Two-stage portal vein ligation facilitates liver regeneration in rats.

BACKGROUND/AIMS: Recent reports have demonstrated that some patients are unable to undergo scheduled liver resection after preoperative portal vein embolization due to insufficient hypertrophy of the future remnant liver. The present study examined whether two-stage portal vein ligation (PVL) increases hypertrophy of the future remnant liver compared to conventional PVL in rats. METHODS: Rats were divided into 3 groups: group A, ligation of left primary branch; group B, ligation of right and left primary branches; group C, ligation of the left primary branch, followed by 2-stage PVL 7 days postoperatively. To evaluate liver regeneration, the proliferating cell nuclear antigen labeling index (LI), mitotic index (MI) in the caudate lobe and weight ratio of caudate lobe to body weight were measured. RESULTS: The weight ratio of caudate lobe to body weight was significantly higher in group C than in groups A or B 14 days postoperatively. In groups A and B, LI and MI in the caudate lobe peaked 2 days postoperatively, then decreased to preoperative levels by 7-8 days postoperatively, but remained significantly elevated until 10-14 days postoperatively in group C. CONCLUSION: Two-stage PVL increases hypertrophy of the future remnant liver compared to conventional PVL in rats.

Costimulatory signals distinctively affect CD20- and B-cell-antigen-receptor-mediated apoptosis in Burkitt's lymphoma/leukemia cells.

CD20 is a B-cell differentiation antigen and known to induce apoptosis in Burkitt's lymphoma/leukemia (BL) cells upon antibody-mediated crosslinking. We examined the biological effect of CD20 crosslinking on BL cell lines and observed that apoptosis induction is accompanied by activation of multiple caspases, including caspase-8, -9, -3, -2, and -7. Further investigation revealed a clear synergism between apoptosis mediated by CD20 and by B-cell antigen receptor (BCR). Examination of the effect of simultaneous crosslinking of other cell surface molecules with crosslinking of CD20 or BCR on apoptosis induction showed that these molecules had either a synergistic or inhibitory effect on induction of apoptosis. It is worth noting that some molecules had a different effect on CD20- and BCR-mediated apoptosis. Simultaneous crosslinking of the molecules CD10, CD22, CD72, and CD80 inhibited BCR-mediated apoptosis, but enhanced CD20-mediated apoptosis. Further studies revealed that regulation of CD20-induced apoptosis by other costimulatory molecules is achieved by modification of caspase activation. CD20-mediated apoptosis in BL cells may provide not only a model for understanding the mechanism regulating clonal selection of B cells but a new therapeutic strategy for BL patients.

Mechanical and magnetic properties of nickel-dispersed tetragonal zirconia nanocomposites.

Effects of Ni dispersions on microstructure and mechanical properties have been studied for Y2O3-stabilized tetragonal zirconia (Y-TZP)/Ni nanocomposites with Ni dispersion up to 10 vol%. Composites were successfully fabricated by reducing and hot-pressing Y-TZP/NiO powder mixtures. Fracture strength was significantly improved from 1.5 GPa for monolithic Y-TZP to 1.9 GPa for nanocomposites with a small addition of Ni (1-2 vol%). Magnetic properties of Y-TZP/Ni nanocomposites were also investigated. Magnetization curves of Y-TZP/Ni nanocomposites showed typical hysteresis loops of soft magnetic materials, whereas coercivity was much larger than that of pure Ni metal. A new function arising from magnetomechanical effects of metallic Ni is also discussed for the present nanocomposites.

Kinetic analysis of binding between Shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance.

Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface and is responsible for hemolytic uremic syndrome. Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown. In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonance analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface. Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased. In addition, this assay was shown to be specific, because neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer. It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the "bivalent analyte" model was found to best fit the interaction between Stxs and Gb3Cer. This shows that the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect. The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, although kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate. The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins.

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.

CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system.

The glycosylphosphatidylinositol-anchored CD24 protein is a B cell differentiation Ag that is expressed on mature resting B cells but disappears upon Ag stimulation. We used Burkitt's lymphoma (BL) cells, which are thought to be related to germinal center B cells, to examine the biological effect of Ab-mediated CD24 cross-linking on human B cells and observed 1) induction of apoptosis in BL cells mediated by cross-linking of CD24; and 2) synergism between the cross-linking of CD24 and that of the B cell receptor for Ag in the effect on apoptosis induction. We also observed activation of mitogen-activated protein kinases following CD24 cross-linking, suggesting that CD24 mediates the intracellular signaling that leads to apoptosis in BL cells. Although CD24 has no cytoplasmic portion to transduce signals intracellularly, analysis of biochemically separated glycolipid-enriched membrane (GEM) fractions indicated enhanced association of CD24 and Lyn protein tyrosine kinase in GEM as well as increased Lyn kinase activity after CD24 cross-linking, suggesting that CD24 mediates intracellular signaling via a GEM-dependent mechanism. Specific microscopic cocapping of CD24 and Lyn, but not of other kinases, following CD24 cross-linking supported this idea. We further observed that apoptosis induction by cross-linking is a common feature shared by GEM-associated molecules expressed on BL cells, including GPI-anchored proteins and glycosphingolipids. CD24-mediated apoptosis in BL cells may provide a model for the cell death mechanism initiated by GEM-associated molecules, which is closely related to B cell receptor for Ag-mediated apoptosis.

Activation of the caspase cascade during Stx1-induced apoptosis in Burkitt's lymphoma cells.

Shiga toxin 1 (Stx1) produced by Escherichia coli has been reported to induce apoptosis in many different cell types, including Burkitt's lymphoma (BL) cells. Since it has been established that the caspases play essential roles as the effector molecules in the apoptotic process in most cases, we examined the kinetics of caspase activation during the process of Stx1-mediated apoptosis of BL cells. Using Ramos BL cells that are highly sensitive to Stx1-mediated cytotoxicity, we observed that multiple caspases, including caspase-3, -7, and -8 were promptly activated following Stx1 treatment, as indicated by both the procaspase cleavages and enhancement of cleavage of the tetrapeptide substrates of the caspases. In addition, the inhibition assay revealed that caspase-8 is located upstream of both caspase-3 and -7, suggesting that Stx1-mediated apoptosis utilizes a similar caspase cascade to that involved in Fas-mediated apoptosis. Neither anti-Fas mAb nor TNF-alpha, however, affected the Stx1-mediated apoptosis of Ramos cells. Although the precise mechanism of Stx1-mediated activation of caspase-8 is still unclear, we have demonstrated that crosslinkage of CD77, a functional receptor for Stx1, with specific antibody is sufficient to induce activation of caspase-8. Our findings should provide new insight into the understanding of the molecular basis of Stx1-mediated cell injury.

Prominent immunogenicity of monosialosyl galactosylgloboside, carrying a stage-specific embryonic antigen-4 (SSEA-4) epitope in the ACHN human renal tubular cell line-a simple method for producing monoclonal antibodies against detergent-insoluble microdomains/raft.

The binding of Shiga toxin (Stx) to Gb3Cer in detergent-insoluble microdomains (DIM)/raft of the ACHN human renal tubular cell line causes the temporal activation of the Src-family kinase Yes [1]. As a strategy for examining signaling mechanisms in DIM/raft, monoclonal antibodies (MAbs) are reliable tools for characterizing the constituent molecules in these microdomains. Thus, we employed DIM/raft suspensions of ACHN cells as an immunogen to develop MAbs. Simply subcutaneous injections of ACHN DIM/raft could elevate the serum titer after several boosts. The first screening was performed using dot-blot immunostaining with culture supernatants on a polyvinylidene difluoride (PVDF) membrane, on which DIM/raft or their chloroform/methanol (C/M) (2:1, v/v) extracts were dot-blotted. The next screening was performed by flowcytometric analysis of ACHN cells treated with or without a permeabilizing reagent. Many of the clones (21/31 clones=68%) thus obtained were also found to recognize to lipid fractions of the DIM/raft. Strikingly, all of the 21 clones that reacted to the lipid fraction were found to recognize monosialosyl galactosylgloboside (MSGG) or GL7, which carries the SSEA-4 epitope. Using DIM/raft as immunogens may enable us to easily obtain MAbs for glycolipids.

Nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, protects against Shiga toxin cytotoxicity in human microvascular endothelial cells.

Infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause microvascular endothelial cell damage, resulting in hemorrhagic colitis and hemolytic uremic syndrome. The prevention of endothelial cell damage is therefore a crucial step in overcoming this disorder. Here, we report that nitrobenzylthioinosine (NBT), a nucleoside transport inhibitor, has a protective effect against the cytotoxicity of Stxs in human microvascular endothelial cells (HMVECs). The relative viability of cells treated with 1.5-15 pM of Stx1 was reduced to 10-20% of that without Stx1. However, the viability of cells treated with NBT (10-100 microM) remained higher than 80%, even in the presence of Stx1. NBT also protected against Stx1 cytotoxicity in sodium butyrate-treated hypersensitive HMVECs. The protective effect of NBT against Stx cytotoxicity may be due to the depletion of ATP in the cells, thereby inhibiting the entry of Stx1.

Globotriaosyl ceramide (CD77/Gb3) in the glycolipid-enriched membrane domain participates in B-cell receptor-mediated apoptosis by regulating lyn kinase activity in human B cells.

The role of CD77 expressed on a fraction of germinal center B cells, also known as glycosphyngolipid Gb3, and as a functional receptor for Shiga toxins (Stx) in B-cell receptor (BCR)-mediated apoptosis was investigated. Using Stx1-sensitive Burkitt's lymphoma Ramos cells as an in vitro model of CD77(+) germinal center B cells, intracellular signaling events mediated by either Stx1 or anti-CD77 antibody were examined immunobiochemically and immunocytologically. We observed prompt activation of Lyn and Syk kinases leading to increased binding of these proteins to surface IgM (sIgM) in Ramos cells after Stx1 treatment. We also observed microscopic colocalization of CD77 and sIgM after stimulation with Stx1. Along with the synergism between the cross-linking of CD77 and that of sIgM in their effect on apoptosis induction, it was highly probable that CD77 cross-linking induces activation of the BCR signaling cascade. Analysis using sucrose density gradient centrifugation suggested that Stx1 binding to CD77 induced recruitment and activation of Lyn in the glycolipid-enriched membrane (GEM) fractions. Once activated, however, Lyn seemed to acquire an increased detergent solubility and moved outside of the GEM fractions. This study describes the participation of the GEM domain in BCR-signaling cascade and suggests a possible role of CD77 as a regulator of BCR-induced apoptosis in human B cells.

Functional conservation of platelet glycoprotein V promoter between mouse and human megakaryocytes.

OBJECTIVE: In an attempt to clarify the megakaryo-specific regulatory mechanism of GPV gene transcription, we characterized the 5'-flanking region of the mouse GPV gene. MATERIALS AND METHODS: The promotor activity of a -481/+22 5'-fragment of the mouse GPV gene was examined in normal mouse bone marrow cells (BMC) and various human cell lines using two distinct reporter gene assay systems, luciferase and green fluorescence protein (GFP). RESULTS: When a DNA construct consisting of this fragment and a GFP reporter gene were transiently expressed in thrombopoietin-supported mouse BMC culture, GFP was identified only in megakaryocytes. The same construct expressed high levels of GFP in the human megakaryocytic Dami line. When assessed by dual luciferase assay, the full -481/+22 fragment could drive variable promoter activity in human as well as mouse megakaryocytic lines but did not work in non-megakaryocytic cells. Sufficient transcriptional activation of this fragment was restricted to the cells expressing apparent GPV mRNA. A deletion and point mutation study indicated that GATA and Ets motifs, typical cis-acting elements for platelet-specific genes, located of -75 and -46, respectively, were essential for promoter function. CONCLUSION: The GPV promoter has the general characteristics found in platelet-specific genes, and the mechanism for megakaryocyte-specific, maturation-dependent regulation of GPV gene transcription is highly conserved between mouse and human. Analysis of GPV transcription mechanism utilizing human lines as well as BMC should provide new information on the final maturational process of megakaryocytes.

A new assay for thyroglobulin concentration in serum using monoclonal antibodies against synthetic peptides.

The concentration of thyroglobulin (Tg) measured by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) is greatly affected by the presence of anti-Tg autoantibodies in sera. We developed a new assay for detecting Tg in the presence of high concentrations of anti-Tg autoantibodies. A 48-kDa fragment was purified from Tg after treatment with V8 protease. This fragment did not appear to bind to two types of monoclonal antibodies (57Ab and 28D3) against a peptide in the C-terminus (amino acids 2735-2748) of Tg and intact Tg, respectively, by ELISA and Western blot analysis. In contrast, anti-Tg autoantibody or anti-Tg polyclonal antibody reacted well with this fragment. Our new ELISA used 57Ab as a solid phase antibody and 28D3 as a antibody conjugated to horseradish peroxidase. Buffer containing purified 48-kDa fragment was used to neutralize autoantibodies against Tg. With this assay, the recovery of Tg was 84.0-89.6% in normal healthy donors (n=5) in the presence of immunoglobulin G (IgG) purified from sera positive for anti-Tg autoantibody, and 76.2-104.4% in patient sera Grave's disease (n=15). Furthermore, the Tg concentrations in sera from patients with Grave's disease (n=20) ranged from 25 to 526 ng/ml, even though the Tg concentration, as measured by a commercial RIA did not exceed 55 ng/ml. There was good agreement between Tg concentrations measured by new Tg-ELISA and commercial Tg-RIA in sera that were negative for anti-Tg autoantibody. Overall, our new ELISA containing a Tg fragment to neutralize the presence of autoantibodies, showed good sensitivity and precision, and may be useful for routine use. Further investigations with the new assay should allow wider assessment of the prevalence and pattern of thyroid autoimmunity or thyroid neoplasms.

Characterization of monoclonal antibodies against mouse and rat platelet glycoprotein V (CD42d).

The mouse- and rat-platelet-specific hamster monoclonal antibody (MAb) 1C2, previously found to react with a thrombin-sensitive 74-kD glycoprotein, was now shown to recognize platelet glycoprotein V (GPV, CD42d). 1C2 reacted with NIH-3T3 cells in which recombinant mouse or rat GPV was expressed. Both 1C2 and 4A5, another mouse-platelet-specific rat MAb, immunoprecipitated GVP, although they recognized different epitopes. Side-by-side comparison confirmed that 1C2 as well as RPM.9, a MAb against rat GPV, recognized the same rat platelet molecule. In a mouse bone marrow culture, 1C2+ megakaryocytes emerged from CD41 (GPIIb)+1C2- megakaryocytes. Because 1C2+ megakaryocytes exhibited higher DNA ploidy distribution than CD41+ cells, GPV likely appears in the late stage of megakaryocyte maturation. This study established 1C2 as a MAb against mouse and rat GPV, namely CD42d, and as useful tool to study rodent megakaryopoiesis.

Correlation analysis between fatty acid compositions of zooplankter individuals, fed on different phytoplankton species by means of pyrolysis-gas chromatography combined with on-line methylation.

Pyrolysis-gas chromatography (Py-GC) combined with on-line methylation was applied to a correlation analysis between the distributions of fatty acid components in the lipids of zooplankter individuals and those of ingested algae using principal component analysis (PCA). Py-GC in the presence of organic alkali, tetramethylammonium hydroxide (TMAH), was used to estimate the apparent distributions of fatty acid components contained in a single individual zooplankter weighing several tens of micrograms and a small sample size of ingested algae samples in the order of 10 microg. The observed fatty acid compositions were used as a database for the PCA in order to discriminate the zooplankton and ingested algae samples. The result obtained indicated that the fatty acid compositions of zooplankton individuals used in this work were significantly reflected in those of their ingested food in spite of some contribution from isomerization and/or elongation of fatty acid components during digestion of the ingested algae phytoplankton in living zooplankters.

Analysis of lipid components in zooplankter individuals by reactive pyrolysis-gas chromatography in the presence of organic alkali.

It has been eagerly requested to develop a highly-sensitive method to characterize extremely minute amounts of natural organic materials occluded in meteorites and/or space dusts in order to confirm the existence of life in the extraterrestrial space. In this article, the reactive pyrolysis-gas chromatography (Py-GC) applied to the analysis of the lipid components contained in every zooplankter individual is introduced for the sake of its potential extension to the characterization of trace amounts of the extraterrestrial organic materials. Here, Py-GC was applied to 1) the discriminative analysis among zooplankter individuals cultured in different food concentrations, and 2) the correlation analysis between the distributions of fatty acid components in the lipids of zooplankter individuals and ingested algae phytoplankton.

[Histopathological study of valvular deposits of amyloid protein in cardiac amyloidosis].

Cardiac amyloidosis is associated with amyloid deposits in cardiac valves which also show the thickening of leaflets and cusps. This study examined amyloid deposits on cardiac valves to investigate the possible involvement in echocardiographic valvular abnormalities in 17 patients with systemic amyloidosis (12 males and 5 females aged 44 to 82, mean 66.4 years) in the autopsy files of the National Cardiovascular Center between 1980 and 1993. All four cardiac valves were examined histologically using hematoxylin-eosin and Congo red stains with polarization. All cusps and leaflets were divided into six segments and all segments of each cusp and leaflet were scored for the proportional area of amyloid deposit (from 0 to 3). The immunohistochemical types of amyloid proteins were immunoglobulin light chain-related (AL) amyloidosis in 16 cases, and amyloid A-related (AA) amyloidosis in one case. Twelve of 16 cases with AL amyloidosis were subclassified as AL lambda type and 4 were subclassified as AL kappa type. In the atrioventricular valve leaflets, the atrial side of basal portion showed the most remarkable amyloid deposits among the six segments. In the semilunar valves, amyloid deposits were mild in the tip and middle portions. Among the patients with AL amyloidosis, those with AL lambda type amyloid appeared to have greater deposits than those with AL kappa type amyloid. Mitral valves appearing abnormal by echocardiography had greater amyloid deposits. However, considering other factors affecting valvular function, the relationship between the localization or the degree of amyloid deposition and endocardiographic valvular abnormalities was unclear.

Pulmonary thromboembolic hypertension in systemic lupus erythematosus with lupus anticoagulant: histopathological analysis of localization and distribution of thromboemboli in pulmonary vasculature.

We report a systemic lupus erythematosus patient with lupus anticoagulant accompanied by pulmonary thromboembolic hypertension possibly due to large vegetation of the tricuspid valve of the heart. Histopathological analysis of localization and distribution of thromboemboli in pulmonary vasculature revealed that the organized thromboembolic occlusion of multiple blood vessels (99 out of 222 arteries), might be responsible for the pulmonary hypertension both in quality and quantity. The contribution of lupus anticoagulant to the pathogenesis of Libman-Sacks endocarditis in systemic lupus erythematosus, and its possible relationship to pulmonary thromboembolic hypertension are discussed.

Glycosaminoglycan components in duodenum with advancing age and in patients with progressive systemic sclerosis.

The glycosaminoglycan (GAG) components in normal human duodenal tissue and in duodenal GAGs of patients with progressive systemic sclerosis (PSS) were characterized at the macromolecular level by electrophoresis combined with various GAG-specific digestion. High-performance liquid chromatographic assay quantified various unsaturated disaccharides derived from chondroitin sulfate and dermatan sulfate isomers (DS). The data obtained showed that on average, the total GAG content was 1.02 mg as uronic acid/g dry tissue weight, and the main GAGs were DS (33% of total GAGs), and heparan sulfates (HS, 27%), followed by hyaluronic acid (HA, 14%) and over-sulfated DS (10%), and small amounts of chondroitin, chondroitin-4- and -6-sulfates (C-4S and C-6S, totally 16%). With advancing age, the proportion of DS, HS and oversulfated DS increased, while HA, chondroitin and C-4S decreased. The composition of duodenal GAGs in PSS patients was similar to that in the aged: a higher proportion of DS isomers and HS and a lower proportion of HA and chondroitin. As the structure of sulfated GAGs differs with advancing age and in patients with PSS, GAGs may have a role in duodenal function and metabolism.

Basal interfalcine approach through a frontal sinusotomy with vein and nerve preservation. Technical note.

The authors describe a new method for a frontal interhemispheric approach when treating craniopharyngiomas of the third ventricle or anterior communicating artery aneurysms. This technique ensures preservation of the bridging veins and the olfactory nerves. This "basal interfalcine approach" involves a craniotomy in the centrobasal portion of the frontal bone (the frontal sinus), removal of the inner tables and the crista galli, and splitting the basal portion of the falx into two leaves, through which the basal interhemispheric fissure is opened. The olfactory nerves are protected by the leaves of the falx, and the bridging veins are preserved because the approach is low enough to spare them. The surgical techniques are described together with a unilateral variation of this approach. The significance of preserving the bridging veins is discussed in connection with avoidance of postoperative contusional hemorrhage.

[Traumatic enucleation of the eye ball--report of a case and considerations concerning the pathogenic mechanism of intracranial complications].

A 48-year-old man had his left eye ball enucleated by fingers of an assailant. The optic nerve, measuring 4 cm in length, was attached to the enucleated eye ball, but there was neither a wound in his eyelids nor cerebrospinal fluid leakage from inside the orbit. He was confused, but responsive and was able to recognize other persons with his right eye. His right-eye vision deteriorated within 24 hours and was almost totally lost for about one month. Three months after the trauma his vision recovered to 0.1, but his visual field showed severe concentric narrowing. An emergency CT on admission showed a small subarachnoid hemorrhage in the suprasellar cistern, and follow-up CT scanning on day 7 demonstrated a small infarction in the left globus pallidus and putamen. Cerebral angiography performed on day 17 showed residual vasospasm of the horizontal portion of left anterior cerebral artery. Left ophthalmic artery was patent and there was no aneurysm formation either on the intracranial or on the intraorbital arteries. Literature review yielded only three cases of eye ball enucleation by an assailant. Intracranial complications reported in the literature, including those associated with eye ball enucleation caused by other mechanisms, are; contralateral visual field defect: seven cases, hypothalamic involvement: two cases, subarachnoid hemorrhage: two cases, cerebrospinal fluid leakage: one case, and meningitis: one case. The optic nerve, from just behind the eye ball to the chiasm, is reported to be 40-50 mm long, and eye ball enucleation with the optic nerve measuring 4 cm or more is quite likely to cause intracranial complications such as are cited above.