Developmental expression of carbonic anhydrase-related proteins VIII, X, and XI in the human brain.

Three cDNA homologues of carbonic anhydrase with unknown biological functions have been reported: carbonic anhydrase-related proteins (CA-RP) VIII, X, and XI. In the present study, we produced monoclonal antibodies to these CA-RPs and studied their regional and cellular distributions in the human adult and fetal brains by immunohistochemical analysis. In the adult brain, CA-RP VIII was expressed in the neural cell body spreading to most parts of the brain. CA-RP X was expressed in the myelin sheath and its expression was shown in the cytoplasm of cultured tumor cells by immunocytochemical analysis. CA-RP XI was expressed in the neural cell body, neurites, and astrocytes in relatively limited regions of the brain. In the fetal brain, CA-RP VIII and XI were expressed in the neuroprogenitor cells in the subventricular zone as early as the 84th day of gestation and subsequently detected in the neural cells migrating to the cortex. CA-RP X first appeared in the neural cells in the cortex at the 141st day. In the choroid plexus, the epithelial cells gave CA-RP VIII and XI expressions in both adult and fetal brains. From the findings in the present study on the distribution and the developmental expression of CA-RP VIII, X, and XI in the human brain we suggest that these CA-RPs play roles in various biological process of the CNS.

cDNA sequence of human carbonic anhydrase-related protein, CA-RP X: mRNA expressions of CA-RP X and XI in human brain.

A full-length cDNA clone of human carbonic anhydrase-related protein (CA-RP) X was obtained and sequenced. The 2720 bp long cDNA sequence was predicted to encode a 328 amino acid polypeptide. The deduced amino acid sequence showed an overall similarity of 25-57% to other CA isozymes and the highest % similarity to a CA-RP XI. Similar to CA-RP XI, CA-RP X lacked two out of three zinc-liganded histidine residues, suggesting no biological activity of CA. Northern blot analysis demonstrated an approx. 2.8 kb transcript in the human brain and kidney. RNA dot blotting showed significant signals for CA-RP X and XI mRNA expressions in the adult total brain and almost all parts of the central nervous system, but no expression in the fetal brain. These results suggest that CA-RP X and XI play some role in human brain, especially in brain development.

The fine form of Helicobacter pylori on the metaplastic duodenal mucosa is associated with recurrent duodenal ulcers.

BACKGROUND/AIM: It is well known that Helicobacter pylori changes its shapes according to various environmental conditions. The aim of this study was to examine the morphological differences between H. pylori in the duodenum and that in the stomach. METHODS: Duodenal biopsy specimens were obtained from patients with duodenal mucosal lesions, and these specimens were then histopathologically examined. Morphological and genetic properties of cultured H. pylori isolates were analyzed. RESULTS: H. pylori was identified in 16 out of 50 duodenal biopsy specimens. Along with the regular form, we found a fine form of H. pylori in the gastric metaplastic mucosa of ten duodenal specimens that was shorter in length and thinner in diameter than the regular helical form. There were no detectable differences between the ureA-ureB polymorphism of the duodenal fine form and that of gastric regular form in a single patient. As compared with patients without H. pylori in the duodenum, the prevalence of recurrent duodenal ulcers significantly increased in patients with the fine form (p < 0.05), but not in patients with the regular form. CONCLUSION: The fine form of H. pylori in the metaplastic duodenal mucosa could result from its adaptation to the duodenal environment and may be associated with the recurrence of a duodenal ulcer.

Altered expression of fatty acid-metabolizing enzymes in aromatase-deficient mice.

Hepatic steatosis is a frequent complication in nonobese patients with breast cancer treated with tamoxifen, a potent antagonist of estrogen. In addition, hepatic steatosis became evident spontaneously in the aromatase-deficient (ArKO) mouse, which lacks intrinsic estrogen production. These clinical and laboratory observations suggest that estrogen helps to maintain constitutive lipid metabolism. To clarify this hypothesis, we characterized the expression and activity in ArKO mouse liver of enzymes involved in peroxisomal and mitochondrial fatty acid beta-oxidation. Northern analysis showed reduced expression of mRNAs for very long fatty acyl-CoA synthetase, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase, enzymes required in fatty acid beta-oxidation. In vitro assays of fatty acid beta-oxidation activity using very long (C24:0), long (C16:0), or medium (C12:0) chain fatty acids as the substrates confirmed that the corresponding activities are also diminished. Impaired gene expression and enzyme activities of fatty acid beta-oxidation were restored to the wild-type levels, and hepatic steatosis was substantially diminished in animals treated with 17beta-estradiol. Wild-type and ArKO mice showed no difference in the binding activities of the hepatic nuclear extracts to a peroxisome proliferator response element. These findings demonstrate the pivotal role of estrogen in supporting constitutive hepatic expression of genes involved in lipid beta-oxidation and in maintaining hepatic lipid homeostasis.

Human carbonic anhydrase XIV (CA14): cDNA cloning, mRNA expression, and mapping to chromosome 1.

A full-length cDNA clone of a human carbonic anhydrase XIV (HGMW-approved gene symbol CA14) was obtained and sequenced. The cDNA sequence was 1757 bp long and was predicted to encode a 337-amino-acid polypeptide with a molecular mass of 37.6 kDa. The deduced amino acid sequence of CA XIV showed an overall similarity of 29-46% to other active CA isozymes. The highest percentage similarity was with a transmembrane CA isoform, CA XII. As observed for CA XII, CA XIV has hydrophobic segments at both termini of the deduced protein for a putative signal sequence and a transmembrane domain. CA XIV showed low activity and was sensitive to acetazolamide, but not to sulfonamide. Northern blot analysis demonstrated an approximately 1.7-kb transcript in the adult human heart, brain, liver, and skeletal muscle. RNA dot-blot analysis for CA XIV mRNA expression showed a strong signal in all parts of the human brain and a weaker signal in the colon, small intestine, urinary bladder, and kidney. RT-PCR analysis showed an intense signal in the liver and spinal cord and a faint signal in the kidney. No CA XIV mRNA was seen in the salivary gland and pancreas. In contrast, CA XII mRNA was expressed in the kidney, salivary gland, and pancreas, but not in the liver or spinal cord. The CA XIV gene was localized to human chromosome 1q21. These findings indicate genetically distinct but closely related isoforms of human transmembrane CAs, CA XII and CA XIV, which have different patterns of tissue-specific expression.

Cholecystokinin A and B receptor mRNA expression in human pancreas.

Little information is available on the expression of cholecystokinin (CCK) receptors in the human pancreas, especially in the developing pancreas. We evaluated expression patterns for the CCK receptors in human pancreas at three different ages: fetus, infant, and adult. Expressions of CCK-A and CCK-B receptor messenger RNA (mRNA) were studied in human midtrimester fetus (14-15 weeks' gestation), infant (50 days old), and adult pancreas by reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot analysis. Expression levels of mRNA for both receptors also were evaluated by Northern blot analysis of adult pancreas. Northern blot analysis showed a strong signal for CCK-B receptor mRNA in adult pancreas, but no detectable signal for CCK-A receptor mRNA. However, RT-PCR/Southern blotting showed the presence of CCK-A receptor mRNA in adult pancreas. This was confirmed by sequencing of the complementary DNA (cDNA). RT-PCR/Southern blot analysis also showed CCK-A and CCK-B receptor mRNA expression in fetal and infant pancreas. These results show that the both CCK receptor types are expressed in human pancreas at stages of early gestation, but there is predominant expression of CCK-B receptor in adult pancreas.

Human mitochondrial carbonic anhydrase VB. cDNA cloning, mRNA expression, subcellular localization, and mapping to chromosome x.

A cDNA clone for a novel carbonic anhydrase (CA) isozyme was isolated from human pancreas and salivary glands. The cDNA sequence of 1182 base pairs encoded a 317-amino acid protein with a predicted mass of 36.4 kDa. The highest similarity of this cDNA and the deduced amino acid sequence is to human CA V (mitochondrial CA), hereafter referred to as CA VA. Recombinant protein expressed in COS-7 cells transfected with this cDNA clone was enriched in a mitochondrial fraction. Confocal fluorescence microscopy showed cytoplasmic granular signals in COS-7 cells expressing a fusion protein of the novel CA and green fluorescent protein. Several lines of evidence suggest that the cDNA clone presented herein encodes a novel human mitochondrial CA isozyme, designated CA VB. CA VB has a hydrophobic N-terminal mitochondrial signal sequence (33 amino acid residues). Western blot analysis showed a 36-kDa protein precursor and a 32-kDa mature protein for CA VB. Similar to CA VA, CA VB is a "low activity" enzyme with a sensitivity to acetazolamide. The CA VB gene is located on Xp22.1. Northern blot analysis in normal human tissues demonstrated expression of a 1.3-kilobase transcript in heart and skeletal muscle, and reverse transcription-polymerase chain reaction analysis showed expression of CA VB in pancreas, kidney, salivary glands, and spinal cord but not in liver. CA VA mRNA expression was observed only in liver. These findings indicate these are two genetically distinct isoforms of human CA V, designated CA VA and CA VB, which have different patterns of tissue-specific distribution, suggest different physiological roles for the two mitochondrial isozymes.

cDNA sequence, mRNA expression, and chromosomal localization of human carbonic anhydrase-related protein, CA-RP XI.

A full-length cDNA clone of a human carbonic anhydrase-related protein, CA-RP XI encoded by CA11, was obtained and sequenced. The cDNA sequence was 1475 bp long and predicted to encode a 328-amino acid polypeptide with a molecular mass of 36200 Da. The deduced amino acid sequence of CA-RP XI showed an overall similarity of 42-53% to the active site residues of other active CA isozymes; however, it lacked three zinc-binding histidine residues, raising questions regarding its CA catalytic activity. Northern blot analysis demonstrated strong expression of an approx. 1.5 kb transcript in the human brain, particularly in the cerebellum, cerebral cortex, and putamen. A single copy of the CA11 gene was localized to the human chromosome 19q13.2-3. These results suggest that CA-RP XI plays a general role in the human central nervous system.

Identification of carbonic anhydrase IV and VI mRNA expression in human pancreas and salivary glands.

Cytoplasmic carbonic anhydrase (CA) II is involved in acid-base balance in a wide variety of tissues. Extracellular CAs, membrane-bound CA IV and excretory CA VI, play a cooperative role with CA II in regulating the luminal pH in kidney and salivary glands, respectively. To extend the evidence into pancreas, we studied messenger RNA (mRNA) expressions of CA IV and CA VI in human pancreas. mRNA expressions of CA II, IV, and VI were studied in human pancreas, kidney, liver, and salivary glands by three different detection methods: reverse transcription-polymerase chain reaction (RT-PCR), RT-PCR-Southern blot, and Northern blot analyses. CA IV mRNA expression was consistently detected in all four tissues except for liver; only RT-PCR-Southern blot successfully identified its expression in liver. In contrast, RT-PCR and RT-PCR-Southern blot identified CA VI mRNA in salivary glands and pancreas, but Northern blot failed to detect its expression in pancreas. There was no detectable signal of CA VI expression in kidney and liver by all detection methods. CA II mRNA expression was consistently detected in all tissues studied. These results indicate that pancreas and salivary glands contain both of CA IV and VI and suggest that the extracellular CA isozymes may form a mutually complementary system with CA II to regulate the luminal pH of the pancreatic duct system.

Mutations in exons 2 and 3 of the cationic trypsinogen gene in Japanese families with hereditary pancreatitis.

BACKGROUND/AIMS: Single-point mutations in the cationic trypsinogen gene have been reported in hereditary pancreatitis kindreds in the white population. The aim of the present study was to investigate whether similar gene mutations are present in Japanese hereditary pancreatitis kindreds. METHODS: All five exons of the cationic trypsinogen gene were amplified by polymerase chain reaction and sequenced in six Japanese families with hereditary pancreatitis. RESULTS: Two types of single-point mutation in the cationic trypsinogen gene, which were identical with those reported in white families with hereditary pancreatitis, were observed in separate Japanese families with hereditary pancreatitis: 21Asn (AAC) to Ile (ATC) (N21I) in exon 2 and 117Arg (CGC) to His (CAC) (R117H) in exon 3. Pancreatitis occurred at more advanced ages in patients with the N21I mutation than in those with the R117H mutation. Besides normal polymorphisms in exons 4 and 5, no mutation was found in patients in the remaining four families with hereditary pancreatitis, 21 patients with sporadic chronic pancreatitis, or five normal subjects. CONCLUSIONS: These results show heterogeneity, but no racial specificity, in the cationic trypsinogen gene mutations in hereditary pancreatitis kindreds. A distinctive clinical feature for each of the mutation types is suggested: adult onset for the N21I mutation and childhood onset for the R117H mutation.

Evidence for a promoter-like activity in the short non-coding region of human papillomaviruses.

A short non-coding region (SNR) commonly exists between the E5 and L2 open reading frames of human papillomaviruses (HPVs). Except for the poly(A) signal for early gene transcripts, no biological functions have been discovered for the SNR. To test a possible promoter-like activity of the SNR, we carried out CAT reporter assays using constructs containing the SNRs from HPV-16, -18 and -33 linked to a promoterless CAT gene. We reproducibly observed enhanced expression of CAT gene by the SNRs. Co-expression of a transcriptional activator (LAP/NF-IL6) or deletion of the poly(A) signal augmented the promoter-like activity of the SNRs. RNase protection assays revealed a LAP-inducible CAT mRNA properly initiated from the HPV-16 SNR. These results may suggest that the SNR has a promoter activity that is regulated by keratinocyte differentiation.