Detection of cerebrospinal fluid leak by dual-isotope spect with In-111 DTPA and Tc-99m HMDP.
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Adult↗
Biomedical subjects
H Nakai
Publications and source records attributed to H Nakai.
Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo.
Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.
Animals↗
Basic science and epilepsy: experimental epilepsy surgery.
Epilepsy surgery, as is employed for the management of intractable seizures, was performed in animals harboring a seizure focus induced by a local application of kainic acid (KA). Amygdalo-hippocampectomy failed to stop spontaneous seizures in the contralateral hippocampus. Callosotomy inhibited seizure propagation to the contralateral sensori-motor cortex. However, epileptic activity ipsilateral to the focus, including subcortical structures, persisted even after the callosotomy. Multiple subpial transection (MST) around the epileptic cortical focus suppressed the seizure activity of the cortex. However, seizure propagations in subcortical structures remained, even after MST. Nefiracetam (a new nootropic agent) was tested in these models, and its promising effect on the intractable extratemporal epilepsy is reported.
Amygdala↗
[Development of new hydrophilic catheter kit for self-catheterization: study in pediatric cases].
PURPOSE: To increase the management of self-catheterization in children of school age, a catheter kit consisting of hydrophilic catheter and a packet containing sterilized water was developed. We evaluated the lubricating characteristic and clinical efficacy of this new catheter kit. MATERIALS AND METHODS: The catheter kit used in the study was a pocket-size plastic container in which a polyurethane catheter coated with hydrophilic polymer and a packet containing sterilized water were packed in combination. The lubricating characteristic of catheter was assessed by the measurement of friction value. For clinical assessment, male children aged over 6 years old who were doing self-catheterization at 17 medical institutions nationwide were selected as the subjects. The 32 children who had given informed consent (mean age: 11.6 years old) were asked to use the catheter kit continuously for 1 week. The results were investigated by a questionnaire survey in which the assessment before and after the use was expressed in scores. At the same time, urinalysis and urine culture were examined. RESULTS: The friction value of hydrophilic catheter was equivalent to or less than that observed by applying a lubricant to the conventional catheter. The comparison of conventional catheter with the kit indicated significantly higher scores (assessment in 5 grades expressed in scores) for the portability and operability of the kit. Though there was no significant difference in the ease of insertion between the two catheters, there were several comments that the kit got stuck in the urethra when it was withdrawn. The global assessment gave a significantly higher score to the kit and 30 (94%) of the 32 children wanted to use the kit continuously. No increase in hematuria which caused a clinical problem or no new apparent urinary tract infection occurred after the use of the kit. CONCLUSIONS: Compared with the conventional catheter, the hydrophilic catheter kit highly satisfied a large number of children at the time of self-catheterization. Depending on the condition of children, the kit is considered useful for continued self-catheterization for a long term.
Child↗
Bisabosquals, novel squalene synthase inhibitors. II. Physico-chemical properties and structure elucidation.
The squalene synthase inhibitor bisabosqual A was isolated from the culture broth of Stachybotrys sp. RF-7260, and its structure was determined on the basis of spectroscopic methods including detailed 2D NMR analyses. The structures of bisabosquals B, C and D isolated from Stachybotrys ruwenzoriensis RF-6853 were determined by spectroscopic methods and chemical reactions. The absolute stereochemistry of bisabosquals A, B and D was determined by X-ray crystallographic analysis. They have novel cis-fused tetracyclic structures with a bisabolane-type sesquiterpene and phenol moieties.
Crystallography, X-Ray↗
[A case of a multiple sclerosis with severe perifocal edema].
We present a case of tumoral multiple sclerosis in a 66-year-old male and emphasize two clinical features: (1) Multiple well-demarcated massive enhanced lesions except for the central area, which was accompanied with severe perifocal edema, occurred within the cerebral hemispheres and should be strongly considered in the diagnosis of mass lesions. The patient underwent partial removal of the mass lesion. The initial histopathological diagnosis of the surgical specimen was astrocytoma grade II. (2) Multiple lesions disappeared completely for one and half months after starting medication using steroid. The reexamination of the surgical specimen was compatible with that of multiple sclerosis. Again, half a month after remission, multiple enhanced lesions without perifocal edema developed in the cerebral and cerebellar hemisphere. The difficulty of the initial diagnosis of multiple sclerosis with severe perifocal edema was discussed, based on our experience.
Aged↗
Involvement of heat shock proteins in the healing of acetic acid-induced gastric ulcers in rats.
The present study examined the expression of 73-kDa of heat shock cognate protein (HSC70), 72-kDa of heat shock protein (HSP70) and 47-kDa of HSP (HSP47) observed in the ulcer healing process in rats. Gastric ulcers were induced by a luminal application of acetic acid in male Donryu rats. During the ulcer healing process, the expression of HSPs in the ulcerated tissue was determined. A high level of HSC70 expression was observed both in the normal mucosa and ulcerated tissue, but the level did not change upon ulceration and ulcer healing. While HSP70 and HSP47 were markedly expressed in the ulcer base during ulceration, and decreased with ulcer healing. HSP70 expression in the ulcer margin was gradually increased with ulcer healing. Omeprazole accelerated the healing of gastric ulcers with strong inhibition of gastric acid secretion, while indomethactin delayed in ulcer healing despite slight inhibition of gastric acid secretion. Omperazole enhanced the expression of HSP70 both in the ulcer margin and base, but it reduced HSP47 expression in the ulcer base Indomethacin markedly enhanced HSP47 expression only in the ulcer base. In conclusion, the expression of HSP70 and HSP47 is changed during ulcer healing. Furthermore, it was suggested that the enhanced expression of HSP70 is involved in acceleration of ulcer healing, but overexpression of HSP47 is involved in delayed ulcer healing.
Animals↗
Design and synthesis of new orally active nonpeptidic inhibitors of human neutrophil elastase.
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Administration, Oral↗
Effects of various saccharides on cycloinulo-oligosaccharide fructanotransferase reaction: production of beta-inulotriosyl-alpha-D-mannopyranoside and 1-O-beta-inulotriosyl-alpha-L-sorbopyranose.
The effects of various saccharides on the reaction of cycloinulo-oligosaccharide fructanotransferase with cycloinu-lohexaose were examined. In addition to beta-D-fructofuranosides and methyl alpha-D-glucopyranoside, D-mannose and L-sorbose were found to be effective acceptors in the reactions, and they enhanced the hydrolytic activity as effectively as methyl alpha-D-glucopyranoside. Hetero-tetrasaccharides were isolated as the major transfer products from both reaction mixtures. The isolates were identified by NMR spectroscopy as beta-inulotriosyl-alpha-D-mannopyranoside and 1-O-beta-inulotriosyl-alpha-L-sorbopyranose. Methyl beta-D-glucopyranoside was slightly effective and methyl alpha-D-mannopyranoside was not effective at all as the acceptor, but these saccharides strongly enhanced the hydrolytic activity. D-Glucosamine inhibited the enzyme activity.
Carbohydrate Sequence↗
Vascular abnormalities in surgical specimens obtained from the resected focus of intractable epilepsy.
The histopathological features, particularly hypervascularity, were examined in specimens resected from 21 patients, 15 with intractable epilepsy accompanying cortical dysplasia or dysembryoplastic neuroepithelial tumor (DNT), and 6 with benign brain tumors, such as ganglioglioma and low-grade glioma. Hypervascularity was found in resected specimens from 15 of the 21 patients (71.4%) and in 10 of the 12 patients (83.3%) who had double pathology. Counting of numbers of vessels by CD31 immunohistochemistry revealed that hypervascularity was prominent, especially in cases of vascular malformation or cortical dysplasia. However, almost all cases were negative for vascular endothelial growth factor (VEGF) staining, except for some cases of benign brain tumors. Moreover, all cases showed low or no proliferative potential in MIB-1 immunohistochemistry. These results suggest that the etiology of hypervascularity in the dysplastic lesions is one of a variety of cerebral malformations, as is the case with abnormal maturation and differentiation in neuroglial elements.
Adolescent↗
A giant urinoma in a neonate without obstructive uropathy.
We report a neonate with a giant urinoma and renal failure. A 7-day-old boy had a giant abdominal mass of 6.5 cm x 8 cm in the right quadrant, gastroesophageal reflux, and renal failure caused by the compression from the mass. Radiological observations revealed a multiseptated cyst and neither kidney could be detected. To relieve these symptoms percutaneous drainage was performed. The contents of the fluid were similar to the patient's urine. The symptoms were improved by the drainage, and we found the left kidney to be absent and the right kidney small. Four prenatal ultrasound scans detected no cystic lesions in his abdomen. Neonatal urinomas are commonly complicated by obstructive uropathy, such as posterior urethral valves or ureteropelvic junction obstruction. These obstructive uropathies were ruled out by retrograde pyelography and voiding cystourethrography. A severely dilated upper pole of a double collecting system was also ruled out by intravenous pyelography and direct observation of the kidney during an open biopsy. The cause of the urinoma is still uncertain, but trauma during delivery and the dysplastic right kidney may be involved.
Cysts↗
Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses.
Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.
Animals↗
Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors.
A major shortcoming to the use of adeno-associated virus (rAAV) vectors is their limited packaging size. To overcome this hurdle, we split an expression cassette and cloned it into two separate vectors. The vectors contained either a nuclear localizing Escherichia coli lacZ transgene (nlslacZ) with a splice acceptor, or the human elongation factor 1alpha ( EF1alpha) gene enhancer/promoter(s) (EF1alphaEP) with a splice donor. We co-injected a promoter-less nlslacZ vector with a vector containing either a single EF1alphaEP or a double copy of the EF1alphaEP in a head-to-head orientation, into the portal vein of mice. Gene expression, measured by both transduction efficiency and quantitation of the recombinant protein, was as much as 60-70% of that obtained from mice that received a single vector containing a complete EFalphaEP/nlslacZ expression cassette. This two-vector approach may allow development of gene therapy strategies that will carry exogenous DNA sequences with large therapeutic cDNAs and/or regulatory elements.
Animals↗
PriA and phage T4 gp59: factors that promote DNA replication on forked DNA substrates microreview.
The initiation of DNA synthesis on forked DNA templates is a vital process in the replication and maintenance of cellular chromosomes. Two proteins that promote replisome assembly on DNA forks have so far been identified. In phage T4 development the gene 59 protein (gp59) assembles replisomes at D-loops, the sites of homologous strand exchange. Bacterial PriA protein plays an analogous function, most probably restarting replication after replication fork arrest with the aid of homologous recombination proteins, and PriA is also required for phage Mu replication by transposition. Gp59 and PriA exhibit similar DNA fork binding activities, but PriA also has a 3' to 5' helicase activity that can promote duplex opening for replisome assembly. The helicase activity allows PriA's repertoire of templates to be more diverse than that of gp59. It may give PriA the versatility to restart DNA replication without recombination on arrested replication forks that lack appropriate duplex openings.
Bacteriophage T4↗
Congenital vesicovaginal fistula.
Congenital vesicovaginal fistula is a very rare entity, the etiology of which has not been clearly elucidated because pathologic features have not been mentioned in previous reports. The case of a 4-year-old girl having incontinence resulting from a congenital vesicovaginal fistula joining with the left ectopic ureter from the hypoplastic kidney is described. This is thought to be the first presentation of congenital vesicovaginal fistula joining with ectopic ureter. A microscopic examination revealed the fistula consisting of transitional cell epithelium, suggesting an abnormal fusion of the ureteral bud and caudal end of the müllerian duct with the urogenital sinus.
Child, Preschool↗
Inflammatory pseudotumor of the bladder in neonates.
Inflammatory pseudotumors of the bladder are rare in children. We describe a 7-day-old male neonate with inflammatory pseudotumor of the bladder. He presented with a 3-day history of macroscopic hematuria. Ultrasonography, computed tomography and cystoscopy showed an intravesical mass arising from the right lateral wall. Pathologic findings obtained by open excision revealed that the tumor had spindle-shaped cells without significant atypia infiltrating into submucosal fibrous tissue. There has been no evidence of recurrent tumor 12 months post-operatively. To our knowledge our case is the first presentation in a neonate among the reported pediatric cases.
Granuloma, Plasma Cell↗
Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.
Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.
Animals↗
Nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: cell cycling does not influence hepatocyte transduction.
Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about approximately 5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only approximately 5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5'-bromo-2'-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic beta-galactosidase. Colabeling for beta-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.
Animals↗