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Objective: To explore the clinical features and plastic treatment of Paget's disease of the scrotum and penis., Methods: We analyzed 11 cases of Paget's disease of the scrotum and penis treated from 1997 to 2007. Extended excision of the focus of infection was performed and the skin absence was repaired by free skin grafting, local random skin flap, and island skin flap., Results: Satisfactory wound healing was achieved in all but 1 case, which was delayed due to infection. All the patients were followed up for 1-4 years. One patient relapsed 2 years after the surgery and received a second operation, and another 1 died of lymphoma complicated by lung infection. The original shape and contractibility of the scrotum and penis were basically preserved and their appearance and function were fairly good., Conclusion: For patients with Pagets disease of the scrotum and penis, it is a desirable method to at once excise the lesion and repair the skin absence by plastic surgery.

Background: Both the role of inducible nitric oxide synthase (iNOS) in the development of inflammatory bowel disease (IBD) as well as the molecular details governing its mucosal induction remain unclear., Methods: In the present study we evaluated the role of the residing intestinal microflora in the induction of epithelial iNOS upon transfer of CD45RB(high) CD4(+) T cells to SCID mice. CB-17 SCID mice were reared with conventional flora (CNV) or germfree CB-17 SCID mice were monoassociated with Helicobacter muridarum, act A(-) mutant Listeria monocytogenes, segmented filamentous bacteria (SFB), or Ochrobactrum anthropi., Results: Within 2 weeks CNV SCID mice injected with CD45RB(high) CD4(+) T cells showed a focal, epithelial iNOS expression on the apical site of villi that preceded the infiltration of CD4(+) T cells and cytokine production followed by extension of this expression to the entire surface along the whole crypt axis as the colitis progressed. SCID mice monoassociated with H. muridarum developed a severe colitis and showed high epithelial iNOS expression. CNV-SCID mice without T cells and SCID mice monoassociated with SFB did not show any iNOS expression, whereas SCID mice monoassociated with act A(-) mutant L. monocytogenes and O. anthropi showed some scattered epithelial iNOS staining on the apical site of a few villi, but none of these mice developed colitis., Conclusions: These findings demonstrate that the expression of epithelial iNOS is highly bacterium-specific and correlates with the severity of disease, suggesting an important role for this enzyme in the development of IBD.

Objective: To establish a mathematical model of hepatitis C virus (HCV) replication and develop a working theory for antiviral therapy in order to understand the dynamics of HCV replication., Methods: Peripheral blood cells of 4 hepatitis C patients were cultured. Quantities of the HCV were detected every 15 min by real-time PCR. The data were analyzed using SPSS software. A mathematical functional relationship between HCV RNA and the time lapse was established., Results: The quantity of HCV RNA declined and it fell into a mathematical model: Y=3E+0.8e(-0.5467x) (r=0.9547). The estimated virion half-life was 45 min on the average., Conclusions: The decline of HCV RNA in the blood is not of a linear trend and the HCV RNA lasts a longer time although the speed of the decline is faster than that in vivo.

Aim: To investigate the effect of IH764-3 on the expression of MMP-13 and TIMP-1 by H2O2-stimulated hepatic stellate cell and the alteration of FAK during this process., Methods: The expression of MMP-13 and FAK mRNA was examined by RT-PCR. TIMP-1 mRNA was analyzed by in-situ hybridization. FAK and TIMP-1 were evaluated at protein level through Western blotting method., Results: Being incubated for 2 h, compared with control group, MMP-13 mRNA was upregulated by IH764-3, but TIMP-1 transcription was reduced in a dose-dependent manner, accompanied with the decrease of FAK mRNA. The expression of TIMP-1 and FAK protein in HSC also decreased after being exposed by IH764-3 for 24 h., Conclusion: IH764-3 can induce the expression of MMP-13 and inhibit the expression of TIMP-1. Down-regulating the expression of FAK mRNA may be one of its mechanisms.

Aim: To study the effect of Arg-Gly-Asp-Ser (RGDS) tetrapeptide on the expression of matrix metalloproteinase-13 (MMP-13) and the tissue inhibitor of metalloproteinase-1(TIMP-1) in hepatic stellate cell(HSC) stimulated by fibronectin (FN)., Methods: The expression of MMP-13 was evaluated by RT-PCR. TIMP-1 was analyzed by in-situ hybridization at mRNA level and by Western blot at protein level., Results: Treated by RGDS tetrapeptide for 2 h, the mRNA expression of MMP-13 was upregulated but the expression of TIMP-1 mRNA and its protein in HSC was inhibited., Conclusion: The expression of MMP-13 can be induced by RGDS tetrapeptide and the expression of TIMP-1 can be inhibited by RGDS tetrapeptide, which is one of the mechanisms of its anti-fibrogenesis.

Background: Direct and indirect evidences have suggested that angiogenesis is a prerequisite for the development of endometriosis. Aiming at offering experimental evidences for anti-angiogenesis therapy, we transplanted the eutopic endometrium from patient with endometriosis into the severe combined immunodeficiency disease (SCID) mice, to evaluate the effect of the endostatin on the growth and angiogenesis of the established endometriosis lesions in SCID mice model., Methods: Eutopic endometrium of women with endometriosis was transplanted into the SCID mice. The mice were randomized into treatment (n = 10) and control groups (n = 10). Two weeks after the implantation of endometrium fragment, the treatment group was injected with recombinant human endostatin YH-16 into the peritoneal cavity (2 mgxkg(-1)xd(-1)), whereas the control group received equivalent volume of PBS (200 microl/d). The volume of endometriotic lesions in SCID mice was measured every three days, and all the treatment lasted for 14 days. Immunohistochemistry was used to determine microvessel density (MVD) and the expression of VEGF. The results were analyzed by t test and chi(2) test to value the treating effect., Results: Compared with the control group, growth of endometriosis lesion was reduced in the mice treated with YH-16. Statistically significant differences in the volume and weight of the ectopic lesions were observed between the treatment and the control groups (P < 0.05). Microscopical examination showed that after being treated with YH-16, the volume of the endometrial tissues decreased, the glands depauperated, and the glandular epithelium partially degenerated. Necrotic debris was observed in the endometrial stroma. MVD and expression of VEGF in the treatment group were significantly lower than those in the control group (P < 0.05)., Conclusions: Recombinant human endostatin affects the maintenance and growth of endometriotic tissues by inhibiting angiogenesis and reducing the expression of VEGF in ectopic lesion. The angiostatic agent may be promising as a therapy for endometriosis.

Objective: To investigate the distribution, phenotype and development of pericytes in infantile hemangioma., Methods: Fifty-two infantile hemangioma samples were included in our study. alpha-SMA was used as the marker antigen to observe the distribution of pericytes. Transmission electron microscope and TUNEL method were used to analyze the apoptosis of pericytes., Results: In the early and middle proliferating stage, there existed many pericytes in hemangioma; Pericytes together with endothelial cells generated vasculogenesis. In the late proliferating stage, many pericytes became apoptotic. In the early involuting stage, there were only a few of pericytes around the microvessels; After that, the microvessels became obstruction progressively and pericytes disappeared finally., Conclusions: The pericyte is one of the major constitutive cells of hemangioma. The vasculogenesis, development and disappearance of microvessels undertaken by pericytes and endothelial cells lead to the pathologic evolution of infantile hemangioma.

Objective: To investigate the expression and role of glucose transporter-1 (Glut1) in infantile hemangioma., Methods: Fifty-two samples from infantile hemangioma, 25 in cavernous venous malformation, 9 in arteriovenous malformation, 2 in capillary malformation and 5 in normal skin samples were involved in this study. The EnVision immunohistochemical stain was used to investigate the expression of Glut1 protein in these samples., Results: In the early proliferating stage, a number of endothelial cells expressed Glut1. In the middle proliferating stage, most of vascular endothelial cells and scattered endothelial cells expressed Glut1. In the late proliferating stage, the expression of Glut1 decreased quickly. In the involuting stage, all hemangioma samples didn't express Glut1. All of the samples from the cavernous venous malformations, arteriovenous malformations, capillary malformations and normal skin had no expression of Glut1., Conclusions: Glut1 may be one of the phenotypes of infantile hemangioma endothelial cells in their development, rather than the inherent character. The expression of Glut1 changes according to the metabolic need of infantile hemangioma cells.

Objective: To explore whether there could be proliferative cells in the cochlea of the newborn rat or not and what kinds of cells should be differentiated from the proliferative cells while to study the effect of the growth factors on the proliferative cells and the ultrastructure of the proliferative cells., Methods: The Corti's organ were dissected from the cochlea of newborn SD rats and cultured. The proliferative condition of cells was tested by infusing the 5-bromo-2-deoxyuridine (BrdU) into the culture medium. And the variety of the spheres and differentiated cells were identified by immunohistochemistry. Corti's organ from forty-eight surface preparations was randomly divided into 4 groups: control group; epidermal growth factor (EGF) group; basic fibroblast growth factor (bFGF) group and EGF + bFGF group, with each group including 12 Corti's organ, and then the number of cell spheres of each Corti's organ was counted. The data was statistically analysed with ANOVE. Finally, the proliferative cells were observed under scanning electron microscope and transmission electron microscope., Results: (1) The cell spheres can be observed in the cell culture of the Corti's organ. In present experiment, 90.1% of cells in spheres were labeled by BrdU, while nestin of spheres, the marker of hair cells--myosin 7A, espin, and phalloidin of the differentiated cells were positive. The marker of neuron-microfilament-M was also positive, and some differentiated cells were labeled by myosin 7A and BrdU, espin and BrdU, NF-M and BrdU at the same time. (2) The average number (x +/- s) of spheres from single Corti's organ was: 45.3 +/- 23.00 in control group, 86.2 +/- 34.1 in EGF group, 96.5 +/- 33.6 in bFGF group and 131.2 +/- 47.00 in EGF + bFGF group. There were significant differences between other groups respectively (P < 0.05) but there was no significant differences between EGF group and bFGF group (P > 0.05). (3) Scanning electron microscopy and transmission electron microscopy showed that cells of the spheres were round and had the same size and many short and thin microvilli on the surface of these cells. The cytoplasm were rich of organellae such as endoplasmic reticulum, mitochondrion, and cytoskeleton such as microfilament, microtube, et al. Tight junction, desmosomes and gap junctions between two adjacent cells were seen., Conclusions: The proliferative cells are observed in the cochlea of the newborn rats and proliferative cells could differentiated into hair cells with bundles-like structure and neuron. Both EGF and bFGF possess the promoting effects for proliferation on the proliferative cells while the proliferative cells have characters of earlier immature cells.

Objectives: To observe the effects of fasudil, a Rho/ROCK signaling pathways inhibitor, on adhesion, migration and proliferation of hepatic stellate cells (HSCs)., Methods: Cultured HSCs were divided into 5 groups. Fasudil was added to 4 groups in a concentration of 12.5, 25, 50, and 100 micromol/L, respectively. A group without fasudil added served as untreated controls. The adhesive inhibition effect of fasudil on HSCs was examined by toluidine blue colorimetric assay, the inhibition of migration of HSCs was evaluated by a modified Boyden chamber, and cell proliferation was assessed by MTT assay. The protein levels of RhoA, p-MLC (Thr18/Ser19) and alpha-SMA were assayed by Western blot., Results: Fasudil inhibited HSC adhesion, proliferation and LPA-induced migration in a concentration-dependent manner; the protein expressions of alpha-SMA and p-MLC (Thr18/Ser19) were significantly decreased in the presence of fasudil., Conclusion: Fasudil can inhibit HSC adhesion, migration and proliferation by suppressing the cytoskeleton regulation function of Rho/ROCK signaling pathways.

Aim: To investigate whether extracellular signal-regulated kinase 1 (ERK(1)) is activated and associated with hepatic stellate cell (HSC) proliferation in fibrotic rat liver tissue., Methods: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were evaluated by hematoxylin and eosin staining, and Masson's trichrome method. ERK(1) mRNA in rat liver tissue was determined by reverse transcription-polymerase chain reaction, while the distribution of ERK(1) was assessed by immunohistochemistry. ERK(1) protein was detected by Western blotting analysis. The number of activated HSCs was quantified after alpha smooth muscle actin (alpha-SMA) staining., Results: With the development of hepatic fibrosis, the positive staining cells of alpha-SMA increased obviously, and mainly resided in the portal ducts. Fiber septa and perisinuses were accompanied with proliferating bile ducts. The positive staining areas of the rat livers in model groups 1-4 wk after ligation of common bile duct (12.88% +/- 2.63%, 22.65% +/- 2.16%, 27.45% +/- 1.86%, 35.25% +/- 2.34%, respectively) were significantly larger than those in the control group (5.88% +/- 1.46%, P < 0.01). With the development of hepatic fibrosis, the positive cells of ERK(1) increased a lot, and were mainly distributed in portal ducts, fiber septa around the bile ducts, vascular endothelial cells and perisinusoidal cells. Western blotting analysis displayed that the expression of ERK(1) and ERK(2) protein was up-regulated during the model course, and its level was the highest 4 wk after operation, being 3.9-fold and 7.2-fold higher in fibrotic rat liver than in controls. ERK(1) mRNA was expressed in normal rat livers as well, which was up-regulated two days after BDL and reached the highest 4 wk after BDL. The expression of ERK(1) was positively correlated with alpha-SMA expression (r = 0.958, P < 0.05)., Conclusion: The expression of ERK(1) protein and mRNA is greatly increased in fibrotic rat liver tissues, which may play a key role in HSC proliferation and hepatic fibrogenesis.

1. The objective of this study was to evaluate whether the oligosaccharide stachyose enhances gastrointestinal tract health by fermentation and proliferation of desirable bacteria species and thus affects growth performance and nutrient digestibility in broilers. 2. A total of 432 1-d-old male Arbor Acres (AA) broilers were randomly allocated to one of 6 treatments, with 12 replicate pens per treatment and 6 birds per pen. Chicks were fed a maize-hamlet protein 300 (HP300) basal diet with 0, 4.0, 8.0, 12.0 or 16.0 g/kg stachyose. A sixth diet contained no HP300 but soybean meal (SBM) and provided 8.7 g/kg stachyose and 3.1 g/kg raffinose. The duration of the study was 42 d. 3. Stachyose contents above 12.0 g/kg depressed group body weights, average daily gain and feed/gain but not feed intake during the whole experimental period. Broiler growth decreased linearly and quadratically with increasing stachyose content. No differences were detected between diets supplemented with 12.0 g/kg stachyose and SBM. 4. Nutrient digestibility tended to decrease but not significantly with increasing stachyose. 5. Stachyose content had no significant positive effects on caecal pH, microflora population and the resulting short-chain fatty acid (SCFA) metabolites during the 42 d experiment, with only butyrate differing significantly in the initial period.

Objective: To evaluate the effects of nerve growth factor (NGF) on the proliferation and apoptosis of hepatic stellate cells (HSCs) of fibrotic rat., Methods: HSCs of fibrotic rat were incubated in medium with different concentrations of NGF (50 ng/ml, 100 ng/ml, and 150 ng/ml respectively) or without NGF for 24, 48 and 72 hours respectively. The proliferation rate of the HSCs was determined by MTT assay. Apoptotic HSCs were detected by TUNEL method. The apoptotic rate of HSCs was studied by flow cytometry. The conformation of HSCs was observed by microscopy., Results: (1) MTT method indicated that the proliferation rates of the HSCs induced with different concentrations of NGF for 24 hours was significantly reduced compared with that of the control group (0.77 +/- 0.03, all P < 0.05). The proliferation rate of the HSCs treated with NGF of the concentration of 100 ng/ml for 24 hours was the lowest (0.63 +/- 0.02, P < 0.05). (2) The proliferation rate of the HSCs treated with NGF of the concentration of 100 ng/ml, the most appropriate concentration, increased time-dependently, and the proliferation rate at the 72 th hour was the lowest in comparison with that of the control group (0.48 +/- 0.03 vs 0.89 +/- 0.01, P < 0.05). (3) After stimulation of the HSCs by NGF of the concentration of 100 ng/ml for 24 hr, TUNEL method showed a significant increased apoptotic rate of HSCs compared with the control group (10.2% +/- 1.2% vs 1.6% +/- 0.1%, P < 0.05). Meanwhile, flow cytometry showed an apoptotic rate of 6.2% +/- 0.2% among the HSCs in the experimental group, but no apoptosis in control group. (4) Microscopy did not show significant morphological change in the HSCs with the proliferation inhibited., Conclusion: NGF can induce apoptosis of activated HSCs. The mechanism may be the interference with the apoptotic pathway.

A family of outer membrane lipoproteins of Neisseria meningitidis, LP2086, has been shown to induce serum bactericidal activity against a broad variety of meningococcal strains. Two sub-families of serologically distinct LP2086 proteins (A and B) have been identified. In the present study, we have shown that polyclonal anti-serum against rLP2086 is protective in vivo in an infant rat passive-protection model. Additionally, the LP2086 protein is displayed on the surface of 91% meningococcal strains as measured in a whole cell ELISA using polyclonal anti-sera raised against these proteins. We also demonstrate based on the reactivity of anti-rLP2086 antibody with recombinantly expressed C- and N-terminal fragments of rLP2086 in a Western blot assay that the C-terminal fragment of LP2086 dictates sub-family specificity and the N-terminal fragment determines the family specificity. A formulation containing family A and B of LP2086 potentially would provide broad protection against a majority of Neisseria meningitidis strains.

Homeostatic proliferation of naive T cells transferred to T cell-deficient syngeneic mice is driven by low-affinity self-MHC/peptide ligands and the cytokine IL-7. In addition to homeostatic proliferation, a subset of naive T cells undergoes massive proliferation in chronically immunodeficient hosts, but not in irradiated normal hosts. Such rapid T cell proliferation occurs largely independent of homeostatic factors, because it was apparent in the absence of IL-7 and in T cell-sufficient hosts devoid of functional T cell immunity. Strikingly, immunodeficient mice raised under germfree conditions supported only slow homeostatic proliferation, but not the marked T cell proliferation observed in conventionally raised immunodeficient mice. Thus, polyclonal naive T cell expansion in T cell-deficient hosts can be driven predominantly by either self-Ags or foreign Ags depending on the host's previous state of T cell immunocompetency.

Mucosal IgA is the most abundantly produced Ig upon colonization of the intestinal tract with commensal organisms in the majority of mammals. The repertoire of these IgA molecules is still largely unknown; a large amount of the mucosal IgA cannot be shown to react with the inducing microorganisms. Analysis of the repertoire of used H chain Ig (V(H)) genes by H-CDR3 spectrotyping, cloning, and sequencing of V(H) genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of V(H) genes and multiple clonally related sequences. The restricted usage of V(H) genes is a very consistent observation, and is observed for IgA plasma cells derived from B-1 or conventional B-2 cells from different mouse strains. Clonal patterns from all analyzed V(H) gene sequences show mainly independently acquired somatic mutations in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer's patches. Our data suggest a model of clonal expansion in which many mucosal IgA-producing B cells develop in the absence of affinity maturation. The affinity of most produced IgA might not be the most critical factor for its possible function to control the commensal organisms, but simply the abundance of large amounts of IgA that can bind with relatively unselected affinity to redundant epitopes on such organisms.

Aim: To investigate the dynamic expression of focal adhesion kinase (FAK) protein and FAK mRNA in fibrotic rat liver tissue, and the relationship between FAK and hepatic stellate cell (HSC) proliferation., Methods: Rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were evaluated by hematoxylin and eosin staining, and by Masson's trichrome method. FAK mRNA in the rat livers was determined by reverse transcription-polymerase chain reaction (RT-PCR), and the distributions of FAK were assessed immunohistochemistrically. The number of activated HSCs was quantified after alpha smooth muscle actin (alpha-SMA) staining., Results: With the development of hepatic fibrosis, the positively stained cells of alpha-SMA increased obviously, which were mainly resided in the portal ducts, fiber septa and perisinuses accompanied with proliferating bile ducts. The positively stained areas of the rat livers in model groups 1 to 4 wk after ligation of common bile duct (12.88+/-2.63%, 22.65+/-2.16%, 27.45+/-1.86%, 35.25+/-2.34%, respectively) were significantly larger than those in the control group (5.88+/-1.46%) (P<0.01). The positive staining for FAK significantly increased, which was mainly situated in portal ducts, fiber septa and around the bile ducts, vascular endothelial cells and perisinusoidal cells. The expression of FAK was positively correlated with alpha-SMA expression (r = 0.963, P<0.05). FAK mRNA expression was obviously up-regulated in the model groups compared to the control group., Conclusion: These data suggest that expressions of FAK protein and mRNA are greatly increased in fibrotic rat livers, which may play an important role in HSC proliferation and hepatic fibrogenesis.

Aim: To observe the dynamic expression of ERK1 in fibrotic rat liver., Methods: The rat hepatic fibrosis was induced by bile duct ligation (BDL). Histopathological changes were evaluated by hematoxylin and eosin staining, and by Masson's trichrome method. ERK1 mRNA in liver was determined by reverse transcription-polymerase chain reaction (RT-PCR), while the distribution of ERK1 was assessed by immunohistochemistry. ERK1 protein was detected by using Western blotting analysis., Results: With the development of hepatic fibrosis, the positive cells of ERK1 increased a lot, they were mainly distributed at portal ducts, fiber septa and around the bile ducts, vascular endothelial cells and perisinusoidal cells. Western blotting analysis results displayed that the expression of ERK1 protein were up-regulated with model course, and its levels were the highest at week 4 after operation, achieving to 3.9-fold of that in normal rat liver. ERK1 mRNA expressed in normal rat livers as well, they were up-regulated at day 2 after BDL and its level was the highest at week 4 after BDL., Conclusion: These data suggest that the expression of ERK1 and its mRNA can be increase greatly in fibrotic rat liver.

Although mechanisms operative in the induction and maintenance of specific, adaptive immunity, including 'cognate' B/T interactions, have been extensively studied and defined, we still know little about the mechanisms operative in developing and maintaining B- and T-cell dependent 'natural' immunity. Particularly, we are still rather ignorant concerning gut microbial/gut or systemic APC, T cell and B cell interactions that lead to lymphoid cell mediated 'natural' immunity: specific or broadly reactive, activation via TCR and BCR and/or via other receptors such as the TLR series, and whether T/B interactions are operative at this level? Here we will address: (1) the general role of gut microbes in the development and maintenance of the intestinal, humoral immune system; (2) the general role of gut microbes in the development of B1 cell mediated, 'natural' gut IgA and the dependence of these B1 cells on bystander T cell help; (3) the relative contributions of B1 versus B2 cells to gut 'natural' and specific IgA responses; (4) the role for particular 'normal' gut microbes in the initiation of inflammatory bowel diseases (IBD) in mice with a dysregulated immune system; and (5) the possible roles of gut microbes in facilitating oral tolerance, a mechanism likely operative in forestalling or ameliorating IBD. A central theme of this paper is to attempt to define the specificities of activated, functional CD4+ T cells in the gut for Ags of particular, usually benign gut microbes. We will also consider the still-unresolved issue of whether the contributions of B1-derived IgA in the gut to the 'natural' Ab pool are Ag-selected and driven to proliferation/differentiation or whether the main stimuli are not via BCRs but rather other receptors (TLRs, etc.). The main experimental approach has been to use antigen-free, germ-free, or gnotobiotic (mono- or oligo-associated with precisely known bacterial species) mice.

Background & Aims: Goblet cells are highly polarized exocrine cells found throughout the small and large intestine that have a characteristic morphology due to the accumulation of apical secretory granules. These granules contain proteins that play important physiologic roles in cellular protection, barrier function, and proliferation. A limited number of intestinal goblet cell-specific proteins have been identified. In this study, we investigate the expression and regulation of RELMbeta, a novel colon-specific gene., Methods: The regulation of RELMbeta messenger RNA expression was determined in LS174T, Caco-2, and HT-29 cell lines in response to stimulation with interleukin 13 and lipopolysaccharide. Quantitative reverse-transcription polymerase chain reaction, immunoblots, and immunohistochemistry were used to examine the expression of RELMbeta in BALB/c and C.B17.SCID mice housed in conventional, germ-free, and gnotobiotic environments., Results: Messenger RNA for RELMbeta is restricted to the undifferentiated, proliferating colonic epithelium. Immunohistochemistry shows that this protein is expressed in goblet cells located primarily in the distal half of the colon and cecum with lower levels detectable in the proximal colon. High levels of RELMbeta can be detected in the stool of mice and humans, where it exists as a homodimer under nonreducing conditions. Interestingly, the secretion of RELMbeta is dramatically reduced in germ-free mice. Furthermore, introduction of germ-free mice into a conventional environment results in enhanced expression and robust secretion of RELMbeta within 48 hours., Conclusions: These studies define a new goblet cell-specific protein and provide the first evidence that colon-specific gene expression can be regulated by colonization with normal enteric bacteria.

Objective: To investigate the possibility and efficacy of allograft transplantation in treating patient with huge tissue defect after radical giant malignant melanoma resection., Methods: A male person received blood type matching was chosen as donor. Immediately after the donor's brain death, allograft was excised with the depth to the layer intervenient between periosteum and epicranial fascia in calvaria, the superficial layer of deep temporal fascia in both sides of temporal regions, close to zygomatic bones and mandibles including masseter and auricles upon in face, and cervical soft tissues including sternocleidomastoid muscles, cervical and external jugular vessels of both sides were excised simultaneously. After being perfused with 4 degrees C UW solution through both common carotid arteries, the homograft was sheared and radiated with X-ray before being preserved in UW solution for further use. During the operation, both sides of external auditory meatus were anastomosed with ears firstly, and vessels were anastomosed end-to-end sequently, at last, the border of skin flap was sutured intermittently. Combined use of MMF, FK506, Prednisone and Zenopax was performed as post-operation immunosuppressive treatment. Clinical observations were made on the signs and symptoms of graft survival or rejection as well as blood FK506 concentrations and immunological indexes were tested in laboratory. Biopsies of graft were also made at 1 h, 4 h, 8 h, 7 d, 14 d and 30 d after operation., Results: The circulation of the graft was satisfactory, and the temperature and color of skin were normal. Primary healing of suture and hair growth about 0.8 cm in a month were observed. Skin Biopsies of every time had no found of hyperacute or acute rejection. The concentration of FK506 was maintained 20 mg/ml 1 month after the operation., Conclusion: Allograft transplantation with compound tissue of head skin flap and ears is a kind of effective and safe treatment in repairing huge tissue defect. Good tissue matching and combined use of currently available immunosuppressants can prevent hyperacute and acute rejection efficiently.

Objectives: To investigate the effects of Arg-Gly-Asp-Ser (RGDS) tetrapeptide on integrin signaling and apoptosis in fibronectin (FN) -stimulated hepatic stellate cells (HSCs)., Methods: 3H-thymidine incorporation, annexin-V/propidium iodide double-labeled flow cytometry (FCM) and transmission electron microscopy were employed to estimate the influence of RGDS on the proliferation and apoptosis of HSCs. And the adhesion rates were observed by toluidine blue colorimetric assay. The expression of focal adhesion kinase (FAK) mRNA and protein in HSCs was detected using RT-PCR and western blotting analysis, respectively., Results: RGDS tetrapeptide at the concentrations of 25 microg/ml, 50 microg/ml and 100 microg/ml inhibited the proliferation of HSCs and induced HSCs apoptosis in dose-dependent and time-dependent manners, with the apoptotic rates of 9.49%, 27.67%, 31.59%, and the necrotic rates of 3.47%, 5.38%, 9.10%, respectively. Both the rates were higher than those in FN group (apoptotic rate: 3.44%; necrotic rate: 2.39%), F=8.02, P<0.05. After adding RGDS tetrapeptide to HSCs for 2 hours, the adhesive inhibition rates were 8.82%, 29.41% and 45.59%, respectively, which were higher than that in FN group (F=20.58, P<0.01). After exposure of HSCs to RGDS tetrapeptide for 24 hours, FAK protein decreased, and FAK mRNA was down-regulated earlier, about 2 hours after exposure to RGDS tetrapeptide., Conclusion: These results suggest that RGDS tetrapeptide may induce apoptosis of HSC in both dose-dependent and time-dependent manners in vitro, which may be related to the disruption of cell matrix adhesion and down-regulation of FAK expression.

Objectives: To explore the effect of IL-18 on peripheral blood monocytes (PBMCs) from chronic hepatitis B (CHB) patients and HBV DNA released by HepG2.2.15 cells, which were transfected with the gene of HBV., Methods: PBMCs were isolated from 25 healthy persons and 25 CHB patients, which were co-cultured with HBcAg and IL-18 at different concentrations for 72 hours. The level of IFN-gamma in the culture supernatant of PBMCs was determined by ELISA. One patient' PBMCs were co-cultured for 96 hours with various concentrations of IL-18 and HepG2.2.15 cells which had been cultured for 24 hours, the supernatant was collected to detect HBV DNA level by PCR., Results: When PBMCs were stimulated by HBcAg and IL-18 at various concentrations, the levels of supernatant IFN-gamma in the CHB group were much higher than those in the normal control group (at 0.2ng/ml: t=11.7, P<0.01; at 1.0ng/ml: t=16.19, P<0.01; at 5.0ng/ml: t=20.12, P<0.01), especially when the PBMCs were stimulated by HBcAg, IL-18 and IL-12 (1313.20pg/ml+-187.76pg/ml vs. 390.75pg/ml+-43.23pg/ml, t=23.94, P<0.01). The IFN-gamma level in the patients who were stimulated by HBcAg alone was much lower than the levels in the patients who were stimulated by HBcAg and IL-18 at various concentrations, and which were lower than those in the patients stimulated by HBcAg, IL-12 and IL-18 at the same concentrations (light: t=2.2, P<0.05; moderate: t=2.97, P<0.05). The HBV DNA content in the supernatant of co-cultivation with HepG2.2.15 cells and PBMCs was much higher than that of the two kinds of cells stimulated by HBcAg and IL-18 at various concentrations or HBcAg, IL-18 and IL-12/IFN-a1b., Conclusion: IL-18 can improve the PBMCs from CHB patients to produce a great deal of IFN-gamma, so it has a good application prospect in two aspects: immunoregulatory effects and increasing the ability to kill the cells infected with virus.

Leptospirosis is a widely spread disease of global concern. Infection causes flu-like episodes with frequent severe renal and hepatic damage, such as haemorrhage and jaundice. In more severe cases, massive pulmonary haemorrhages, including fatal sudden haemoptysis, can occur. Here we report the complete genomic sequence of a representative virulent serovar type strain (Lai) of Leptospira interrogans serogroup Icterohaemorrhagiae consisting of a 4.33-megabase large chromosome and a 359-kilobase small chromosome, with a total of 4,768 predicted genes. In terms of the genetic determinants of physiological characteristics, the facultatively parasitic L. interrogans differs extensively from two other strictly parasitic pathogenic spirochaetes, Treponema pallidum and Borrelia burgdorferi, although similarities exist in the genes that govern their unique morphological features. A comprehensive analysis of the L. interrogans genes for chemotaxis/motility and lipopolysaccharide synthesis provides a basis for in-depth studies of virulence and pathogenesis. The discovery of a series of genes possibly related to adhesion, invasion and the haematological changes that characterize leptospirosis has provided clues about how an environmental organism might evolve into an important human pathogen.

Aim: To investigate the inhibiting role of Salvia miltiorrhiza monomer IH764-3 in proliferation and collagen synthesis and the effect on the expression of focal adhesion kinase in hepatic stellate cells (HSCs) stimulated by H2O2 in vitro, so as to provide evidences for preventing hepatic fibrosis., Methods: The effects of IH764-3 on the proliferation and collagen synthesis of HSCs were examined by 3H-TdR and 3H-proline incorporation assay, respectively. The expression of FAK mRNA was examined by RT-PCR in lysates of cultured HSCs., Results: The proliferation and collagen synthesis were inhibited in dose-dependent and time-dependent manners. The expressions of FAK mRNA in the IH764-3 (10 microg/ml, 20 microg/ml, 30 microg/ml, 40 microg/ml) groups were significantly lower than that in the control group., Conclusion: The present results demonstrate that IH764-3 can inhibit HSC proliferation and collagen synthesis. Meanwhile the mRNA transcription of FAK is reduced significantly, FAK as a signal molecule, can be involved in the effect of IH764-3. This may be one of the anti-fibrotic mechanisms of Salvia Miltiorrhiza.

Objective: To review the research progress and medical application of nano-materials., Methods: The literature review and comprehensive analysis, methods were used in this study., Results: The Nanotechnology is a typical crossing knowledge. It could be extensively applied in the fields of novel biomaterials, effective transmission of bioactive factor; the detection of functions for all vital organ systems, vascular circulation condition, the control of repair of burn trauma wounds will be monitored by the varied methods of nano technology combined with molecular biological engineering., Conclusion: The application of Nanotechnology will play important roles in clinical medicine, wound repair and basic research for the traditional Chinese medicine.

A main feature of the common mucosal immune system is that lymphocytes primed in one mucosal inductive site may home to distant mucosal effector sites. However, the mechanisms responsible for such cross-protection remain elusive. To address these we have used a model of local mucosal infection of mice with reovirus. In immunocompetent mice local duodenal priming protected against subsequent respiratory challenge. In the upper respiratory tract this protection appeared to be mainly mediated by specific IgA- and IgG2a-producing B cells, whereas ex vivo active effector memory CTL were found in the lower respiratory tract. In accordance with these findings, clearance of reovirus from the lower respiratory tract, but not from the upper respiratory tract, of infected SCID mice upon transfer of gut-primed lymphocytes depended on the presence of T cells. Taken together this study reveals that intestinal priming leads to protection of both the upper and lower respiratory tracts, however through distinct mechanisms. We suggest that cross-protection in the common mucosal immune system is mediated by trafficking of B cells and effector memory CTL.

Aim: To investigate the effects of IH764-3 on HSC apoptosis and the expression of caspase-3 protein in HSC apoptotic process., Methods: HSCs were cultured in medium with different IH764-3 doses(10 microg.mL(-1) 20 microg.mL(-1) 30 microg.mL(-1) 40 microg.mL(-1)) and without IH764-3 and HSC proliferation was quantitatively measured by (3)H-thymidine incorporation. The morphological changes of HSCs were observed with transmission electron microscope after exposure to the dose of 40 microg.mL(-1) of IH764-3 for 48 hr. The apoptosis rates were detected by annexin V/PI and TdT-mediated dUTP nick end labeling (TUNEL). The expression of caspase-3 protein was determined by flow cytometry., Results: (1) HSC proliferation rates induced with different IH764-3 doses (10 microg.mL(-1) 20 microg.mL(-1) 30 microg.mL(-1) 40 microg.mL(-1)) were significantly reduced compared with that of the control group (P<0.01). (2)With the doses above,IH764-3 dose-dependently produced HSC apoptosis rates of 6.7%(9.4%) 9.3%(21.6%) 15.1%(27.2%) and 19.0%(28.4%) respectively by annexin V and PI-labeled flow cytometry assay or TUNEL while it was only 2.3%(6.7%) in the control. (3) The expression of caspase-3 protein in IH764-3 groups was significantly higher than that of the control (P<0.05)., Conclusion: Within the dose range used in present study IH764-3 can inhibit HSC proliferation as well as enhance HSC apoptosis. Furthermore IH764-3 can significantly increase the caspase-3 protein expression.

Background & Aims: Recently, a number of animal models for different aspects of inflammatory bowel disease (IBD) have been developed. The aim of this study was to use one of these to determine whether particular, ostensibly innocuous, intestinal bacteria could provoke or exacerbate IBD., Methods: Conventionally reared C.B17 SCID mice were compared with germ-free and gnotobiotic mice, monoassociated with 1 of 5 intestinal bacteria, after transfer of CD45RB(high) CD4(+) T cells from conventionally reared congenic BALB/c mice. Recipient mice were monitored over 7-12 weeks for clinical signs of IBD, and tissues were analyzed by histology/flow cytometry for abnormal inflammation and CD4(+) T cell outgrowth., Results: Neither germ-free mice nor mice monoassociated with segmented filamentous bacteria, Ochrobactrum anthropi, a nonpathogenic mutant of Listeria monocytogenes, or Morganella morganii developed any signs of IBD. In contrast, mice monoassociated with Helicobacter muridarum displayed an accelerated development of IBD in 5-6 weeks compared with 8-12 weeks observed in conventionally reared mice. The outgrowth of CD4(+) T cells in spleen and large intestine of H. muridarum monoassociated mice, as well as in conventionally reared mice was significantly higher than that in the other monoassociated mice., Conclusions: Among the intestinal bacteria tested, H. muridarum can serve as a provocateur of IBD in this model.

Objective: To modify the design of the Achilles tendon-scar composite flap into the Z-shape and to apply it into clinic situations to correct the tendon-scar contracture after burn and other injures., Methods: According to degree of contracture and strephenopedia, the central limb of Z-shaped design lay in contracture line of posterior ankle area just over the Achilles tendon and extended 8 to 12 cm in length. Lateral limbs extended 5 to 8 cm and usual angles of the Z-shape was 60 degrees. The two tendon-scar composite flaps were made and slipped along the central limb between them. From March 1994 to August 1999, seven patients with Achilles tendon scar contracture were operated with this method., Results: Excellent relieving of the Achilles tendon-scar contractue was achieved. No such complications happened as ischemia and necrosis of the flaps., Conclusion: The Z-shaped tendon-scar composite flap is practical in clinics. It is characterized by abundant blood supply, easily procedure and reliable result.

We have identified LASS2, a previously unknown human homologue of the yeast longevity assurance gene LAG1. The LASS2 transcript is highly expressed in liver and kidney, which is very different from the expression of the previously identified human LAG1 homologue LAG1Hs-1. Radiation hybrid mapping studies indicated that LASS2 is located on chromosome 1q11. Yeast two-hybrid screening and glutathione S-transferase pull-down assays showed that the LASS2 protein interacts with several membrane-associated receptors or transporters. Furthermore, LASS2 protein was able to inhibit the colony formation of human hepatoma cells in vitro, which suggests that this gene may be involved in the regulation of cell growth.

Using a combination of hybridization of PAC to a cDNA library and RACE technique, we isolated a novel cDNA, designated as C17orf25 (Chromosome 17 open reading frame 25, previously named it HC71A), from the deletion region on chromosome 17p13.3. The cDNA encodes a protein of 313 amino acids with a calculated molecular mass of 34.8 kDa. C17orf25 is divided into 10 exons and 9 introns, spanning 23 kb of genomic DNA. Northern blot analysis showed that the mRNA expression of C17orf25 was decreased in hepatocellular carcinoma samples as compared to adjacent noncancerous liver tissues from the same patients. The transfection of C17orf25 into the hepatocellular carcinoma cell SMMC7721 and overexpression could inhibit the cell growth. The above results indicate that C17orf25 is a novel human gene, and the cloning and preliminary characterization of C17orf25 is a prerequisite for further functional analysis of this novel gene in human hepatocellular carcinoma.

As a member of the indigenous gut mucosal microbiota, segmented filamentous bacteria (SFB) colonize the guts of a variety of vertebrates and invertebrates. They are potent microbial stimuli of the gut mucosal immune system. In the small intestines of mice and rats, it has been observed that SFB are absent during the suckling period and appear in high numbers shortly after weaning, then quickly retreat to the cecum and large intestine. In this study, we explored whether this microecological phenomenon resulted from the interaction between SFB and the passively acquired maternal mucosal immunity and/or the actively acquired mucosal immunity. We set up a mouse model by reciprocal crossings and backcrossings of SFB-monoassociated, formerly germ-free, immunocompetent (+/+) BALB/c mice and immunodeficient (scid/scid) mice to produce pups which are either immunocompetent (scid/+) or immunodeficient (scid/scid) and are born either to immunocompetent (scid/+) mothers or to immunodeficient (scid/scid) mothers. We monitored the number of SFB on the mucosa of the small intestine in the four different groups of mice after birth, as well as the level of passively acquired antibodies, the active gut mucosal immune responses, and immunoglobulin A (IgA) coating of SFB in the gut. The results showed that, irrespective of whether the pups were scid/scid or scid/+, SFB could be found earlier on the mucosa of the small intestine in pups born to scid/scid mothers, appearing from day 13 and rapidly reaching a climax around weaning time on day 28, compared to the significantly delayed colonization in the pups of scid/+ mothers, starting from day 16 and peaking around days 28 to 32. After the climax, SFB quickly declined to very low levels in the small intestines of scid/+ pups of either scid/scid mothers or scid/+ mothers, whereas they remained at high levels in scid/scid pups at least until day 70, the last observation time in this study. The dynamic changes in SFB colonization of the small intestines of the different groups of pups may be related to the dynamic changes in the levels of SFB coated with secretory IgA (sIgA), which resulted from the significantly different levels of sIgA obtained from the mothers' milk during the suckling period and, later, of self-produced sIgA in the small intestine. Nevertheless, it is evident that the timing, localization, and persistence of colonization of the neonatal gut by SFB depends on the immune status of both mothers and pups.

Segmented filamentous bacteria (SFB) are autochthonous bacteria inhabiting the intestinal tracts of many species, including humans. We studied the effect of SFB on the mucosal immune system by monoassociating formerly germfree C3H/HeN mice with SFB. At various time points during 190 days of colonization, fragment cultures of small intestine and Peyer's patches (PP) were analyzed for total immunoglobulin A (IgA) and SFB-specific IgA production. Also, phenotypic changes indicating germinal center reactions (GCRs) and the activation of CD4(+) T cells in PP were determined by using fluorescence-activated cell sorter analyses. A second group of SFB-monoassociated mice was colonized with a gram-negative commensal, Morganella morganii, to determine if the mucosal immune system was again stimulated and to evaluate the effect of prior colonization with SFB on the ability of M. morganii to translocate to the spleen and mesenteric lymph nodes. We found that SFB stimulated GCRs in PP from day 6 after monoassociation, that GCRs only gradually waned over the entire length of colonization, that natural IgA production was increased to levels 24 to 63% of that of conventionally reared mice, and that SFB-specific IgA was produced but accounted for less than 1.4% of total IgA. Also, the proportion of CD4(+), CD45RBlow T cells, indicative of activated cells, gradually increased in the PP to the level found in conventionally reared mice. Secondary colonization with M. morganii was able to stimulate GCRs anew, leading to a specific IgA antibody response. Previous stimulation of mucosal immunity by SFB did not prevent the translocation of M. morganii in the double-colonized mice. Our findings generally indicate that SFB are one of the single most potent microbial stimuli of the gut mucosal immune system.

Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors. GV1, GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex. Using pSV2-beta-galactosidase as a reporter gene, it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro. In vivo experiments, exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO, human malignant melanoma A375 and human hepatoma graft in nude mice. This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice. These results are correlated with the relevant receptors (flt-1, flk-1/KDR) expression on the targeted cells and tissues.

Cell-free yeast extract has been successfully used to catalyze the enzymatic formation of 11 out of the 14 naturally occurring modified nucleotides in yeast tRNAPhe(anticodon GAA). They are m2G10, D17, m22G26, Cm32, Gm34,psi39, m5C40, m7G46, m5C49, T54 andpsi55. Only D16, Y37 and m1A58 were not formed under in vitro conditions. However, m1G37was quantitatively produced instead of Y37. The naturally occurring intron was absolutely required for m5C40formation while it hindered completely the enzymatic formation of Cm32, Gm34and m1G37. Enzymatic formation of m22G26,psi39, m7G46, m5C49, T54 andpsi55were not or only slightly affected by the presence of the intron. These results allow us to classify the different tRNA modification enzymes into three groups: intron insensitive, intron dependent, and those requiring the absence of the intron. The fact that truncated tRNAPheconsisting of the anticodon stem and loop prolonged with the 19 nucleotide long intron is a substrate for tRNA: cytosine-40 methylase demonstrates that the enzyme is not only strictly intron dependent, but also does not require fully structured tRNA.

A patient with an infection with the L-form of Staphylococcus aureus occurring in burn wounds is reported. The authors conclude that the infection was attributable to a decrease in the host immunological functions and repeated administration of antibiotics resulting in the development of bacterial L-forms.

This is the first report to describe the presence of an HBV-like DNA sequence in two hepatoma and their tumor surrounding liver tissues, one precancerous and one non-malignant liver tissue of ducks collected from Qidong County of China. The HBV-like sequences were either in an episomal form of 3.2 kb or in an integrated form of various sizes, while the DHBV DNA sequences (3.0 kb) were either present or absent in these tissues and in different size pattern. Furthermore, there was no evidence of cross-hybridization between HBV-like DNA sequences in duck and DHBV DNA. A 3.2 kb HBV-like DNA sequence has been cloned from one duck hepatoma (40 K), designated as pDKHBV. The 3218 bp full-length nucleotide sequence of this clone has been determined, which had no apparent homology with Duck Hepatitis B Virus (DHBV) genome, but was highly homologous to human HBV adw2 subtype (99.0%). The sequence was composed of four open reading frames for HBV gene Pre-S/S, X, C and P respectively. In addition to multiple sites of point mutation, one nt and two nt deletion were detected downstream the initiation codon of Pre-S1 gene and at the 3' end of C gene respectively, thereby suggesting a frameshift mutation in Pre-S gene and C gene. Based on these results, it was implicated that an unusual HBV variant might exist in Qidong County, which might have transmitted into duck through some yet unknown mechanisms. The possibility from exogenous contamination could be thus excluded.

Acute volume loading produced by autogenous reinfusion of ascitic fluid provides an ideal volume expansion model for studying hormonal regulation and it was carried out in 10 cirrhotic patients with massive ascites. The basal plasma ANP level in the cirrhotic patients with ascites was 1776.00 +/- 160.72 ng/L, which was significantly elevated as compared with the level in normal controls (378.36 +/- 39.58 ng/L, P less than 0.01), PRA was significantly higher in patients with ascites (5.13 +/- 0.18 micrograms/l/h) than in healthy volunteers (1.46 +/- 0.31 micrograms/l/h, P less than 0.01). During reinfusion of ascitic fluid there was a significant natriuresis and diuresis; ANP rose from a basal mean value to a peak value of 2166.00 +/- 195.70 ng/l (P less than 0.05) at periinfusion. Subsequently, ANP dropped at 1 hour after infusion (1819.00 +/- 165.92ng/L, P less than 0.05), PRA dropped progressively from a mean basal level to that of 2.48 +/- 0.58 micrograms/l/h (P less than 0.05) at periinfusion. These data demonstrate that there is no evidence for absolute deficiency of ANP in cirrhosis with ascites. The immediate diuresis and natriuresis were associated with a rise in ANP, but the sustained renal consequences may be possibly connected with suppression of RAAS.

From a subtracting cDNA library constructed from normal liver versus human primary hepatic cancer (PHC) a cDNA clone pG8 was isolated. Using it as a probe, RNA extracted from one human liver and 9 PHC samples were analyzed by Northern hybridization. As expected, its mRNA was highly expressed in liver; however, the expression was strikingly suppressed in PHC. Only weak signal was observed in 2 out of 9 PHC, while no signal was detectable in the other 7 samples. Utilizing pG8 as a probe, DNA from the same PHC specimens was analyzed after MspI digestion and Southern hybridization. Deletion of DNA fragment was observed in 4 out of 9 samples. In further study of cancer and non-cancerous liver from other 7 PHC patients, similar deletion of DNA fragments in cancer was observed in 4 out of 7 samples. After sequencing of the clone of 572 bp, it was unexpectedly found that pG8 was completely homologous to the coding sequence of transthyretin, TTR gene, as TTR (or prealbumin) gene has been known to be linked to a hereditary disorder, familial amyloidosis (FAP), and related to thyroxine transport and binding to retinol-RBP (the retinol binding protein) complex. This is the first report of a study on TTR in human primary hepatic cancer. Since TTR gene was strikingly suppressed in mRNA expression and possibly defective in its gene structure, it was strongly implicated that TTR might be an important gene marker or a candidate of anti-oncogene for human PHC. The biological activity of TTR gene is under study.

The transfection of NIH 3T3 cells was performed with DNAs from 2 duck primary hepatic carcinomas (DHC 40K, 9K) and 1 tumor-adjacent liver tissue (TAL, 9N). Transfectants were found from 40K, 9K and 9N DNAs. The secondary transfectants were obtained after transfection of RAT-1 cells with DNAs from primary transfectants. After hybridization with Ha-ras, Ki-ras, N-ras and mht oncogenes, it was found that duck mht (5.2 and 3.2 kb EcoRI fragments) and duck Ha-ras (3.4 kb EcoRI fragments) were present in all these transformants. This is the first report on transforming genes in duck primary hepatic cancer as well as tumor-adjacent liver tissue.

DNA was extracted from NIH 3T3 cells transformed with DNAs from human primary hepatic cancer (PHC) and Hepatoma 7402 cell line. The transformant DNA was analyzed by Southern transfer and hybridization with 32P-labeled probes of various oncogenes. The EcoRI 7.2 and 9.0 kb bands characteristic of human N-ras gene were identified in transformed NIH 3T3 cells derived both from PHC and 7402 DNA. The BamHI 6.6 kb band characteristic of human c-Ha-ras I was present only in 7402 transformants, but not in PHC transformants. Using 35S-methionine incorporation, immunoprecipitation with anti-p21 monoclonal antibodies, SDS-PAGE and autoradiography, it was demonstrated that p21 synthesis was remarkably enhanced in 7402 cells as well as in transformed cells derived from both 7402 and PHC DNA. Taking the data together, it strongly implies that N-ras is one of the transforming genes for human liver cancer.

Hepatitis B virus (HBV) DNA in leucocytes from 50 hepatitis patients with various patterns of HBV serological markers and serum HBV DNA and 13 normal controls were examined by Southern blot hybridization with 32P-labeled 3.2 Kb HBV DNA. A free form of HBV DNA was observed in leucocytes of 8 patients, 7 of whom were positive for serum HBeAg, and in 6 patients an integrated form of HBV DNA was identified. HBV DNA was not identified in leucocytes from 13 normal controls. The free form of HBV DNA in leucocytes existed as a heterogeneous smear from 2.0 to 3.2 Kb, similar to the pattern in liver and hepatocellular carcinoma cells but different from serum HBV DNA in which the 3.2 Kb band was absent. The banding pattern of the integrated form of HBV DNA in leucocytes varied among different patients. During preparation of white blood cells and purification of HBV DNA probes, it was important to remove plasma contamination and traces of pBR322, respectively. The presence of extrachromosomal DNA sequences partially homologous to pBR322 could cause false results. The presence of a free and integrated form of HBV DNA in leucocytes is important for explaining the biology of HBV, the harbouring and replication sites of extrahepatic origin, the mechanism of recurrent infection, and the rationale of the treatment of hepatitis B.