouse type XIII collagen chain was determined by cDNA cloning. Comparison of the mouse amino acid sequences with the previously determined human sequences revealed a high identity of 90%. Surprisingly, the mouse cDNAs extended further in the 5' direction than the previously identified human clones. The 5' sequences contained a new in-frame ATG codon for translation initiation which resulted in elongation of the N-terminal noncollagenous domain by 81 residues. These N-terminal sequences lack a typical signal sequence but include a highly hydrophobic segment that clearly fulfills the criteria for a transmembrane domain. The sequence data thus unexpectedly suggested that type XIII collagen may be located on the plasma membrane, with a short cytosolic N-terminal portion and a long collagenous extracellular portion. These sequence data prompted us to generate antipeptide antibodies against type XIII collagen in order to study the protein and its subcellular location. Western blotting of human tumor HT-1080 cell extract revealed bands of over 180 kDa. These appeared to represent disulfide-bonded multimeric polypeptide forms that resolved upon reduction into 85-95-kDa bands that are likely to represent a mixture of splice forms of monomeric type XIII collagen chains. These chains were shown to contain the predicted N-terminal extension and thus also the putative transmembrane segment. Immunoprecipitation of biotinylated type XIII collagen from surface-labeled HT-1080 cells, subcellular fractionation, and immunofluorescence staining were used to demonstrate that type XIII collagen molecules are indeed located in the plasma membranes of these cells.
Amino Acid Sequence↗
Collagen XVIII is localized in sinusoids and basement membrane zones and expressed by hepatocytes and activated stellate cells in fibrotic human liver.
Type XVIII collagen is a recently discovered nonfibrillar collagen associated with basement membranes in mice and expressed at high levels in human liver. We studied the origin, distribution, and RNA levels of type XVIII collagen in normal and fibrotic human livers by in situ hybridization, immunohistochemistry, and Northern and dot blots and compared procollagen alpha1(XVIII) RNA levels with those of procollagen alpha1(IV) and laminin gamma1, the two major components of liver basement membranes. In normal liver, type XVIII collagen was heavily deposited in perisinusoidal spaces and basement membrane zones. The major source of type XVIII collagen was hepatocytes and, to a lesser extent, endothelial, biliary epithelial, and vascular smooth muscle cells and peripheral nerves. In cirrhosis, type XVIII collagen formed a thick deposit along capillarized sinusoids. Grain counts after in situ hybridization showed myofibroblasts to increase their expression 13-fold in active and twofold in quiescent fibrosis, whereas hepatocytes increased their expression only twofold in both active and quiescent fibrosis. Activated stellate cells in vitro expressed type XVIII collagen at high levels. These data indicate that type XVIII collagen is a component of the perisinusoidal space and is associated with basement membrane remodeling. Hepatocytes and activated stellate cells are important sources of type XVIII collagen in normal and fibrotic liver respectively, which suggests tissue-specific regulation of its expression.
Basement Membrane↗
Role of the cytoskeleton in stimulation of Na+ channels in A6 cells by changes in osmolality.
Permeable supports with A6 cell monolayers were mounted in an Ussing chamber and bilaterally bathed with Ringer solution at room temperature. Short-circuit current (Isc) was recorded continuously, and noise analysis revealed microscopic channel current characteristics. Our investigation focuses on the stimulation of apical Na+ entry caused by exposing the serosal surface of the A6 cell monolayers to hyposmotic Ringer solution. To evaluate the possible role of the cytoskeleton in the regulation of Na+ channels in response to a change in osmolality we used four different experimental approaches. In the control group, which were not exposed to any cytoskeleton-influencing drugs, there was a 1.5-fold increase in Isc and in the number of open Na+ channels after osmotic stimulation. For the second group cytochalasin D (0.1 microg/ml) was present on the serosal side during the experiments. Neither Isc nor the number of open Na+ channels increased after osmotic stimulation. In the third group colchicine (0.2 mM) or nocodazole (20 microM) was present on the serosal side, which resulted in 1.8-fold and 1.5-fold increases in Isc as well as 3-fold and 2-fold increases in the number of Na+ channels, respectively. In the fourth experimental group erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA, 0.5 mM), a dynein inhibitor, was present on the serosal side. In this group Isc decreased to about 0.4 microA/cm2, and subsequent application of amiloride abolished Isc completely. Under hyposmolar conditions EHNA abolished entirely the sensitivity of Isc to the osmotic challenge. Because of the EHNA-induced down-regulation of Isc, the density of apical Na+ channels in this experimental group could not be determined. These results show that the cytoskeleton is dominantly involved in osmotic channel regulation at the apical membrane, and that actin filaments, microtubules and molecular motors are involved in the recruitment of additional Na+ channels.
Actins↗
The short and long forms of type XVIII collagen show clear tissue specificities in their expression and location in basement membrane zones in humans.
Two N-terminal ends of human type XVIII collagen chains have recently been identified. The two chains have different signal peptides and variant N-terminal noncollagenous NC1 domains of 493 (NC1-493) and 303 (NC1-303) amino acid residues, respectively, but share 301 residues of their NC1 domains as well as the collagenous and C-terminal noncollagenous portions of the molecule. Antibodies were produced against the NC1 region common to both human alpha1(XVIII) chain variants and against NC1 sequences specific to the long variant and were used in combination with in situ hybridization to localize this collagen in a number of human tissues. They were also used for Western blotting, which resulted in detection of overlapping high-molecular weight bands above the 200-kd standard in a kidney extract. Heparin lyase II and heparin lyase III digestions of kidney and placenta extracts indicated that at least in these tissues, type XVIII collagen contains heparin sulfate glycosaminoglycan side chains. Type XVIII collagen was found to be a ubiquitous basement membrane component, occurring prominently at vascular and epithelial basement membranes throughout the body. Comparison of the expression of the NC1-493 and NC1-303 variants revealed marked differences. The short variant was found in most conventional basement membranes, including blood vessels and the various epithelial structures, and around muscular structures. The long variant was expressed very strongly in liver, where it was virtually the only variant in the liver sinusoids, and it occurred only in minor amounts elsewhere. Thus, the 192 N-terminal residues specific to the long variant apparently confer some functional property needed above all in the liver sinusoids, but also at certain other locations.
Antibodies↗
Complete primary structure of two variant forms of human type XVIII collagen and tissue-specific differences in the expression of the corresponding transcripts.
We report on full-length human type XVIII collagen cDNAs that encode 1516- or 1336-residue alpha 1 (XVIII) chains. The two chains have different signal peptides and variant N-terminal non-collagenous NC1 domains of 493 (NC1-493) and 303 (NC1-303) amino acid residues, respectively, but share 301 residues of their NC1 domains, a 688-residue highly interrupted collagenous portion, and a 312-residue C-terminal non-collagenous portion. Alternative splicing affecting a 43-residue stretch at the junction of the NC1 domain and the beginning of the collagenous portion was identified. The amino acid sequences of the human and previously characterized mouse alpha 1 (XVIII) chains exhibit an overall identity of 79%. The highest homology between these chains was observed in their last 184 residues, corresponding to the proteolytic fragment endostatin, which is capable of inhibiting endothelial cell proliferation, angiogenesis and tumor growth (O'Reilly, et al., Cell 88: 277-285, 1997). Northern analysis of several adult and fetal tissues with a probe for the NC1-493 variant revealed marked amounts of the corresponding 6.2 and 5.0 kb mRNAs in liver, while other tissues contained only faint or undetectable signals. Hybridizations with a probe specific for the NC1-303 variant virtually lacked the liver signal but revealed clear 5.6 and 4.5 kb bands in heart, kidney, placenta, prostate, ovaries, skeletal muscle and small intestine, and faint signals in several other tissues. Thus mRNAs for the long variant occur prominently in liver, while those for the short variant appear to be the major ones in the other tissues analyzed.
Adult↗
The frizzled motif: in how many different protein families does it occur?
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Amino Acid Sequence↗
Successful abdominal delivery in a woman with sonographic diagnosis of diffuse cavernous hemangioma of the uterus.
Diffuse hemangioma of the pregnant uterus is a serious lesion. We report the first case of a successful cesarean section at term following expectant management of pregnancy in a patient with presumed isolated diffuse cavernous hemangioma of the uterus and protein S deficiency. The sonographic diagnosis and clinical management of this condition is described. The presented successful pregnancy underlines that, under close surveillance, consideration should be given to a conservative approach to this sonographic finding during pregnancy, as even an abdominal delivery does not imply hysterectomy inevitably.
Adult↗
Alternative splicing of mouse alpha1(XIII) collagen RNAs results in at least 17 different transcripts, predicting alpha1(XIII) collagen chains with length varying between 651 and 710 amino acid residues.
The alpha1(XIII) collagen chain has three collagenous domains (COL1-COL3) and four noncollagenous domains (NC1-NC4). A hydrophobic sequence in the extreme amino-terminal noncollagenous domain suggests that type XIII collagen is a transmembrane protein. The alpha1(XIII) collagen RNA is characterized by complex alternative splicing. In this study, expression of the alpha1(XIII) collagen chain was detected in 12 mouse tissues using reverse transcription (RT) and the polymerase chain reaction (PCR). Alternative splicings affecting the COL1, NC2, and COL3 domains were first evaluated separately. Subsequently, sequences spanning from the NC1 domain to the NC4 domain were studied for the first time to elucidate how the alternative splicing of type XIII collagen transcripts affects the structures of the entire mRNAs. A total of 10 alternatively spliced exons, which were freely combinatory, and 9 new exon combinations encoding parts of the COL1, NC2, and COL3 domains have been found. The sequences for the COL1 domain involved two common variants, one containing all the known COL1 exons and the other lacking exon 4B. Exons 12 and 13, encoding most of the NC2 domain, were subject to an alternative splicing that was found to display marked tissue-specific differences. The most common variant of the COL3 sequences lacked exons 28B and 33, or only the exon 33, which was found to be 100% identical to the corresponding human sequences. A total of 17 splice combinations of nine exons were characterized. The results suggest that the predicted length of the corresponding polypeptide varies between 710 and 651 residues.
Alternative Splicing↗
Characterization of the mouse gene for the alpha 1 chain of type XVIII collagen (Col18a1) reveals that the three variant N-terminal polypeptide forms are transcribed from two widely separated promoters.
The mouse gene for the alpha 1 chain of type XVIII collagen (Col18a1) is more than 102 kb and consists of 43 exons. Type XVIII collagen transcripts encode polypeptides that differ with respect to three variant N-terminal noncollagenous domains that are 301 (NC1-301), 517 (NC1-517), or 764 (NC1-764) residues in length. Characterization of genomic clones revealed that the three variant NC1 domains result from the use of two alternative promoters, separated by a distance of 50 kb. The upstream promoter, promoter 1, directs the synthesis of the NC1-301 domain in conjunction with exons 1 and 2, whereas the downstream promoter, promoter 2, directs that of the NC1-517 and NC1-764 domains in conjunction with exon 3, with the latter two variants differing with respect to alternative splicing of the exon 3 sequences. Exons 4-9 encode a portion of the NC1 domain shared by all three polypeptide variants, and exons 9-43 encode the common collagenous and C-terminal noncollagenous sequences. The marked differences previously observed in the expression of variant type XVIII collagen transcripts in mouse tissues thus result from tissue-specific use of these two promoters.
Alternative Splicing↗
Transvaginal ultrasonography of pelvic masses: evaluation of B-mode technique and Doppler ultrasonography.
OBJECTIVE: The purpose of this study was to evaluate pelvic masses by B-mode and Doppler ultrasonography for identification of ovarian malignancies. STUDY DESIGN: A previously described scoring system for pelvic masses was applied in 310 women, and the lesions were classified into four groups according to the ultrasonographic structure. Pulsatility index values of vessels within the tumor and the contralateral ovary and of both uterine arteries were determined. RESULTS: A total of 259 masses were benign and 51 were malignant. The scoring system and classification into morphologic groups revealed similar sensitivity, specificity, and positive and negative predictive values. Intratumoral and opposite ovarian blood flow and ipsilateral and contralateral uterine blood flow did not differ in benign and malignant masses except in cases of benign lesions in postmenopausal women. In malignant lesions a lower pulsatility index (0.94 +/- 0.4) was measured than in benign lesions (1.06 +/- 0.4, p < 0.05), although a remarkable overlap was found. CONCLUSION: The results suggest that further refinement of assessment of pelvic masses with Doppler ultrasonography is needed.
Adnexal Diseases↗
Identification of three N-terminal ends of type XVIII collagen chains and tissue-specific differences in the expression of the corresponding transcripts. The longest form contains a novel motif homologous to rat and Drosophila frizzled proteins.
Transcripts for the alpha 1 chain of mouse type XVIII collagen were found to be heterogeneous at their 5'-ends and to encode three variant N-terminal sequences of the ensuing 1315-, 1527-, or 1774-residue collagen chains. The variant mRNAs appeared to originate from the use of two alternate promoters of the alpha 1(XVIII) chain gene, resulting in the synthesis of either short or long N-terminal non-collagenous NC1 domains, the latter being further subject to modification due to alternative splicing of the transcripts. As a result, the 1527- and 1774-residue polypeptides share the same signal peptide, and the lengths of their NC1 domains are 517 or 764 amino acid residues, respectively, while the 1315-residue polypeptide has a different signal peptide and a 301-residue NC1 domain. The longest NC1 domain was strikingly characterized by a 110-residue sequence with 10 cysteines, which was found to be homologous with the previously identified frizzled proteins belonging to the family of G-protein-coupled membrane receptors. Thus, it is proposed that the cysteine-rich motif, termed fz, represents a new sequence motif that can be found in otherwise unrelated proteins. Tissues containing mainly one or two NC1 domain mRNA variants or all three NC1 domains were identified, indicating that there is tissue-specific utilization of two alternate promoters and alternative splicing of alpha 1(XVIII) transcripts.
Amino Acid Sequence↗
Surveillance of pregnant diabetics--comparison between outpatient and inpatient management.
From 1982 to 1993 67 diabetic women attended the University Women's Hospital obstetric clinic at Würzburg. These women were separated into two groups: group 1 (inpatient group) delivered between 1/82 and 7/87, group 2 (outpatient group) delivered between 8/87 and 4/93. Between 1982 and 1987 we used extensive inpatient care. But did not so after 1987. The fetus was monitored by ultrasound scans, cardiotocography, oxytocin stress test and doppler measurements. Nearly all patients used home blood glucose monitoring. We compared blood glucose levels, mode of delivery and fetal outcome for the two groups. There was no significant difference in the average blood glucose levels between the two groups. Cesarean section rates (37%) and gestational age at delivery (37 weeks) were similar in both groups. Fetal macrosomia (birth weight > 4000 g) occured more frequently in the outpatient group (group 1: 4%, group 2: 25%, P < 0.01). This fact did not effect perinatal morbidity. Postnatal metabolic disorders did not increase. Fetal anomalies occurred less frequently in the outpatient group (group 1: 7%, group 2: 2%, P < 0.05).
Adult↗
Two new collagen subgroups: membrane-associated collagens and types XV and XVII.
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Amino Acid Sequence↗
Structure and expression of the human gene for the alpha subunit of prolyl 4-hydroxylase. The two alternatively spliced types of mRNA correspond to two homologous exons the sequences of which are expressed in a variety of tissues.
Prolyl 4-hydroxylase, an alpha 2 beta 2 tetramer, plays a central role in collagen synthesis as it catalyzes the formation of 4-hydroxyproline residues by the hydroxylation of proline in X-Pro-Gly sequences. We report here that the human gene for the catalytically important alpha subunit is more than 69 kilobase pairs and consists of 16 exons. The exons that encode solely protein sequences vary from 54 to 240 base pairs (bp), and the introns vary from 750 to more than 16,000 bp. The 133 bp of 5'-untranslated sequences of the mRNA are coded by two exons, and these sequences contain inverted repeats with a potential for stem-loop formation, which may be involved in translational control of the expression of this gene. The 5'-flanking region contains a TATa motif at -29 relative to the major transcription site but no CCAAT motif. The 5'-flanking region and the downstream sequences contain several motifs that may act as binding sites for various transcription factors. Evidence has previously been reported for a mutually exclusive alternative splicing of RNA transcripts of this gene. The present data indicate that the mutually exclusive sequences found in the mRNAs are coded by two consecutive, homologous 71-bp exons 9 and 10. These exons are identical in their first 5 bp and the overall identity between them is 61% at the nucleotide level and 58% at the level of the coded amino acids. Both types of mRNA were found to be expressed in all of the tissues studied, but in some tissues the type coding for exon 9 or 10 sequences was more abundant than the other type.
Alternative Splicing↗
cDNA cloning and amino acid sequence of human mitochondrial delta 3 delta 2-enoyl-CoA isomerase: comparison of the human enzyme with its rat counterpart, mitochondrial short-chain isomerase.
We report the isolation of a cDNA encoding a mature human monofunctional delta 3 delta 2-enoyl-CoA isomerase and the determination of its nucleotide sequence. The purified uncleaved protein, as well as several internal tryptic and CNBr fragments, were subjected to N-terminal peptide sequencing. The deduced amino acid sequence of the mature protein consists of 260 amino acids with a predicted M(r) of 28735. The human mitochondrial isomerase exhibits a 74% (78%) sequence identity with the corresponding rat counterpart at amino acid (nucleotide) level(s). Many basic amino acid residues in rat isomerase have been changed to acidic or neutral residues in human enzyme, explaining the differences observed between these proteins.
Amino Acid Sequence↗
Primary structure of the alpha 1 chain of mouse type XVIII collagen, partial structure of the corresponding gene, and comparison of the alpha 1(XVIII) chain with its homologue, the alpha 1(XV) collagen chain.
We have isolated cDNAs that complete the elucidation of the primary structure of the mouse alpha 1(XVIII) collagen chain, a polypeptide homologous to the alpha 1(XV) collagen chain. The 1315-residue alpha 1(XVIII) chain includes a 25-residue signal peptide, a 301-residue NH2-terminal non-collagenous domain (NC1), a 674-residue collagenous sequence with nine interruptions of 10-24 residues, and a 315-residue COOH-terminal noncollagenous domain (NC11). Seven of the collagenous domains and both flanking noncollagenous domains share homology with the alpha 1(XV) chain. The COOH-terminal noncollagenous domains are unique to the alpha 1(XVIII) and alpha 1(XV) chains, and they contain a homologous beginning, a variable portion, and a highly homologous COOH-terminal half with 4 conserved cysteines. The differences in the collagenous sequences probably preclude the existence of the two chains in the same molecule, however. A 12.5-kilobase pair genomic sequence was found to contain the 12 extreme 3'-exons of the alpha 1(XVIII) gene, covering 40% of the coding sequences. Exons start with either a complete codon or a split codon for the glycines of Gly-Xaa-Yaa repeats, and seven exons completely cover the NC11 domain. Comparison of the sequences encoded by these seven exons with the corresponding region of the alpha 1(XV) gene indicated conserved exon-intron organization, suggesting that the two genes derive from a common ancestor.
Amino Acid Sequence↗