p120(ctn) acts as an inhibitory regulator of cadherin function in colon carcinoma cells.

p120(ctn) binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin-dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120(ctn) to a faster position. To investigate whether p120(ctn) plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120(ctn) deletion constructs. Notably, expression of NH2-terminally deleted p120(ctn) induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120(ctn)-binding site was introduced into Colo 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120(ctn). These findings suggest that in Colo 205 cells, a signaling mechanism exists to modify a biochemical state of p120(ctn) and the modified p120(ctn) blocks the cadherin system. The NH2 terminus-deleted p120(ctn) appears to compete with the endogenous p120(ctn) to abolish the adhesion-blocking action.

Regulation of neuregulin expression in the injured rat brain and cultured astrocytes.

In this report, we investigated whether reactive astrocytes produce neuregulins (glial growth factor 2/heregulin/acetylcholine receptor-inducing activity or neu differentiation factor) and its putative receptors, ErbB2 and ErbB3 tyrosine kinases, in the injured CNS in vivo. Significant immunoreactivities with anti-neuregulin, anti-ErbB2, and anti-ErbB3 antibodies were detected on astrocytes at the injured site 4 d after injury to the adult rat cerebral cortex. To elucidate the mechanisms for the upregulation of neuregulin expression in astrocytes, primary cultured astrocytes were treated with certain reagents, including forskolin, that are known to elevate the intracellular level of cAMP and induce marked morphological changes in astrocytes. Western blot analysis showed that the expression of a 52 kDa membrane-spanning form of a neuregulin protein was enhanced in cultured astrocytes after administration of forskolin. The upregulation of glial fibrillary acidic protein was also observed in astrocytes treated with forskolin. In contrast, inactivation of protein kinase C because of chronic treatment with phorbol ester 12-O-tetradecanoyl phorbol 13-acetate downregulated the expression of the 52 kDa isoform, although other splice variants with apparent molecular sizes of 65 and 60 kDa were upregulated. These results suggest that the enhancement of neuregulin expression at injured sites is induced, at least in part, by elevation in intracellular cAMP levels and/or a protein kinase C signaling pathway. The neuregulin expressed on reactive astrocytes may stimulate their proliferation and support the survival of neurons surrounding cortical brain wounds in vivo.

The D13C variant of Bacillus schlegelii 7Fe ferredoxin is an 8Fe ferredoxin as revealed by 1H-NMR spectroscopy.

The N-terminal cluster binding motif Cys8XXXXXXXCys16....Cys49 of Bacillus schlegelii 7Fe ferredoxin, which provides the ligands to the [Fe3S4]+ cluster, was modified by the mutation Asp13 --> Cys. The mutant D13C is expressed in Escherichia coli as an 8Fe ferredoxin, with NMR properties similar to those of clostridial-type ferredoxins. The full assignment of the hyperfine shifted resonances indicates that Cys13 serves as ligand to the new fourth iron atom in the N-terminal cluster despite the atypical binding sequence CysXXXXCysXXCys....Cys. The C alpha-C beta-S-Fe dihedral angles of all cysteine ligands to the two [Fe4S4]2+ clusters of the D13C variant are similar to those observed in other 8Fe and 4Fe ferredoxins.

Transient expression of juvenile-type neurocan by reactive astrocytes in adult rat brains injured by kainate-induced seizures as well as surgical incision.

Neurocan is one of the major chondroitin sulfate proteoglycans expressed in nervous tissues. The expression of neurocan is developmentally regulated, and full-length neurocan is detected in juvenile brains but not in adult brains. In the present study, we demonstrated by western blot analysis that full-length neurocan transiently appeared in adult rat hippocampus when it was lesioned by kainate-induced seizures. Immunohistochemical studies showed that neurocan was detected mainly around the CA1 region although the seizure resulted in neuronal cell degeneration in both the CA1 and CA3 regions of the hippocampus. Double-labeling for neurocan mRNA and glial fibrillary acidic protein demonstrated that many reactive astrocytes expressed neurocan mRNA. The re-expression of full-length neurocan was also observed in the surgically injured adult rat brain. In contrast, the expression of other nervous tissue chondroitin sulfate proteoglycans, such as phosphacan and neuroglycan C, was not intensified but rather was either reduced in the kainate-lesioned hippocampus or in the surgically injured cerebral cortex. These observations indicate that induction of neurocan expression by reactive astrocytes is a common phenomenon in the repair process of adult brain injury, and therefore, it can be postulated that juvenile-type neurocan plays some roles in brain repair.

High-molecular-weight cadmium-binding proteins in rat liver cytosol: isolation and partial characterization of an approximately 50-kDa protein.

The time-dependent changes in the chromatographic pattern of subcutaneously injected cadmium associated with non-metallothionein cadmium-binding proteins were studied in the rat liver cytosol. Prior to the induction of cadmium-thionein (less than 3 h), cadmium appeared in three major peaks (P-1 with the void volume, P-2 and P-3) on Sephacryl S-300 column chromatography. Accompanied with the emergence of apo-metallothionein (about 3 h after administration), the amount of P-3 decreased and instead a cadmium-thionein peak (P-4) increased. Ion-exchange chromatography of P-3 with a combination of CM and DEAE Bio-Gel columns showed the existence of three major cadmium-binding proteins with molecular sizes of 46 kDa (in the CM Bio-Gel column eluate), 50 kDa (in the DEAE Bio-Gel column eluate), and 41 kDa (in the non-adsorbed fraction). The cadmium-binding protein in the CM Bio-Gel column eluate was purified to apparent homogeneity. The purified protein (CM-CdP) was 47 or 53 kDa in molecular size as determined by SDS-polyacrylamide gel electrophoresis or gel filtration chromatography, respectively. The apparent dissociation constant and maximum binding for cadmium were about 1 microM and 1 mol of the metal/mol of protein, respectively. The isoelectric point was estimated to be 8.8. The amino acid composition showed that the protein was relatively rich in glutamyl (including its amide) and alanyl residues. The N-terminal amino acid sequence was determined as Ala-Pro-Ile-Ala-Gly-Lys-Lys-Ala-Lys-Ala-Gly-Ile-Leu-Leu-Gly-. In-vitro experiments revealed that cadmium bound to CM-CdP could be easily transferred to apo-metallothionein, confirming that the affinity for the metal of the former protein was lower than that of the latter.

Purification and characterization of a 7Fe ferredoxin from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii.

A ferredoxin (Fd) was purified from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii. This ferredoxin was a monomer with apparent molecular weight of 13,000 and contained 7 mol Fe/mol ferredoxin. The oxidized ferredoxin showed the characteristic EPR spectrum for [3Fe-4S]1+ (1.2 spin/mol Fd). This signal disappeared upon reduction with dithionite and new signals due to [3Fe-4S]0 and [4Fe-4S]1+ (0.7 spin/mol Fd) appeared. The quantitation of EPR signals and the iron content reveal that B. schlegelii ferredoxin contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster. The ferredoxin has the characteristic distribution of cysteines (-Cys8-X7-Cys16-X3-Cys20-Pro-) for 7Fe ferredoxins in the N-terminus.

Cloning and chromosomal mapping of the human gene of neuroglycan C (NGC), a neural transmembrane chondroitin sulfate proteoglycan with an EGF module.

Neuroglycan C (NGC) is a 150 kDa transmembrane chondroitin sulfate proteoglycan with a 120 kDa core glycoprotein that was originally isolated from the developing rat brain. A rabbit antiserum, raised against a recombinant polypeptide representing a protein of the rat NGC core protein, recognized an NGC homolog in homogenates of brains of various vertebrates including humans. Because of the possible involvement of this proteoglycan in the etiology of a human neuronal disease, we cloned a complete coding sequence from a human brain cDNA library using a rat NGC cDNA as a probe. The predicted protein contains 539 amino acids and shows 86% homology with the rat counterpart. The domain structure characteristic of rat NGC was completely conserved in human NGC, which consisted of an N-terminal signal sequence, a chondroitin sulfate-attachment domain, an acidic amino acid cluster, an EGF-like domain, a transmembrane domain and a cytoplasmic tail. Northern blot analysis revealed that a single transcript of 2.4 kb was detectable in the brain, but not in other human tissues. By fluorescence in situ hybridization (FISH) analysis, the human NGC gene was assigned to the chromosomal 3p21.3 band, where the Sotos syndrome has been mapped. Involvement of the NGC gene in the etiology of the Sotos syndrome remains to be examined.

Occurrence of a N-terminal proteolytic fragment of neurocan, not a C-terminal half, in a perineuronal net in the adult rat cerebrum.

Neurocan is a nervous tissue-unique chondroitin sulfate proteoglycan (CSPG) whose expression and proteolytic cleavage are developmentally regulated. In the adult rat brain, neurocan is completely cleaved into some proteoglycan fragments including the C-terminal half known as neurocan-C and a N-terminal fragment with a 130 kDa core glycoprotein (neurocan-130). We describe here the differential distribution of these two neurocan-derived CSPGs in the adult rat cerebrum and the occurrence of neurocan-130 as a new member of a perineuronal net-constituting molecule. At the light microscopic level, neurocan-130 exhibited pericellular localization around a subset of neurons in addition to diffuse distribution in the neuropil. In contrast, neurocan-C was distributed only diffusely in the neuropil. Double staining with anti-neurocan-130 and anti-synaptophysin antibodies suggested that neurocan-130 was localized in the vicinity of the synapses, but not at the synapses. Immunoelectron microscopy showed that neurocan-130 was mainly localized in the cytoplasm of glial cell processes, the so-called glial perineuronal net, encompassing the cell bodies of certain neurons. The presence of neurocan-130 in a limited number of glial cells may reflect some functional heterogeneity of the glia.

Two cases of inflammatory bowel disease with multiple myeloma.

A significant increase has been reported in reticuloendothelial neoplasms in patients with inflammatory bowel diseases. We present two rare cases of multiple myeloma in patients with inflammatory bowel diseases. One was in a 58-year-old woman with ulcerative colitis, and the other was in a 59-year old woman with Crohn's disease. In both patients, multiple myeloma occurred during long-term observation of inflammatory bowel disease and during the inactive stage of intestinal inflammation. The multiple myeloma appeared to have resulted from monoclonal gammopathy of undertermined significance in both patients, and was diagnosed by characteristic serum and bone marrow findings. Our findings suggested that multiple myeloma should be particularly considered in women of middle or advanced age with ulcerative colitis or Crohn's colitis and serum monoclonal gammopathy.

Plasma fructosamine assay in children with insulin-dependent diabetes mellitus.

Plasma concentrations of fructosamine, an indicator of glycated plasma proteins, were measured in non-diabetic children and children with insulin-dependent diabetes mellitus (IDDM) to see if they also correlate with glycemic control in children as well as in adults. Non-diabetic children aged less than 4 yr had significantly lower plasma fructosamine than non-diabetic children aged 4 or more. Total plasma protein in these children was slightly lower or close to that of older children. There was no difference in fructosamine between non-diabetic children aged 4 or more and healthy adult subjects. Plasma fructosamine in children with IDDM was twofold that of age-matched controls. In children with IDDM, correlations between fructosamine and HbAI (r = 0.799) or HbAIc (r = 0.841) were high. The measurement of plasma fructosamine, which is practical in children because of the small sample volume needed and no influence of HbF, is useful in the management of children with IDDM.

Relationship between hypoglycemic symptoms and blood glucose levels due to self-monitoring in summer camp for diabetic children in Japan.

In a summer camp for 47 diabetic children in Kinki district, Japan, in 1984, the relationship between hypoglycemic symptoms and blood glucose levels by self-monitoring was analyzed. During the 7-day camp, self-monitoring of blood glucose (SMBG) was carried out 599 times in total, 12.7 times per camper. SMBG due to hypoglycemic complaints amounted to 371. 154 measurements out of 371 indicated blood glucose levels under 80 mg/dl, but 78 monitorings were found to be over 200 mg/dl. Fatigue or weakness were the most frequent hypoglycemic symptoms, as was hunger sensation, each reaching approximately 40% in frequency. In most complaints of tremor, the blood glucose level was critically low. Prompt measurement of blood glucose is indeed necessary to properly treat diabetic children with 'hypoglycemic' symptoms.

Heterogeneity of human islet cell antibodies in terms of cross-species reactivity.

There has been no consistent view about the cross-species reactivity of islet cell antibodies (ICA), and no report about their target antigens in the pancreatic islet cells of different species. Therefore, we examined the cross-reactivity of human ICA against rodent pancreatic islet cells, and found at least two types of ICA, one having a comparatively strong cross-reactivity and the other lacking it. Furthermore, using human as well as some rodent pancreatic tissues that had been modified chemically, we came to suspect that the target antigens of ICA were sialic acid residues of glycolipids. Therefore, we suggest that there are at least two types of ICA recognizing sialic acid residues of glycolipids, one reacting with antigen(s) commonly present in human and rat islets, and the other with antigen(s) only present in human islets.

Marriage rate and number of children among young adults with insulin-dependent diabetes mellitus in Japan.

The main purpose of our study was to identify the social circumstances and lifestyle of IDDM patients in Japan. The present study focused on the marriage status of both men and women with IDDM as well as the number of children of women with IDDM. A questionnaire was sent to hospitals across the country. Doctors handed it or mailed it to IDDM patients aged 18 years or older. Unsigned answer sheets were returned directly by the patients. Data on the marriage rate and number of children were obtained, and possible factors affecting these indices were assessed. One thousand and thirteen patients (354 men and 659 women) answered the questionnaire. Both men and women with IDDM were less likely to be married in comparison with age-matched Japanese. The number of children of married IDDM women in various age groups was also lower in comparison with the general Japanese female population. Several factors other than diabetes complications including job discrimination, high medical costs, and psychological pressures, were thought to be responsible for these results.

Control of CooA activity by the mutation at the C-terminal end of the heme-binding domain.

A constitutively active mutant of a CooA, in which Met131 was replaced by Leu, was isolated by random mutagenesis. Site-directed mutagenesis at position 131 revealed that M131R-CooA was also constitutively active even in the absence of CO and that M131P-, M131D-, and M131E-CooA were constitutively inactive regardless of the presence or absence of CO. While M131L- and M131E-CooA showed almost the same electronic absorption spectra as those of the wild type in the ferric, ferrous, and CO-bound forms, M131D-CooA showed the typical spectrum of a five-coordinate heme protein in the ferric form. The conformational change around the heme induced by CO binding, which triggers the activation of CooA, is thought to be linked to the rearrangement of the conformation around the hinge region between the heme-binding and DNA-binding domains and/or of the relative orientation of the two domains to activate CooA.

CO sensing and regulation of gene expression by the transcriptional activator CooA.

The transcriptional activator CooA from Rhodospirillum rubrum contains a six-coordinate protoheme that acts as a CO sensor in vivo. CO is a physiological effector of CooA and replaces one of the axial ligands of the ferrous heme to form the CO-bound CooA that is active as the transcriptional activator. Cys75 or His77 is coordinated to the ferric and ferrous hemes in CooA, respectively. The redox-controlled ligand exchange between Cys75 and His77 proceeds during the change in the redox state of the heme. The reduction and oxidation midpoint potentials of CooA have been determined to be -320 and -260 mV, respectively. The properties of a functional chimera derived from CRP and CooA suggest that CooA activates the transcription by a similar mechanism to that for CRP at Class II CRP-dependent promoters. Alanine-scanning mutagenesis has revealed that Arg24 and Arg53 of CooA, which will be concerned with the protein-protein interaction with RNA polymerase, are critical amino acid residues for the transcriptional activator activity of CooA, and that Lys26 and Asp94 modulate the activity of CooA.

Effect of (-)N-[alpha-phenyl-beta-(p-tolyl)ethyl] linoleamide on lipid levels in serum and liver in cholesterol-fed rats.

The effect of (-)N-[alpha-phenyl-beta-(p-tolyl)ethyl] linoleamide (PTLA) on lipid levels in serum and liver was compared with that of sitosterols in rats maintained on a diet supplemented with 1% of cholesterol, 0.5% of ox bile extracts, and 10% of hydrogenated coconut oil for 8 weeks. When PTLA was added to the diet at a level of 0.1%, the mean liver cholesterol level of male rats was reduced to 41% of the control and that of female rats was reduced to 19% of the control. In female rats, which showed higher cholesterol levels in serum and liver than male rats after cholesterol feeding, PTLA lowered the liver cholesterol level even at 0.0008% in the diet. Serum cholesterol was lowered by PTLA but not so markedly as liver cholesterol. The inhibition of cholesterol deposition in the liver suggests that the interference with cholesterol absorption is one of the main actions of PTLA. Sitosterols showed a similar pattern of lipid-lowering action, but the potency was far less than that of PTLA.

Persistent organochlorines in minke whale (Balaenoptera acutorostrata) and their prey species from the Antarctic and the North Pacific.

Persistent organochlorines such as polychlorinated biphenyls (PCBs), DDTs, chlordane compounds (CHLs), hexachlorocyclohexanes (HCHs) and hexachlorobenzene (HCB) were determined in the blubber of minke whale and its diet collected from the Antarctic and the North Pacific Oceans. Residue levels of these compounds (except HCB) in minke whale from the Antarctic were apparently lower than those from the North Pacific. This is due to the lower levels of these pollutants in the southern hemisphere than in the northern hemisphere and the specific feeding habit of the minke whale from the Antarctic which feeds on lower trophic organisms, primarily euphausiids. The north-south difference for HCB residue levels was small, reflecting its dispersible nature through long-range atmospheric transport. Compositions of DDT and CHL compounds in minke whale from the Antarctic were similar to those from the North Pacific. However, the composition of HCH isomers was different between the Antarctic and the North Pacific as was observed in their diet, suggesting a larger or on-going usage of lindane in the southern hemisphere countries. In minke whale from the Antarctic, the elevated level of PCBs residues was noted during a period of 1984 to 1993, implying a continuous discharge of PCBs in the southern hemisphere. A similar discharge was also suggested in the North Pacific, while a decreasing contamination by DDTs was apparent.

Warfarin-induced eosinophilic pleurisy.

A 51-year-old man was admitted to our hospital because of dry cough and low grade fever with right-sided pleural fluid and blood eosinophilia. Warfarin had been prescribed following coronary artery bypass grafting. After the discontinuation of warfarin the clinical and chest X-ray findings improved; readministration of the drug caused recurrent blood eosinophilia and pleural effusion in the other lung. Since no other specific etiologies for eosinophilia and pleural effusion were determined by extensive evaluation, warfarin seemed to be associated with his illness. This is the first report of warfarin-induced eosinophilic pleurisy.

[Management of short bowel syndrome in infants--experience of adjunctive surgical therapy in three cases].

During the past nine years period, we managed 7 cases with short bowel syndrome resulting from massive intestinal resection in the neonatal period. Six children, aged 2 to 8 years are alive, and one died from cardiomyopathy caused by selenium deficiency at the age of one year. The carbohydrate-free milk was used for initial enteral nutrition, and the result was encouraging in 4 cases. In 3 cases in which residual small bowel varied between 6.5 to 18 cm, the enteral feeding was not incapable. These cases underwent adjunctive surgical therapy. Tailoring jejunoplasty was performed in the first case, and bowel lengthening in the second case. In both cases enteral nutrition was advanced postoperatively. The third patient who had rapid intestinal transit time underwent isoperistaltic colon interposition. Intestinal transit time was increased and enteral nutrition was advanced gradually.