Prevalence and risk factor analysis of microalbuminuria in Japanese general population: the Takahata study.

Microalbuminuria, an indicator of glomerular injury, is associated with increased risk of progressive renal deterioration, cardiovascular disease, and mortality. However, the prevalence of microalbuminuria in Japanese general population is less certain. Thus, we examined the prevalence of microalbuminuria and its associated risk factors in Japan. Subjects of this cross-sectional study were asymptomatic individuals over 40 years in Takahata, Japan. Urine albumin-creatinine ratio was calculated from a single-spot urine specimen collected in the morning. Creatinine clearance (CCr) was obtained by Cockcroft-Gault equation. Multivariate logistic regression analysis was used to determine which risk factors (i.e., age, hypertension, diabetes, obesity, and salt intake) might predict the presence of microalbuminuria. A total of 2321 subjects (mean age, 64 years; men, 1034; women, 1287) were entered into the final analysis. Among them, the prevalence of microalbuminuria, macroalbuminuria, and proteinuria by dipstick test (> or = 1+) were 317 (13.7%), 39 (1.7%), and 103 (4.4%), respectively. Age, hypertension, and diabetes were independently associated with microalbuminuria in men. In addition to the classical risk factors detected in men, estimated 24-h urinary sodium excretion and uric acid were also independently associated with microalbuminuria in women. Among the 668 subjects with renal insufficiency (CCr <60 ml/min/1.73 m(2)), the prevalence of microalbuminuria and macroalbuminuria were 119 (17.8%) and 18 (2.7%), respectively. In conclusion, microalbuminuria is prevalent across all age groups and is associated with lifestyle-related risk factors in Japanese general population. However, there are a substantial number of subjects with renal insufficiency accompanying no microalbuminuria.

Knockout of mouse beta 1,4-galactosyltransferase-1 gene results in a dramatic shift of outer chain moieties of N-glycans from type 2 to type 1 chains in hepatic membrane and plasma glycoproteins.

To understand the contribution of beta 1,4-galactosyltransferase (beta 4Gal-T)-1 to galactosylation in vivo, N-glycans of hepatic membrane glycoproteins and plasma glycoproteins from beta 4Gal-T1 wild-type (beta 4Gal-T1(+/+)) and beta 4Gal-T1 knockout mice were compared. Unexpectedly, glycoproteins from the knockout mice were found to express considerable amounts of sialylated, galactosylated N-glycans. A striking contrast was that galactose residues were largely beta 1,4-linked to GlcNAc residues in the beta 4Gal-T1(+/+) mouse glycans but beta 1,3-linked in the knockout mouse glycans, thus resulting in the shift of the backbone structure from type 2 chain (Gal beta 1-->4GlcNAc) to type 1 chain (Gal beta 1-->3GlcNAc). Detailed analysis of plasma glycoproteins revealed that the expression of sialyl linkage in N-glycans was shifted from the Sia alpha 2-->6Gal to the Sia alpha 2-->3Gal, and oversialylated type 1 chains were, remarkably, found in the knockout mouse glycans. Thus beta 4Gal-T1 deficiency was primarily compensated for by beta1,3-galactosyltransferases, which resulted in different sialyl linkages being formed on the outer chains and altered backbone structures, depending on the acceptor specificities of sialyltransferases. These results suggest that beta 4Gal-T1 in mouse liver plays a central role in the synthesis of type 2 chain and is also involved in the regulation of sialylation of N-glycans. The knockout mice may prove useful in investigation of the mechanism which regulates the tissue-dependent terminal glycosylation.

Autoimmune hepatitis with membranous glomerulonephritis.

We report on a case of autoimmune hepatitis (AIH) associated with membranous glomerulonephritis. A 61-year-old woman was admitted because of peripheral edema, proteinuria and abnormal liver function test findings. A diagnosis of AIH was made on the basis of an elevation of aminotransferase and serum IgG levels, the presence of positive antinuclear antibody and the characteristic histological features of chronic active hepatitis. Histological examination of a renal biopsy specimen disclosed membranous glomerulonephritis with granular deposits of IgG, IgM, C3 and C1q along the capillary walls. This condition is rare in AIH and should be carefully distinguished from systemic lupus erythematosus.

O-Glycosylation of G-protein-coupled receptor, octopus rhodopsin. Direct analysis by FAB mass spectrometry.

In addition to the N-glycan that is evidently conserved in G-protein-coupled receptors (GPCRs), O-glycans in the N-terminus of GPCRs have been suggested. Using a combination of enzymatic and manual Edman degradation in conjunction with G-protein coupled receptor mass spectrometry, the structure and sites of O-glycans in octopus rhodopsin are determined. Two N-acetylgalactosamine residues are O-linked to Thr4 and Thr5 in the N-terminus of octopus rhodopsin. Further, we found chicken iodopsin, but not bovine rhodopsin, contains N-acetylgalactosamine. This is the first direct evidence to determine the structure and sites of O-glycans in GPCRs.

Detection of carbohydrate recognition molecules on the plasma membrane of boar sperm by dextran-based multivalent oligosaccharide probes.

Two kinds of molecules, one recognizing the sialo-/asialo-N-acetyllactosamine structures and the other recognizing the Lewis X structure in a divalent cation-independent manner, were detected on the head of boar sperm prepared from cauda epididymis by fluorescence-labeled or biotinylated dextran-based multivalent oligosaccharide probes. The N-acetyllactosamine recognition molecule(s) is weakly detected on uncapacitated sperm and becomes strongly detectable on capacitated sperm. On the other hand, the Lewis X recognition molecule is detected at a moderate level before capacitation and at a high level after capacitation. Both molecules disappear from the sperm head after induction of acrosome reaction and also by mild detergent treatment. Thus, the two kinds of carbohydrate molecules are expressed on the plasma membrane of boar sperm depending on their physiological state. Inhibition study of the oligosaccharide-dextran probe binding to isolated sperm plasma membrane by various glycoproteins, oligosaccharides, and sulfated polysaccharides also supported the occurrence of the two distinct kinds of molecules.

Three-dimensional observations of the human hepatic artery (Arterial system in the liver).

BACKGROUND/AIMS: We examined the three-dimensional structures of the hepatic artery. MATERIALS/METHODS: A 39-year-old man who died of brain hemorrhage was autopsied. The liver was perfused with physiological saline and 20% formalin from the hepatic artery and portal vein. More than 700 serial sections were obtained from a paraffin-embedded block, and vascular reconstruction was performed under a light microscope. RESULTS: The hepatic artery divides into the axial artery and the peribiliary branch given off from it. These two systems also connect to each other by a few anastomoses. The former systematically supplies arterial blood to all the parenchymal liver cells. The latter forms two layers of plexes around the bile duct. The inner capillary layer is afferent and the outer vascular layer is efferent to the bile duct. CONCLUSION: To maintain constant sinusoidal blood flow, the terminal portions of the axial arteries may contract and thereby divert blood to peribiliary branches through bifurcations and anastomoses. The blood flow of the peribiliary capillary plexus may affect bile flow. The hepatic artery may act as a functional mediator between portal flow and bile excretion.

A new beta-1,2-N-acetylglucosaminyltransferase that may play a role in the biosynthesis of mammalian O-mannosyl glycans.

Recent studies have shown that O-mannosyl glycans are present in several mammalian glycoproteins. Although knowledge on the functional roles of these glycans is accumulating, their biosynthetic pathways are poorly understood. Here we report the identification and initial characterization of a novel enzyme capable of forming GlcNAc beta 1-2Man linkage, namely UDP-N-acetylglucosamine: O-linked mannose beta-1,2-N-acetylglucosaminyltransferase in the microsome fraction of newborn rat brains. The enzyme transfers GlcNAc to beta-linked mannose residues, and the formed linkage was confirmed to be beta 1-2 on the basis of diplococcal beta-N-acetylhexosaminidase susceptibility and by high-pH anion-exchange chromatography. Its activity is linearly dependent on time, protein concentration, and substrate concentration and is enhanced in the presence of manganese ion. Its activity is not due to UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT-I) or UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,2-D-acetylglucosaminyltransferase II (GnT-II), which acts on the early steps of N-glycan biosynthesis, because GnT-I or GnT-II expressed in yeast cells did not show any GlcNAc transfer activity against a synthetic mannosyl peptide. Taken together, the results suggest that the GlcNAc transferase activity described here is relevant to the O-mannosyl glycan pathway in mammals.

Molecular and immunological approaches to mammalian fertilization.

By means of hybridoma technology, we obtained six hydriboma cell lines producing monoclonal antibody (mAb) to porcine zona pellucid (ZP), two of which recognizes the steric structure of common antigens between porcine ZP and humans. Furthermore, we have analyzed all or partial structures of N- and O-linked sugar chains of ZP glycprotein from porcine or murine oocytes. Then, we have clarified that beta-galactose and Le(X) residues on ZP played the binding roles to sperm cells in porcine and murine fertilization. We have also succeeded Sp38 cDNA cloning from cDNA library of porcine testis. We found that Sp38 protein bind to porcine ZP2 and expressed in murine and human sperm cells. Corresponding to the presence of major histocompatibility complex (MHC) class II on murine sperm, CD4 on the murine egg plasma membrane was clearly shown by indirect IIF and immunoprecipitation test. Furthermore, the transcriptional expression of CD4/p56(lck) in eggs was confirmed by RT-PCR method. In addition, the p56(lck) associated with CD4 underneath the plasma membrane of eggs was autophosphorylated after cross-linking of CD4 with anti CD4 mAb. The binding between eggs or Sf9-CD4 cells labeled with anti-CD4 mAb and sperm cells labeled with anti-monomorphic region of class II mAb was completely blocked. Considering these findings together with the fact that an interspecies' heterogeneity is present in CD4 amino acid sequence at the interactive site with class II, we elucidated that one of species' specific intercellular adhesions between two gametes at the fusion step in fertilization is definitely mediated by class II located on the posterior region of sperm head and CD4/p56(lck) complex on the plasma membrane of egg.

Postoperative hyponatremia in a patient with ACTH-producing Merkel cell carcinoma.

Merkel cell carcinoma is characterized by specific neuroendocrine features and the expression of several neuropeptides. We report a case of Merkel cell carcinoma with post-surgical hyponatremia in an 85-year-old Japanese woman. A tumor on the left cheek histopathologically showed the characteristics of Merkel cell carcinoma together with Bowen's disease. Although an increased level of ACTH was found both in the tumor and in the peripheral blood, the postoperative hyponatremia in our patient seems more likely to have been caused by the stress of the operation and indapamide, considering that the ACTH level in the tumor was much lower than those in other ectopic ACTH-producing tumors in previous reports.

Autoimmune forms of chronic hepatitis associated with hepatitis C virus (HCV) infection with and without HCV-RNA: histological differences from pure autoimmune hepatitis and chronic hepatitis C.

Liver biopsy specimens of pure autoimmune hepatitis (pAIH), autoimmune forms of chronic hepatitis with positivity for anti-hepatitis C virus (anti-HCV) and negativity for HCV-RNA (cAIH-RNA(-)), autoimmune forms of chronic hepatitis with positivity for anti-HCV and HCV-RNA (cAIH-RNA(+)), and chronic hepatitis C (CHC) were compared histologically and statistically to clarify the histological character of the autoimmune form of chronic hepatitis with HCV infection. The following representative histological features were used to investigate: inflammation, fibrosis, plasma cell infiltration, lymphoid aggregates/follicles, non-suppurative destructive cholangitis, and the shape of the enlarged portal tracts. While a considerable overlap in histological features between the pAIH and cAIH-RNA(-) groups and between the CHC and cAIH-RNA(+) groups was recognized, the overlap between the pAIH and CHC groups was small. Significant differences were found between cAIH-RNA(-) and cAIH-RAN(+) groups, especially in necroinflammatory findings. In conclusion, most cases of cAIH-RNA(-) with histological features similar to those of pAIH were shown to be AIH. The remaining cases might be CHC with subsidence of viral duplication. Conversely, many cases of cAIH-RNA(+) with histological findings similar to those of CHC were shown to be CHC clinically mimicking pAIH. The remaining cases might represent coexistence of pAIH and HCV infection.

CD56 directly interacts in the process of NCAM-positive target cell-killing by NK cells.

The role of CD56 in the process of target cell killing by NK cells has been investigated. Addition of NK cells to HuH28 cells, a CD56-expressing cell line, led to inhibition of the growth of the target cells, which exhibited morphological features of apoptosis. These changes were prevented by the addition of a polyclonal anti-NCAM to the cultures. Since neither Fas antigen expression nor apoptotic changes were induced by addition to a mixed culture supernatant of NK and target cells, both the Fas-Fas ligand system and soluble factors do not seem to participate in apoptosis in these circumstances. Increased secretion of interferon-gamma and tumour necrosis factor-alpha by NK cells must therefore have been suppressed by the presence of the polyclonal antibody. These results lead us to conclude that CD56, through homophilic binding, plays an important role in the process of target cell killing by an apoptosis mechanism.

Calcium ion-independent recognition of sialyl and nonsialyl N-acetyllactosamine and Le(x) structures by boar sperm.

Recognition of defined carbohydrate structures by boar sperm was studied on the basis of oligosaccharide structures of porcine zona pellucida glycoproteins so far elucidated. Boar sperm abundantly adhered to fetuin-Sepharose beads, moderately to asialofetuin-Sepharose beads, but not at all to galactosidase (beta1-4-linkage-specific)-digested asialofetuin-Sepharose beads. The sperm also adhered to Le(x) oligosaccharide probe-coupled avidin-Sepharose beads. These adhesive activities were retained in the medium containing EDTA instead of calcium ion but abolished after induction of acrosome reaction by preincubation of sperm with calcium ionophore. Inhibition study of sperm adhesion to the beads by soluble ligands demonstrated that boar sperm express at least two kinds of carbohydrate recognition molecules, one recognizing both sialyl and nonsialyl N-acetyllactosamines but not the Le(x) structure and the other recognizing the Le(x) structure but not N-acetyllactosamines. Sperm binding to the zona pellucida on fixed porcine oocytes was inhibited by N-glycans of fetuin and their asialo form but not by the asialo, agalacto-N-glycans. Finally, dextran-based multivalent oligosaccharide polymers were prepared and their inhibitory activities in sperm-oocyte binding were examined. The result indicated that the polymer composed of fetuin N-glycans, its asialo-N-glycans, or lacto-N-fucopentaose III causes a remarkable inhibition at the oligosaccharide-based concentration of 50 microM. Thus, boar sperm are suggested to express multiple carbohydrate recognition molecules which may be involved in the sperm-egg interaction.

Microscale synthesis of dextran-based multivalent N-linked oligosaccharide probes.

We developed a convenient method for the synthesis of dextran-based multivalent probes containing N-linked oligosaccharides which is efficient even in a small scale. Oligosaccharides were derivatized with succinic dihydrazide and dimethylamine borane under a mild acidic condition. The derivatized oligosaccharides were then conjugated in a good yield to periodate-oxidized dextran (500 kDa). Thus, the conjugates containing 120 to 140 oligosaccharide chains per dextran molecule were successfully synthesized. Their practical advantage was shown by the example that the asialofetuin oligosaccharide-dextran conjugate has much higher affinity to Ricinus communis agglutinin (RCA-I) than asialofetuin oligosaccharide itself or asialofetuin. The conjugates were further labeled with fluorescent reagent or biotinylation reagent containing a hydrazino group by the use of the unreacted aldehyde groups of the oxidized dextran, yielding probes with similar densities of fluorophores or biotin groups. Direct binding of the biotinylated asialofetuin oligosaccharide-dextran probe to RCA-I coated on the titer plate at a concentration of 50 ng/50 microl was easily detected using 50 fmol (as oligosaccharides) of the probe. The method for the synthesis of dextran-based oligosaccharide probes will facilitate the investigation of carbohydrate-mediated molecular interactions based on the native oligosaccharide structures.

Three-dimensional image analysis of hepatic restructuring in liver cirrhosis.

OBJECTIVE: To investigate the three-dimensional structure, including the angioarchitecture, of the cirrhotic liver and clarify morphogenesis of the cirrhotic nodule. STUDY DESIGN: The three-dimensional liver structure of nontumor areas in two partially hepatectomized cases of hepatitis C virus-positive liver cirrhosis with hepatocellular carcinoma was examined by computerized reconstruction from serial tissue sections. RESULTS: Our image analysis revealed the following: (1) The parenchyma consisted of two kinds of cirrhotic nodules. The first was the nodule centrifugally formed around the portal veins, and their flows drained into the hepatic veins inside and around the nodule. The second was the nodule derived from the first. The latter was divided into the former by bridging fibrosis-induced intranodular septation. (2) The stroma consisted of the newly formed fibrovascular tissue--i.e., the septum and intranodular inflow and outflow vascular systems and the preexisting one. CONCLUSION: Our computerized reconstruction suggested, from an angioarchitectural point of view, that the first and second kind of cirrhotic nodule might be named the stable and the unstable nodule, respectively, and that the first kind of cirrhotic nodule could be derived from the regenerative nodule appearing in the course of chronic hepatitis.

Three-dimensional image analysis of the hepatic restructuring process in chronic active hepatitis.

OBJECTIVE: To elucidate the process of hepatic restructuring in the course of chronic hepatitis from a morphologic viewpoint. STUDY DESIGN: The three-dimensional (3-D) liver structure was investigated by computer-aided reconstruction in five cases (one autopsy and four surgical cases) of chronic active hepatitis (type C), including early to late stages of restructuring. RESULTS: Our 3-D reconstruction revealed the following. At an early stage, portal and periportal inflammation and fibrosis widened the portal tracts, giving rise to the formation of portal-to-portal and portal-to-hepatic venous connections, although most central veins were still located at an almost normal site in the hepatic lobules. In a middle stage, bridging fibrosis developed to create a network of interstitium where the central veins were rather decreased in number, with regenerative nodules multiplying in the parenchyma. At the late stage, the lobular structure was destroyed, and the parenchyma consisted uniformly of regenerative nodules, with remaining but rearranged lobules among them. CONCLUSION: The above changes of liver structure suggest that in cirrhogenesis from chronic hepatitis, a combination of nodular regeneration and formation of an interstitial network come to replace the normal lobular structure, hastening the development of liver cirrhosis.

Impaired galactosylation of core 2 O-glycans in erythrocytes of beta1,4-galactosyltransferase knockout mice.

O- and N-glycans included in erythrocyte membrane glycoproteins from beta1,4-galactosyltransferase I (GalT-I) knockout mice were analyzed to examine how this enzyme deficiency affects glycosylation of proteins in erythroid cells. The results indicated that greater than 80% of core 2 O-glycans from GalT-I-/- mice are not galactosylated by beta1,4 linkage, resulting in the expression of Neu5Acalpha2 --> 3Galbeta1 --> 3(GlcNAcbeta1 --> 6)GalNAc, while core 2 O-glycans from GalT-I+/+ mice are fully galactosylated and occur as Neu5Acalpha2 --> 3Galbeta1 --> 3(Neu5Acalpha2 --> 3Galbeta1 --> 4GlcNAcbeta1 --> 6)GalNAc. On the other hand, beta1, 4-galactosylation of N-glycans of the mutant was approximately 60% that of the wild type. Thus, it is suggested that GalT-I is predominantly responsible for beta1,4-galactosylation of the core 2 O-glycan branch in erythroid cells.

Squamous cell carcinoma in a renal transplant recipient with linear porokeratosis.

A 40-year-old man developed squamous cell carcinoma on a perianal lesion of linear porokeratosis after renal transplantation. The tumor metastasized to the left inguinal lymph node 25 months after the primary tumor was excised. p53 overexpression was observed in the tumor cells, but not in the porokeratotic lesion. Interestingly, continuous subcutaneous infusion of peplomycin for the lymph node metastasis significantly improved the warty lesions of porokeratosis. In this patient, immunosuppressive agents might have accelerated the development of carcinoma on a skin area with malignant potential.

Analysis of 2-aminobenzamide-labeled oligosaccharides by high-pH anion-exchange chromatography with fluorometric detection.

To establish high-pH anion-exchange chromatography with fluorometric detection (HPAEC-FD) for the sensitive oligosaccharide analysis, the system for HPAEC with pulsed amperometric detection (PAD) was modified by placing the anion micromembrane suppressor between PAD and fluoromonitor to neutralize the alkaline eluate and connecting the flow path. N-Linked oligosaccharides were released from various glycoproteins by hydrazinolysis followed by labeling with 2-aminobenzamide, and their asialooligosaccharides were analyzed by HPAEC-FD using an isocratic elution with 0.3 M NaOH solution. The results indicated that the new system using FD offers fine separation of oligosaccharides at the subpicomole level. Coinjection of reduced dextran oligomers as internal standards which are detected by PAD permits us to express elution positions of the fluorescent oligosaccharides as glucose units, resulting in a much more reliable analysis. The system was also effectively used for structural analysis of oligosaccharides by sequential glycosidase digestion. Thus, HPAEC-FD promises to be an alternative tool for the sensitive analysis of oligosaccharides.

Structural study of N-linked sugar chains of sheep erythrocyte membrane glycoproteins.

Our previous study showed that non-reducing terminal galactose residues of N-linked sugar chains present in sheep erythrocyte membrane glycoproteins are important for rosette formation with T lymphoblastic cells [Ogasawara et al. (1995) Immunol Lett 48: 35-38]. As a first step to elucidate the significant structures of sugar chains involved in rosette formation, we analysed N-linked sugar chains released from the membrane glycoproteins by hydrazinolysis. The oligosaccharides were labeled with NaB3H4 and fractionated using columns of Aleuria aurantia lectin-Sepharose, MonoQ and Bio-Gel P-4. Structural analyses of oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis revealed that the membrane glycoproteins contain bi- (19%), tri- (33%), and tetraantennary (44%) complex-type oligosaccharides and that the oligosaccharides having exposed galactose residues amount to 40% of the total.