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.     

Identification of the calmodulin-binding domain of neuron-specific protein kinase C substrate protein CAP-22/NAP-22. Direct involvement of protein myristoylation in calmodulin-target protein interaction

Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated. Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain. In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain. Phosphorylation of a single serine residue in the N-terminal domain (Ser5) by protein kinase C abolished the binding. Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.     

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.     

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L

Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants.     

1H-NMR and raman studies on perforating trauma-induced cataract formation in a mouse lens

In order to provide new insight into the molecular mechanism of perforating trauma-induced cataract formation in an 8-week-old ddY mouse lens, we performed an in situ investigation into changes in the water-protein and/or protein-protein interactions by using 500 MHz (1)H-NMR spectroscopy, and into structural alterations in lens proteins by using Raman spectroscopy. Cross-relaxation times of water protons in the perforated opaque lens were considerably shorter than those in the intact transparent lens, whereas there was no significant difference in water content, suggesting a drastic change in water-protein and protein-protein interactions in the perforated lens. In addition, there was no significant difference in the intensity ratios of several key Raman bands between intact and perforated lenses, indicating that no significant local and overall conformational changes in lens protein itself occur in the perforated lens. The present (1)H-NMR and Raman results lead us to the conclusion that changes leading to lens opacification in the perforating trauma-induced cataract appear to involve the rapid formation of immobile large lens protein aggregates without formation of intra- and intermolecular disulfide linkages, and rapid increase in a fraction of bound water associated with large protein aggregates.     

Flavonoid and benzophenone glycosides from Coleogyne ramosissima

A benzophenone glucoside and two flavonol glycosides were isolated together with 27 known polyphenols from the aerial parts of Coleogyne ramosissima, and their structures were elucidated by spectroscopic and chemical methods as iriflophenone 2-O-beta-glucopyranoside, isorhamnetin 3-O-2G-rhamnopyranosylrutinoside-7-O-alpha-rhamnopyranoside and limocitrin 3-O-rutinoside-7-O-beta-glucopyranoside, respectively.     

Immunocytochemistry of Rhamnogalacturonan II in Cell Walls of Higher Plants

A polyclonal antibody against a borate-RG-II complex is raisedin rabbits. The antibody recognized RG-II exclusively in cellwall polysaccharides. Immunocytochemical studies demonstratedthat the epitope is ubiquitous in cell walls of all the cellsin radish and rice roots, cultured tobacco cells, red cloverroot nodules, and lily growing pollen tubes. The label was denserin proximal to plasma membrane, and not detected in middle lamella,suggesting that borate may cross-link newly secreted pecticpolysaccharides at the membrane-cell wall interface. (Received October 13, 1997; Accepted February 16, 1998)