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We report here the expression of a bacterial sphingomyelinase in mammalian cells as a functionally active form. A chimeric Pseudomonas sphingomyelinase fused with the lysosomal sorting motif of lysosomal acid phosphatase was sorted to lysosomes in mammalian cells. As expected, the chimeric SMase hydrolyzed sphingomyelin in vivo to produce ceramide, part of which was converted to glucosylceramide.

Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebrafish whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. Ionomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebrafish.

Background: Full-thickness skin defects with exposed bone are often hard to heal. The lack or delayed re-vascularization is considered one of the major causes, and the periosteum is also suggested to have an important role in tissue regeneration., Materials and Methods: Full-thickness skin defect wounds with exposed bone were made in the parietal region of Wister rats. The periosteum of the exposed parietal bone was removed in the periosteum-lacking group, but maintained in the control group (periosteum-intact group). The wound was covered by an artificial dermis made of collagen. The wound healing process was histologically compared. Double immunostaining of alpha-smooth muscle actin (SMA) and von Willebrand factor (vWF) was used for re-vascularization examination, and the blood vessel density in the artificial dermis was quantified., Results: The density of the blood vessels in the uninjured parietal tissue was approximately 80 vessels/mm(2). To reach this density, 7 and 21 days were required for the control (periosteum-intact) and the periosteum-lacking groups, respectively. This coincided with complete revascularization, fibroblast migration and the reentry of blood vessels to the upper layer of the wound were observed., Conclusion: The described results support the importance of the periosteum in the full-thickness skin defect healing process.

Most cellular events are regulated by protein phosphorylation mediated by protein kinases, whose malfunction is involved in the etiology of various disorders. The elucidation of the biochemical properties of the protein phosphorylation reaction will lead not only to a better understanding of the signal transduction mechanism, but also to developing new therapeutic agents. In this review, we briefly summarize the technologies to detect or characterize protein kinases with special emphasis on recently developed and/or commercially available techniques.

Background: The aim of this study was to explain the role of monocyte chemoattractant protein-1 (MCP-1) in biliary atresia (BA)., Methods: Concentrations of serum MCP-1 and collagen type IV were measured in 38 patients with BA by using commercially available kits. MCP-1 was also assessed in liver biopsy specimens by using immunohistochemistry. Subjects were classified into groups. Group 1 comprised BA patients with normal liver function (n = 13), group II comprised BA patients with moderate liver dysfunction (n = 18), group III comprised BA patients older than 20 years awaiting liver transplantation (n = 7), and the control group comprised age-matched patients without evidence of liver disease (n = 23)., Results: Serum MCP-1 levels were significantly increased in group II compared with group I (P < .0001) and the control group (P < .0001). Serum MCP-1 levels in group III were lower than in the control group (P < .0001). There was a significant linear correlation between serum MCP-1 levels and type IV collagen levels in group II. Group II subjects with portal hypertension (PH) had higher MCP-1 levels than those without PH (P = .0009). Biopsy specimens showed MCP-1 was expressed mainly on biliary epithelial cells, vascular endothelial cells, and hepatocytes in group II., Conclusions: These findings suggest that MCP-1 probably plays a significant role in the development of progressive liver fibrosis in BA.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation. A chimera mutant, a fusion of the N-terminal domain of CaMKP and the catalytic domain of PP2Calpha, exhibited similar substrate specificity to CaMKP but not to PP2Calpha, suggesting that the N-terminal region of CaMKP is crucial for its unique substrate specificity. Point mutations at Arg-162, Asp-194, His-196, and Asp-400, highly conserved amino acid residues in the catalytic domain of PP2C family, resulted in a significant loss of phosphatase activity, indicating that these amino acid residues may play important roles in the catalytic activity of CaMKP. Although CaMKP(1-412), a C-terminal truncation mutant, retained phosphatase activity, it was found to be much less stable upon incubation at 37 degrees C than wild type CaMKP, indicating that the C-terminal region of CaMKP is important for the maintenance of the catalytically active conformation. The results suggested that the N- and C-terminal sequences of CaMKP are essential for the regulation and stability of CaMKP.

We studied 247 strains of Proteus mirabilis collected during the 6 months from November 2003 to April 2004 from 12 clinical laboratories in the Kinki region of Japan for the production of extended-spectrum beta-lactamase (ESBL). Eighteen strains (7.3%) showed MICs for cefpodoxime of > or = 2 microg/mL and 13 strains (5.2%) were positive for the double-disk synergy test. Susceptibility depended on genotype. MICs for cefepime, cefozopran, and cefpirome were high (> or = 8 microg/mL), and that for ceftazidime was low (0.12-0.5 microg/mL). Meropenem showed the lowest MIC (< or = 0.03-0.25 microg/mL) of the calbapenems, while other calbapenems showed somewhat higher values (0.5-2 microg/mL). The MIC of tazobactam/piperacillin was also relatively low (< or = 0.25-1 microg/mL). Analysis of the ESBL genotype by the polymerase chain reaction showed that 12 of 13 strains were CTX-M2 types. CTX-M9 was detected in a single laboratory. The clinical background showed 5 strains in urine samples. Twelve of 13 strains were detected in patients with minimal devices use. No symptoms were found in most cases of established syndrome. Analysis of PCR fingerprint profiles of random amplified polymorphic DNA patterns showed that 6 of 7 strains from hospital 1 showed the same pattern, and 5 of 5 strains from hospital 13 showed the same pattern, suggesting the nosocomial spread of P. mirabilis in each hospital.

Aim: We showed previously that adult small bowel could be transplanted successfully in rats without vascular reconstruction by removing the graft serosa. In this study, we assessed if granulocyte colony-stimulating factor (G-CSF) or basic fibroblast growth factor (bFGF) could improve graft survival in the same rat model., Method: A 10-mm-long adult small bowel graft from an adult 12-week-old Lewis rat was transplanted into a pouch created in the omentum of a 5-week-old Lewis rat (syngeneic bowel transplantation [SBTx], n = 49). Graft serosa was removed just before SBTx in the serosectomy group (n = 29) and left intact in the nonserosectomy group (n = 20). Each group was divided into 3 subgroups (sG): sG-1 had no G-CSF or bFGF; sG-2 had daily subcutaneous injections of G-CSF; and sG-3 had continuous infusion of bFGF around the graft in the omentum. All grafts were harvested 14 days after SBTx and studied histologically. A mucosal surface expansion score (MSES) was used where 0 = no mucosa on the graft, 1 = mucosa on one fourth of the graft, 2 = mucosa on one half of the graft, 3 = mucosa on three fourths of the graft, and 4 = mucosa on the whole graft. The density of CD34-positive capillaries per 1000 nuclei was also measured., Results: Serosectomy group MSES were significantly higher than nonserosectomy group MSES indicating that grafts survived (P < .0001). CD34-positive capillaries in serosectomy group subgroups for mucosa were 103.9 +/- 34.2, 130.2 +/- 52.0, and 132.3 +/- 37.7, respectively; for muscle, 74.4 +/- 38.0, 86.2 +/- 32.9, and 82.4 +/- 30.3, respectively; and for omentum, 73.8 +/- 30.1, 151.3 +/- 60.3, and 140.0 +/- 49.0, respectively. Mucosal surface expansion score and overall CD34-positive capillaries for sG-2 and sG-3 were significantly higher than for sG-1 (both, P < .05)., Conclusion: Our results suggest that G-CSF and bFGF enhance angiogenesis and mucosal surface expansion.

To investigate expression profiles of the entire family of protein tyrosine kinases (PTKs), we attempted to generate an antibody that detects a variety of PTKs. For production of the antibody, antigenic peptides corresponding to amino acid sequences of a highly conserved region (subdomain VIB) of PTKs were synthesized and immunized to BALB/c mice. Among various antigens, a peptide with 11 amino acids, CYVHRDLRAAN, efficiently produced a polyclonal antibody with a broad cross-reactivity to PTKs. We established a hybridoma cell line producing a monoclonal antibody, YK34, which appeared to cross-react with at least 68 PTKs in the human genome, as evidenced by its reactivity with the recombinant Src tyrosine kinases whose subdomain VIB had been replaced by those of the other PTKs. When differentiation of HL-60 cells was induced by 12-O-tetradecanoylphorbol-13-acetate, on Western blotting we observed significant changes in immunoreactive bands with YK34 in HL-60 cell extracts along with changes in the morphology of the cells. These results suggest that the YK34 antibody will be a powerful tool for analysis of a variety of cellular PTKs.

Purpose: The aim of this study was to assess whether adult small bowel grafts (ASBGs) can survive transplantation without vascular reconstruction if graft serosectomy (SS) is performed., Methods: Syngeneic ASBG transplants were performed in 85 Lewis rats. The entire serosa was removed just before transplantation in the SS group (n = 50) and left intact in the nonserosectomy group (n = 35). Transplanted ASBG was harvested 1, 3, 5, 7, 14, 21, or 28 days after transplantation and studied using staining with hematoxylin-eosin, immunohistochemistry for protein gene product 9.5, S-100, CD34 and vascular endothelial growth factor (VEGF), and quantification of VEGF messenger RNA (mRNA). Adult small bowel graft viability was assessed blindly using a mucosal surface expansion score (0, no mucosa; 1, mucosa on one fourth of graft; 2, mucosa on one half of graft; 3, mucosa on three fourths of graft; and 4, circumferential mucosa on graft)., Results: No rejection was identified in any ASBG. Average mucosal surface expansion score and VEGF mRNA expression were significantly higher in the SS group (both P < .01). Vascular endothelial growth factor protein was detected in enterocytes from day 3 posttransplant in the SS group. Distribution of protein gene product 9.5 and S-100 was normal in SS-group ASBG., Conclusions: Our results suggest that SS allows VEGF mRNA and, subsequently, VEGF protein in ASBG to be induced very soon after transplantation, which may contribute to the survival of ASBG transplanted without vascular reconstruction.

The chymotryptic fragment of Ca2+/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active enzyme that phosphorylates a variety of protein substrates in vitro. Although 30K-CaMKII is an often used and powerful tool for protein phosphorylation, the efficient production of catalytically active 30K-CaMKII in Escherichia coli has not yet been successfully realized, probably due to its toxicity in host cells. In this study, we found that a high-level expression of 30K-CaMKII as an insoluble form was attained when the N-terminal 43 amino acid residues of Xenopus CaMKI were fused to the N-terminal end of 30K-CaMKII (CX-30K-CaMKII). The inactive CX-30K-CaMKII thus expressed in E. coli was reactivated by simple denaturation/renaturation processes and purified on a Ni2+-chelating column. The renatured CX-30K-CaMKII exhibited specific activity similar to that of rat brain CaMKII, and phosphorylated various proteins such as histones, myosin light chain, myelin basic protein, and synapsin I, as in case of 30K-CaMKII or purified CaMKII. Thus, CX-30K-CaMKII, an autonomous CaMKII, can be obtained with a simple procedure using E. coli expression system.

GM1a [Gal beta1-3GalNAc beta1-4(NeuAc alpha2-3)Gal beta1-4Glc beta1-1Cer] is known to support and protect neuronal functions. However, we report that alpha-linolenic acid-containing GM1a (C18:3-GM1a), which was prepared using the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase, induced apoptosis in neuronal cells. Intranucleosomal DNA fragmentation, chromatin condensation, and caspase activation, all typical features of apoptosis, were observed when mouse neuroblastoma Neuro2a cells were cultured with C18:3-GM1a but not GM1a containing stearic acid (C18:0) or oleic acid (C18:1). The phenotype of Neuro2a cells induced by C18:3-GM1a was similar to that evoked by lyso-GM1a. However, lyso-GM1a caused a complete disruption of lipid microdomains of Neuro2a cells and hemolysis of sheep erythrocytes, whereas C18:3-GM1a did neither. C18:3-GM1a, but not lyso-GM1a, was found to be abundant in lipid microdomains after the removal of loosely bound GM1a by BSA. The activation of stress-activated protein kinase/c-Jun N-terminal kinase in Neuro2a cells was observed with lyso-GM1a but not C18:3-GM1a. These results indicate that the mechanism of apoptosis induced by C18:3-GM1a is distinct from that caused by lyso-GM1a. This study also clearly shows that fatty acid composition of gangliosides significantly affected their pharmacological activities when added to the cell cultures and suggests why naturally occurring gangliosides do not possess polyunsaturated fatty acids as a major constituent.

Three hundred seventy five isolates of Streptococcus pneumoniae were collected from 14 medical institutions in the Kinki region of Japan between November 2003 and February 2004. We determined the minimum inhibitory concentration (MIC) of penicillin G (PCG) and 25 of other antimicrobial agents against these isolates according to the National Committee for Clinical Laboratory Standards (NCCLS). Overall, 71.5% of all isolates were resistant to PCG (intermediate and resistant categories were 51.7% and 19.8%, respectively). For the carbapenems and penem, the rank order of activity was PAPM (MIC90, 0.12 microg/ml) > IPM (0.25 microg/ml) > MEPM (0.5 microg/ml) = FRPM (0.5 microg/ml). For the cephems, the overall rank order of activity was CPR (MIC90, 0.5 microg/ml) = CDTR (0.5 microg/ml) > CTRX (1 microg/ml) = CTX (1 microg/ml) = CZOP (1 microg/ml) = CFPN (1 microg/ml). Rank order activity for six of fluoroquinolones was TFLX = MFLX (MIC90, 0.25 microg/ml) > GFLX (0.5 microg/ ml) = SPFX (0.5 microg/ml) > LVFX (1 microg/ml) > PZFX (4 microg/ml). The rate of resistance to fluoroquinolones per the NCCLS criteria were very low, ranging from 0.7% to 2.6%. Rate of resistance to other antimicrobiotics were CAM, 77.0%; CLDM, 41.7%; TEL, 0%; VCM, 0%; ST, 32.7%, and CP, 21.4%.

Long-term histopathologic changes after bladder augmentation (BA) in rats using living-related partial bladder transplantation (LPBTx) or conventional ileocystoplasty (ICP) were compared. In this study, BA (n = 37), LPBTx (n = 18), and ICP (n = 19) were performed in 16-wk-old Lewis rats. Five donors and seven nontransplanted normal Lewis rats (controls) were also studied. Rats that survived >10 mo after BA were killed after blood biochemistry and neobladder imaging. Harvested bladders were examined with hematoxylin and eosin and proliferating cell nuclear antigen (PCNA). When the rats were killed, there were 16 rats in the LPBTx group and 12 rats in the ICP group; ICP rats were significantly smaller than LPBTx rats (p < 0.05). Mean duration of follow-up for the LPBTX group was 17.3 mo, for the ICP group was 13.7 mo, for the donor group was 16.1 mo, and for the control group was 19.7 mo. Mean serum pH in the LPBTx group was 7.41 +/- 0.78 and in the ICP group was 7.25 +/- 0.38. Mean base excess in the ICP group was significantly lower than in the LPBTx group (p < 0.05). Incidence of bladder calculi in the LPBTx group (6.3%) was significantly lower than in the ICP group (33.3%; p < 0.05). There was no dysplasia/malignancy/increase in PCNA in the LPBTx group. PCNA increased in the ICP group, compared with controls (p < 0.05); two (16.7%) of 12 of ICP rats had dysplasia with mitosis. Bladder capacity increased in LPBTx and ICP compared with controls (both p < 0.05). We hope to show that BA using LPBTx may result in a neobladder with fewer complications than BA using ICP; LPBTx may also decrease the risk for malignancy.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a unique protein phosphatase that specifically dephosphorylates and regulates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs). To clarify the physiological significance of CaMKP, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate aldolase as major binding partners of CaMKP in a soluble fraction of rat brain using the two-dimensional far-Western blotting technique, in conjunction with peptide mass fingerprinting analysis. We analyzed the affinities of these interactions. Wild type CaMKP-glutathione S-transferase (GST) associated with GAPDH in a GST pull-down assay. Deletion analysis suggested that the N-terminal side of the catalytic domain of CaMKP was responsible for the binding to GAPDH. Further, anti-CaMKP antibody coimmunoprecipitated GAPDH in a rat brain extract. GAPDH was phosphorylated by CaMKI or CaMKIV in vitro; however, when CaMKP coexisted, the phosphorylation was markedly attenuated. Under these conditions, CaMKP significantly dephosphorylated CaMKI and CaMKIV, which had been phosphorylated by CaMK kinase, whereas it did not dephosphorylate the previously phosphorylated GAPDH. The results suggest that CaMKP regulates the phosphorylation level of GAPDH in the CaMKP-GAPDH complex by dephosphorylating and deactivating CaMKs that are responsible for the phosphorylation of GAPDH.

We studied the antimicrobial susceptibility of AmpC beta-lactamase-producing Escherichia coli isolates collected at ten medical institutions in the Kinki area of Japan during a 6-month period (November 2002 through April 2003). Of 2845 E. coli isolates tested, 29 (1.0%) showed a minimum inhibitory concentration (MIC) for cefazolin of more than 8 microg/ml and were three-dimensional extract test positive. In standard inoculum susceptibility tests against these 29 strains, the MIC90s for the four carbapenems tested ranged from 0.06 microg/ml to 0.5 microg/ml, and these compounds were more active than the other beta-lactams, with meropenem being the most active. The MIC90s for beta-lactams, except carbapenems, ranged from 4 microg/ml to 32 microg/ml, with cefepime being the most active. In high inoculum susceptibility tests against these strains, the MIC90s for the four carbapenems and cefepime were 8 microg/ml or less, and these compounds were more active than other beta-lactams. The MIC90s for beta-lactams, except carbapenems and cefepime, were 32 microg/ml or more. The MIC90s for the five quinolones tested ranged from 4 microg/ml to 16 microg/ml, and the order of increasing susceptibility was ciprofloxacin > levofloxacin, gatifloxacin and pazufloxacin > prulifloxacin.

To investigate protein kinases expressed in Lotus japonicus, a cDNA expression library of the root-nodule of L. japonicus was immunologically screened with monoclonal antibodies directed to a highly conserved region in protein serine/threonine kinases (Ser/Thr kinases). Among 178 positive clones obtained from the lambdaZAPII cDNA library, 164 clones were found to encode novel proteins possessing the subdomain VIB sequences characteristic of Ser/Thr kinases. By phylogenetic analysis on the basis of deduced amino acid sequences, the isolated clones could be classified into five different families of Ser/Thr kinases : the SnRK family, GSK-3 family, Ndr kinase family, Ark family, and receptor kinase family. These results suggest that this expression cloning using the kinase-specific antibodies will provide new clues for investigations of a wide variety of known and novel protein kinases in higher plants.

Purpose: Is laparoscopic injection of 2-octyl-cyanoacrylate tissue adhesive (Dermabond: Db) into the inguinal hernia sac (IHS) effective for inguinal hernia repair?, Methods: Thirty male 4-week-old Lewis rats were used as subjects for this study. In the right Db (R-Db) group (n = 10), a fine catheter was passed through an 18-guage indwelling intravenous cannula inserted in the right lower quadrant, and 0.2 mL Db was injected into the right IHS under laparoscopic control. The left side was not treated. Both IHSs were treated in the bilateral Db (B-Db) group (n = 10). In the no Db (N-Db or control) group (n = 10), only laparoscope insertion was performed. Herniography was performed before death. B-Db and N-Db rats were mated 50 days after treatment. Half of all rats were killed 2 months after treatment and the remaining half 12 months after treatment., Results: All rats survived until killing. Macroscopic findings postdeath confirmed herniography results; treated IHS were closed, and untreated IHS were patent. There were minor adhesions in 3 of 20 treated rats. Sperm were identified in the vaginas of all mated rats., Conclusions: These results suggest that our new technique is simple, safe, and reliable as an alternative to standard operative repair for inguinal hernia.

A total of 19,753 strains of gram-negative rods collected during two 6-month periods (October 2000 to March 2001 and November 2001 to April 2002) from 13 clinical laboratories in the Kinki region of Japan were investigated for the production of metallo-beta-lactamases (MBLs). MBLs were detected in 96 (0.5%) of the 19,753 isolates by the broth microdilution method, the 2-mercaptopropionic acid inhibition test, and PCR and DNA sequencing analyses. MBL-positive isolates were detected in 9 of 13 laboratories, with the rate of detection ranging between 0 and 2.6% for each laboratory. Forty-four of 1,429 (3.1%) Serratia marcescens, 22 of 6,198 (0.4%) Pseudomonas aeruginosa, 21 of 1,108 (1.9%) Acinetobacter spp., 4 of 544 (0.7%) Citrobacter freundii, 3 of 127 (2.4%) Providencia rettgeri, 1 of 434 (0.2%) Morganella morganii, and 1 of 1,483 (0.1%) Enterobacter cloacae isolates were positive for MBLs. Of these 96 MBL-positive strains, 87 (90.6%), 7 (7.3%), and 2 (2.1%) isolates carried the genes for IMP-1-group MBLs, IMP-2-group MBLs, and VIM-2-group MBLs, respectively. The class 1 integrase gene, intI1, was detected in all MBL-positive strains, and the aac (6')-Ib gene was detected in 37 (38.5%) isolates. Strains with identical PCR fingerprint profiles in a random amplified polymorphic DNA pattern analysis were isolated successively from five separate hospitals, suggesting the nosocomial spread of the organism in each hospital. In conclusion, many species of MBL-positive gram-negative rods are distributed widely in different hospitals in the Kinki region of Japan. The present findings should be considered during the development of policies and strategies to prevent the emergence and further spread of MBL-producing bacteria.

Background: Transforming growth factor-beta (TGF-beta) has been implicated in the development of renal fibrosis induced by unilateral ureteral obstruction (UUO). However, there is little information on signaling pathways mediating TGF-beta activity involved in molecular and cellular events leading to renal fibrosis induced by UUO. In this study, we sought to determine whether Smad3, a major signaling component of TGF-beta, mediated renal fibrosis induced by UUO., Methods: Renal fibrosis, inflammation, and apoptosis induced by UUO were macroscopically and histologically compared between wild-type mice and Smad3 null mice., Results: Gross appearance of the kidney after UUO showed relatively intact kidney in Smad3 null mice [Smad3(-/-) mice] when compared with that of wild-type mice [Smad3(+/+) mice]. Renal interstitial fibrosis based on the interstitial area stained with Aniline-blue or Sirius red solution was significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Deposition of type I and type III collagens were also significantly reduced in the obstructed kidney of Smad3(-/-) mice. In addition, the numbers of myofibroblasts, macrophages, and CD4/CD8 T cells infiltrated into the kidney after UUO were significantly attenuated in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Furthermore, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining after UUO showed significantly reduced number of tubular apoptotic cells in the obstructed kidney of Smad3(-/-) mice when compared with that of Smad3(+/+) mice. Endogenous Smad pathway was activated in the obstructed kidney after UUO in wild-type mice as judged by the increase of phosphorylated Smad2 or phosphorylated Smad2/3-positive cells in renal interstitial area., Conclusion: Smad3 deficiency attenuated renal fibrosis, inflammation, and apoptosis after UUO, suggesting that Smad3 was a key molecule mediating TGF-beta activity leading to real fibrosis after UUO.

Several lines of evidence suggest that neutral ceramidase is involved in the regulation of ceramide-mediated signaling. Recently, the enzymes from mouse and rat were found to be localized at plasma membranes as a type II integral membrane protein, occasionally being detached from the cells after proteolytic processing of the NH(2)-terminal anchoring region (Tani, M., Iida, H., and Ito, M. (2003) J. Biol. Chem. 278, 10523-10530). We report here that conserved hydrophobic amino acid residues in the COOH-terminal tail are indispensable for the correct folding and localization, and enzyme activity of neutral ceramidase. Truncation of four, but not three, amino acid residues from the COOH terminus of rat neutral ceramidase resulted in a complete loss of enzyme activity as well as cell surface expression in HEK293 cells. Point mutation analysis revealed that Ile(758), the 4(th) amino acid residue from the COOH terminus, and Phe(756) are essential for the enzyme to function. The truncated and mutated enzymes were found to be retained in the endoplasmic reticulum (ER) and rapidly degraded without transportation to the Golgi apparatus. Treatment of the cells expressing the aberrant COOH-terminal enzyme with MG-132, a specific inhibitor for the proteasome, increased the accumulation of the enzyme in the ER, indicating that the misfolded enzyme was degraded by the proteasome. It was also found that the COOH-terminal tail was indispensable for the enzyme activity and correct folding of the prokaryote ceramidase from Pseudomonas aeruginosa, indicating that the importance of the COOH-terminal tail of the enzyme has been preserved through evolution.

In order to investigate protein kinases expressed in the different developmental stages of Xenopus laevis, recently developed expression cloning was carried out. When two different expression libraries, Xenopus oocyte and Xenopus head (embryonic stage 28/30) cDNA libraries, were screened by kinase-specific monoclonal antibodies, cDNA clones for various known and novel protein serine/threonine kinases (Ser/Thr kinases) were isolated. In addition to well-characterized Ser/Thr kinases, one cDNA clone for a putative kinase was isolated from the Xenopus head library. The sequence of the open reading frame of the cDNA encoded a protein of 337 amino acid residues with a predicted molecular weight of 38,404. Since the deduced animo acid sequence of this protein was 75% identical to that of rat Ca(2+)/calmodulin-dependent protein kinase I (CaMKI), it was designated as CaMKIx. Although recombinant CaMKIx expressed in Escherichia coli showed no protein kinase activity against syntide-2, a synthetic peptide substrate, it was activated when phosphorylated by mouse Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha). Activated CaMKIx significantly phosphorylated various proteins including synapsin I, histones, and myelin basic protein. CaMKIx could not be detected in the early stages of embryogenesis, but was detected in late embryos of stages 37/38 and thereafter when examined by Western blotting using a specific antibody. This kinase was found to be highly expressed in adult brain and heart, and an upstream kinase that could activate CaMKIx was detected in these tissues. These results suggest that CaMKIx plays some critical role in the late stages of embryogenesis of Xenopus laevis.

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.

We examined whether non-hematopoietic BM cells can migrate into the intestinal graft after fetal small intestinal transplantation (FSITx). Fetal small intestine from donor C57BL/6 mice was transplanted into the rectus abdominis of recipient C57BL/6 mice with only green fluorescent protein (GFP) BM cells (syngeneic FSITx). Intestinal grafts were harvested on days 5, 10, and 30 after FSITx and stained immunohistochemically using anti-CD45 antibody (a marker for hematopoietic BM cells). Although there were no GFP-positive cells identified in the epithelium of the graft intestinal villi, there were a few cells positive for both GFP and CD45 in the lamina propria on day 5 after FSITx, and many present on days 10 and 30. In some grafts there were only cells that were GFP positive/CD45 negative (i.e., non-hematopoietic BM cells) found in the lamina propria on days 10 and 30. These data indicate that non-hematopoietic BM cells as well as hematopoietic BM cells can migrate from the recipient's bone marrow, suggesting that recipient mesenchymal stem cells may be strongly implicated in graft regeneration and development after FSITx.

Purpose: The aim of this study was to assess the feasibility of allogenic penile transplantation (PTx) for creating a source of viable penile tissue for use in penile reconstruction., Methods: The entire penis from an adult Brown-Norway rat was transplanted into a pouch created in the omentum of an adult Lewis rat (fully allogenic PTx, n = 23). Recipients were divided into 2 groups according to immunosuppressant (FK506) usage: in the FK+ group, FK506 (0.6 mg/kg/d) was administered intraperitoneally until a predetermined day (day 3, 5, 7, 10, 14, or 21) after PTx, and then the grafts were harvested. No FK506 was used in the FK- group. Syngeneic PTx (n = 8) patients were used as controls. All grafts were stained with H&E for histologic examination., Results: At laparotomy, each successfully transplanted penis appeared as a cylindrical mass in the omentum. Grafts could be mobilized to the genital area because of a long omental pedicle. Graft survival in the control and FK+ groups was 100%. Rejection was minimal to moderate in FK+ grafts harvested on days 3 and 5 after PTx and minimal or absent in FK+ grafts harvested on days 7, 10, 14, and 21. Penile structure on H&E staining was normal in FK+ and control specimens. Rejection with massive cellular infiltration was observed in all FK- grafts., Conclusions: FK506 successfully prevented rejection in allogenic PTx, and the authors' technique has potential for creating viable penile tissue that could be used as an option for penile reconstruction.

To explore the protein kinase family enzymes expressed in cells, we attempted to generate antibodies that could detect a wide variety of protein kinases. For the production of such antibodies, synthetic peptides corresponding to amino acid sequences of a highly conserved subdomain (subdomain VIB) of the protein kinase family were used for immunization. Among the various peptide antigens, a peptide with 16 amino acids, CVVHRDLKPENLLLAS, effectively produced polyclonal antibodies with broad cross-reactivities to protein kinases. Two monoclonal antibodies, designated M8C and M1C, detected a variety of protein kinases such as calmodulin-dependent protein kinase II, calmodulin-dependent protein kinase IV, cAMP-dependent protein kinase, and mitogen-activated protein kinases, on Western blotting. The antibodies also immunoprecipitated various protein kinases in cell extracts. Furthermore, these antibodies could be used for detection of positive clones in the expression cloning of various protein kinases. Among 39 positive clones obtained from mouse brain cDNA library, 36 clones were identified as cDNA clones for various known and novel protein serine/threonine kinases, suggesting that the antibodies reacted highly specifically with various protein kinases. These results indicate that the present monoclonal antibodies directed to multiple protein kinases will be a powerful tool for the detection of a variety of known and novel protein kinases in cells.

Recently, it has been suggested that neural stem cells and neural progenitor cells exist in the ependyma that forms the central canal of the spinal cord. In this study, we produced various degrees of thoracic cord injury in adult rats using an NYU-weight-drop device, assessed the degree of recovery of lower limb motor function based on a locomotor rating scale, and analyzed the kinetics of ependymal cell proliferation and differentiation by proliferating cell nuclear antigen (PCNA), nestin, glial fibrillary acidic protein (GFAP), or GAP-43 immunostaining. The results showed that the time course of the ependymal cell proliferation and differentiation reactions differed according to the severity of injury, and that the responses occurred not only in the neighborhood of the injury but in the entire spinal cord. An increase in the locomotor rating score was related to an increase in the number of PCNA-positive cells, and the differentiation of ependymal cells into reactive astrocytes was involved in injury repair. No apoptotic cells in the ependyma were detectable by the TUNEL method. These results indicate that the ependymal cells of the spinal central canal are themselves multipotent, can divide and proliferate according to the severity of injury, and differentiate into reactive astrocytes within the ependyma without undergoing apoptosis or cell death.

Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a --> asialo GM1 --> asialo GM2 --> lactosylceramide --> glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, beta-galactosidases, and beta-hexosaminidases. TS12 also produced beta-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.

Recently, lyso-sphingolipids have been identified as ligands for several orphan G protein-coupled receptors, although the molecular mechanism for their generation has yet to be clarified. Here, we report the molecular cloning of the enzyme, which catalyzes the generation of lyso-sphingolipids from various sphingolipids (sphingolipid ceramide N-deacylase). The 75-kDa enzyme was purified from the marine bacterium, Shewanella alga G8, and its gene was cloned from a G8 genomic library using sequences of the purified enzyme. The cloned enzyme was composed of 992 amino acids, including a signal sequence of 35 residues, and its molecular weight was estimated to be 109,843. Significant sequence similarities were found with an unknown protein of Streptomyces fradiae Y59 and a Lumbricus terrestris lectin but not other known functional proteins. The 106-kDa recombinant enzyme expressed in Escherichia coli hydrolyzed various glycosphingolipids and sphingomyelin, although it seems to be much less active than the native 75-kDa enzyme. In vitro translation using wheat germ extract revealed the activity of a 75-kDa deletion mutant lacking a C terminus to be much stronger than that of the full-length enzyme, suggesting that C-terminal processing is necessary for full activity.

We have reported previously that the ceramidase from Pseudomonas aeruginosa AN17 isolated from a patient with atopic dermatitis requires detergents for hydrolysis of ceramide (Cer) [Okino, Tani, Imayama and Ito (1998) J. Biol. Chem. 273, 14368--14373]. In the present study, we report that some glycerophospholipids strongly activated the hydrolysis of Cer by Pseudomonas ceramidase in the absence of detergents. Among the glycerophospholipids tested, cardiolipin was most effective in stimulating hydrolysis of Cer followed by phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol, whereas phosphatidylcholine, lysophosphatidic acid and diacylglycerol were less effective. Interestingly, Staphylococcus aureus-derived lipids, which contain cardiolipin and phosphatidylglycerol as major lipid components, also strongly enhanced the hydrolysis of normal Cer, as well as the human skin-specific omega-hydroxyacyl Cer, by the enzyme in the absence of detergents. It was confirmed that several strains of P. aeruginosa, including AN17, secrete a significant amount of staphylolytic proteases to lyse S. aureus cells, resulting in the release of cardiolipin and phosphatidylglycerol. Since both P. aeruginosa and S. aureus are suspected of being present in microflora of atopic skin, we speculate that S. aureus-derived glycerophospholipids stimulate the hydrolysis of Cer in atopic skin by bacterial ceramidase.

We report here the purification, characterization, and cDNA cloning of a novel N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera. The purified lectin showed 19-kDa, 41-kDa, and 60-kDa protein bands on SDS-PAGE, possibly corresponding to a monomer, homodimer, and homotrimer. Interestingly, on 4-20% native PAGE the lectin showed at least nine protein bands, among which oligomers containing six to nine subunits had potent hemagglutination activity for sheep erythrocytes. The hemagglutination activity of the lectin was specifically inhibited by N-acetylgalactosamine, Tn antigen, and blood group A trisaccharide, but not by N-acetylglucosamine, galactose, galactosamine, or blood group B trisaccharide. The specificity of the lectin was further examined using various glycosphingolipids and biotin-labeled lectin. The lectin was found to bind to Gb5Cer, but not Gb4Cer, Gb3Cer, GM1a, GM2, or asialo-GM2, indicating that the lectin specifically binds to the terminal alpha-GalNAc at the nonreducing end. The hemagglutination activity of the lectin was completely abolished by chelation with EDTA or EGTA and completely restored by the addition of CaCl(2). cDNA cloning of the lectin showed that the protein is composed of 168 amino acids, including a signal sequence of 18 residues, and possesses the typical C-type lectin motif. These findings indicate that the protein is a C-type lectin. The recombinant lectin, produced in a soluble form by Escherichia coli, showed binding activity for asialomucin in the presence of Ca(2+) but no hemagglutination.

We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn(2+). The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.

Background/purpose: Idiopathic hypertrophic pyloric stenosis (IHPS) is a common infantile disorder characterized by enlargement of the pylorus and gastric outlet obstruction. Its complete etiology is still not fully understood, but recent research has focussed on abnormalities of nerve distribution. The authors used confocal laser scanning microscopy to perform 3-dimensional studies of pylorus biopsy specimens taken from cases of IHPS and present their findings., Methods: Pylorus biopsy specimens obtained at pyloromyotomy from 6 infants with IHPS were studied using confocal microscopy and compared with 6 control pylorus biopsy specimens from patients without gastrointestinal disease. Biopsy specimens were pretreated to enhance nerve expression by using protein gene product 9.5 (PGP9.5) polyclonal antibody to identify enteric nerve system fibers. Double staining immunofluorescence was used to detect alpha smooth muscle actin (SMA), a smooth muscle marker., Results: Control pylorus biopsy specimens showed many thin PGP9.5-positive nerve fibers in the circular and longitudinal muscle layers that communicated with each other to create a 3-dimensional meshlike network. Muscle cells stained by alpha SMA antibody were thin. In contrast, muscle cells from IHPS patients were fat and round. The PGP9.5 staining nerve fibers from IHPS patients formed numerous, thick, and contorted bundles that did not communicate., Conclusions: By using confocal laser microscopy the authors were able to identify abnormally thick contorted nerve bundles in the pyloric muscle layers of infants with IHPS. These anormal nerve bundles have not been described previously because of the limitations of 2-dimensional microscopy. The authors suspect that the etiology of IHPS may be related to these abnormal fibers., (Copyright 2001 by W.B. Saunders Company.)

Background/purpose: Ncx/Hox11L.1-deficient (Ncx-/-) mice specifically created by the authors had mega-ileo-ceco-colon (mega-ICC) with a caliber change in the proximal colon. The authors studied the nerve distribution in the bowel of these Ncx-/- mice to determine the cause of their bowel dysmotility., Methods: Four-week-old Ncx-/- mice (n = 10; 5 with mega-ICC, 5 without mega-ICC) were killed and the bowel harvested. Half of each specimen was snap frozen for AchE and NADPH-diaphorase histochemistry, and the other half were fixed with 10% formalin for H&E staining and immunohistochemistry using PGP9.5 antibody (a marker for neurons), C-kit antibody (a marker for intestinal pacemaker cells), and stem cell factor antibody (a marker for C-kit ligand). Age-matched wild-type normal mice (n = 5) served as controls., Results: In the ileum, cecum, and proximal colon from all Ncx-/- mice (irrespective of the association of mega-ICC), typical findings of human intestinal neuronal dysplasia (IND) ie, obvious hyperganglionosis in neuronal plexuses on PGP9.5 immunohistochemistry, ectopic ganglia in the mucosal and muscular layers on AchE histochemistry, and ghostlike ganglia on NADPH-diaphorase histochemistry were found. Likewise, in normal caliber distal colon from these mice, the distribution of ganglion cells, C-kit, and stem cell factor was normal. In control specimens, there was no ectopic ganglia or hyperganglionosis., Conclusions: These findings suggest that the Ncx/Hox11L.1 gene is required for the proper innervation of the enteric nervous system in mice, and our deficient strain may be useful as a model for studying IND in humans., (Copyright 2001 by W.B. Saunders Company.)

Purpose: The aim of this study was to see if allogeneic transplantation (Tx) of newborn esophagus can create viable esophageal tissue that may be used for treating long gap esophageal atresia., Methods: Specimens of thoracic esophagus from newborn Brown-Norway rats, each were transplanted into a pouch created in the distal omentum of 5-week-old Lewis rats. In group I no immunosuppressant was used. FK-506 was used in group II (0.2 mg/kg), group III (0.6 mg/kg), and group IV (1.2 mg/kg) until a predetermined day of graft harvesting (1, 2, 3, 4, 5, 6, and 8 weeks after Tx). FK-506 was used for only 2 weeks in group V (0.6 mg/kg), and group VI (1.2mg/kg), and transplanted esophageal grafts were harvested 1, 2, 3, and 4 weeks after cessation of 2 weeks course FK-506. Syngeneic esophagus transplants were used as controls. All grafts were examined by H&E staining to assess graft viability and degree of rejection., Results: Each successfully transplanted esophagus appeared macroscopically as a tube like mass. Each graft could be mobilized to the thoracic cavity, because of the long omental pedicle. Graft survival in the control group was 100%. Rejection was observed in all grafts from groups I, II, V, and VI. In contrast, grafts from groups III and IV showed only minimal or no rejection. There was no evidence of side effects of FK-506 in rats in groups III and IV, except significantly slower weight gain compared with controls (P <.05)., Conclusions: FK-506 successfully prevented rejection, although immunologic tolerance was not achieved. These observations suggest that the authors' procedure has the potential to produce viable esophageal tissue that could be a new option for treating long gap esophageal atresia., (Copyright 2001 by W.B. Saunders Company.)

Purpose: We created viable bladder tissue by transplantation with immunosuppression., Materials and Methods: For bladder transplantation the bladder of newborn Brown-Norway rats was excised and each was transplanted into a pouch created in the distal omentum of a 5-week-old Lewis rat. In 15 group 1 rats no immunosuppressive agent was used. In 20 group 2 rats 0.6 mg./kg. FK-506 daily were given intramuscularly until a predetermined day of harvest. Recipient rats were sacrificed on day 3, 5, 7 or 14 after bladder transplantation, and the bladder grafts were harvested and formalin fixed. Hematoxylin and eosin staining was done to examine bladder graft survival and the degree of rejection, and immunohistochemical testing was performed for assessing the vesical nervous system. In 5 rats in the control group bladder augmentation was performed by anastomosing the bladder graft to the native bladder. Each augmented bladder was harvested 21 days later for histopathological assessment., Results: Overall bladder graft survival was 96.4%. Each successfully transplanted bladder graft appeared macroscopically as a thin walled cyst. In group 1 all bladder grafts showed rejection with cellular infiltration. In group 2 there was mild rejection in 5 rats and no evidence of rejection in the remaining 15. All group 2 bladder grafts had intact nerve distribution. Bladder augmentation was successful in all 5 cases and the mucosa was normal throughout each augmented bladder., Conclusion: Because FK-506 successfully prevents rejection, our technique would appear to have the potential for creating viable bladder tissue that may be used for bladder augmentation in cases of vesical exstrophy or neurogenic bladder.

Background: Our purpose was to evaluate whether bladder transplantation (BTx) can be used for bladder augmentation (BA)., Methods: Bladders from infantile Brown-Norway rats (less than 21 days old) were excised and each transplanted into a pouch created in the distal omentum of a 6-week-old Lewis rat (fully allogeneic BTx). No immunosuppressant was used in group I (n=12). Intramuscular FK506 was used daily from the day of BTx in group II (n=16; 0.2 mg/kg), group III (n=22; 0.6 mg/kg), and group IV (n=16; 1.2 mg/kg) until harvesting 3, 4, 5, or 6 weeks after BTx. FK506 was used for only 2 weeks in group V (n=12; 0.6 mg/kg/day) and group VI (n=12; 1.2 mg/kg/day). Syngeneic bladder transplants acted as controls (n=16). Hematoxylin and eosin staining was used to examine all grafts. In six rats from group III, BA was performed by anastomosing the graft to the recipient bladder 10 days after BTx., Results: Each successfully transplanted graft appeared macroscopically as a thin-walled cyst. Rejection was seen in all grafts from groups I, II, V, and VI, and was minimal or absent in groups III and IV. On medium to long-term follow-up the only side effect of FK506 observed was reduced weight gain. Graft survival in the control group was 100%. BA was successful in all six cases, and the mucosa was normal throughout each augmented bladder., Conclusions: This is the first report of the successful transplantation of infantile tissue without vascular anastomosis. Because of the efficient, safe immunosuppression possible with FK506, our BTx technique could find clinical application for creating viable vesical tissue that could be used for BA.

Purpose: The aim of this study was to show that a tubed latissimus dorsi musculocutaneous flap (tubed LDMCF) may be useful for treating circumferential esophageal defects., Methods: A segment of esophagus 3 vertebrae long was excised through a right thoracotomy in each of 6 puppies, and a tubed LDMCF was interposed between the cut ends of esophagus. The puppies were sacrificed after a mean follow-up period of 6.6 years. The tubed LDMCF was examined histologically. Functional integrity was assessed using barium meal and endoscopy before sacrifice., Results: All puppies survived and grew normally on a normal diet, although 4 vomited occasionally. There was smooth passage of barium through the tubed LDMCF without stenosis, although endoscopy showed regrowth of hair. Histologically, no metaplasia, dysplasia, or malignancy was observed in any tubed LDMCF., Conclusions: The tubed LDMCF is technically safe, obviates the necessity for laparotomy, and long-term follow-up would suggest that it is histologically stable. Thus, it could become an alternative procedure for bridging esophageal defects.

Background/purpose: The etiology of idiopathic gastric perforation (IGP) in neonates is unclear. Interstitial cells of Cajal (ICC) express tyrosine kinase receptor C-kit, and act as gastrointestinal pacemaker cells. Stem cell factor (SCF) is a C-kit ligand and plays an important role in immune system homeostasis in the gastrointestinal tract. The authors hypothesized that abnormal distribution of ICC or SCF in the gastric wall (ie, abnormal motility or impaired immunity) could predispose the stomach to IGP., Methods: Stomachs obtained at postmortem from neonates who died of IGP (n = 7) and other causes (control group; n = 10) were used. Biopsy sections were taken at random from various sites in the stomach, including macroscopically intact areas, and labeled immunohistochemically using antibodies to C-kit(a marker for ICC) and SCF., Results: In all control specimens, ICC were present between the muscle layers and around the myenteric plexuses of the stomach wall. In contrast, ICC were absent in all biopsy sections from 3 of the 7 IGP stomachs. In the remaining 4 IGP stomachs, there were fewer ICC in the muscle layers compared with controls, and ICC were absent around the myenteric plexuses. The distribution of SCF immunoreactivity in IGP and control specimens was similar., Conclusion: The findings suggest that a lack of ICC (ie, gastric hypomotility) may be implicated in the etiology of IGP in neonates.

Background/purpose: The proto-oncogene c-kit encodes a receptor tyrosine kinase C-KIT. W/Wv mice, which are devoid of C-KIT+ mast cells as a result of mutations in the c-kit gene, develop spontaneous gastric ulceration or perforation after day 7 of life at a high frequency, whereas normal litter mates do not. The authors hypothesized that a lack of C-KIT+ mast cells may be implicated in the development of idiopathic gastric perforation (GP) in neonates., Methods: Postmortem gastric wall specimens were taken from neonates who died of GP (idiopathic, n = 6; secondary, n = 4), and other causes (controls, n = 6). Specimens were taken at random from various sites in the stomach and labeled with antibody to C-KIT. The number of C-KIT+ mast cells from five random fields per specimen were compared under light microscopy (200x)., Results: Overall, the number of C-KIT+ mast cells was significantly lower in gastric wall specimens from cases of idiopathic GP when compared with controls or cases of secondary GP irrespective of the sites of sampling (P<.01, analysis of variance test) with the distribution of cells being uniform and unique for each stomach., Conclusion: A lack of C-KIT+ mast cells may underlie the development of idiopathic GP in neonates.

Background/purpose: In the gut, C-KIT is important for immune system homeostasis, and C-KIT+ cells are known to increase during inflammation. Recently the authors identified that spontaneous intestinal mucosal erosion develops in C-KIT-depleted W/Wv mice after day 14 of life at a high frequency, whereas genotypically normal litter mates do not. The authors hypothesized that a lack of C-KIT may be implicated in the development of necrotizing enterocolitis (NEC)., Methods: Bowel specimens were taken during surgery or postmortem from nine cases of NEC (mean gestational age, 32.0 weeks), six age-matched cases of enteritis, and 10 age-matched controls. Specimens were formalin fixed, paraffin embedded, and labeled with antibody to C-KIT. The number of C-KIT+ cells from five random fields per specimen were compared under light microscopy (200x). Results were expressed as the mean +/- SD and compared using the analysis of variance (ANOVA) test., Results: In enteritis, the number of C-KIT+ cells in the lamina propria and submucosa was significantly higher than in controls (P<.01) indicative of their involvement in inflammation. However, in NEC, the number of C-KIT+ cells in the lamina propria and submucosa was significantly lower than in controls (P<.05) despite histological evidence of inflammation., Conclusion: A lack of C-KIT+ cells may exert a causal influence on the development of NEC.

A 15-year-old boy with hypertrophic cardiomyopathy died of congestive heart failure with progressive left ventricular wall thinning with poor systolic function. Microscopic examination revealed patchy fibrosis in the ventricular myocardium with wall thinning, and immunohistochemical evaluation of apoptosis showed apoptotic cells and bodies in the destroyed myocytes along the border between the fibrotic area and myofibril.

Programmed cell death or apoptosis plays a major role in the modification of morphologic and functional maturity in various normal organ systems. However, it is also related to certain diseases. We conducted a pathological study of the apoptosis of cardiomyocytes in six cases with myocarditis (three of acute myocarditis and three of chronic or persistent myocarditis) using histochemical methods. In normal hearts obtained from autopsy cases, apoptosis was seen in endocardial cells. There was no apoptosis in myocardial cells, except for a few in myocytes with two nuclei. In myocarditis, although the myocytes of all cases with acute myocarditis did not show apoptosis, one of the three cases with chronic or persistent myocarditis showed many apoptotic myocytes. Apoptosis may be one of the mechanisms causing myocyte damage in myocarditis.

Background: Immunohistochemical study of proliferating cells in normal or neoplastic tissues has its advantages over other methods in understanding cell kinetic information. Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin section was reported as an index for cellular proliferation and well-correlated with the histopathological grading of certain systemic malignancies., Methods: By using immunohistochemical staining of PCNA on formalin-fixed, paraffin-embedded sections, the anti-PCNA monoclonal antibody (PC10) staining scores were counted in 32 intracranial gliomas and 32 intracranial meningiomas., Results: The PC10 scores ranged from 7.8% to 24.1% (m = 15.7 +/- 4.5%) in 12 anaplastic astrocytomas, and from 15.1% to 51.3% (m = 38.4 +/- 11.7%) in 9 glioblastomas. The difference in between was significant. The mean PC10 scores of benign meningiomas, malignant meningiomas, and hemangiopericytomas were 13.8%, 50.2%, and 50.8% respectively. The difference of PC10 scores was also significant between benign and malignant meningioma, as well as between benign meningioma and hemangiopericytoma., Conclusions: The PC10 scores have a good correlation with the histopathological grading of both intracranial glioma and meningioma.

The case of a 64-year-old woman with multiple intracranial aneurysms and abnormal ophthalmic arteries arising from the bifurcation of the internal carotid artery is described. It is believed that this type of anomaly of the ophthalmic artery has not previously been reported. The neuroradiological and operative findings of this case are presented.