Bolivian kindred with combined spinocerebellar ataxia types 2 and 10.

BACKGROUND: Spinocerebellar ataxias (SCA) are a group of rare hereditary neurodegenerative disorders. Rare cases of two SCA mutations in the same individual have been reported in the literature, however, family descriptions are lacking. AIMS: To characterize a family with combined SCA2 and SCA10 mutations. MATERIALS & METHODS: Analysis of the clinical features and genetic findings of a Bolivian family expressing both SCA2 and SCA10 mutations. RESULTS: The index case and his mother had both SCA2 and SCA10 mutations with a combined clinical phenotype of both disorders, including slow saccades (SCA2) and seizures (SCA10). The uncle of the index case had only an SCA10 mutation. DISCUSSION: Although the presence of two SCA mutations in the same individuals may be coincidental, the low probability of having both mutations suggests that these mutations might be particularly prevalent in Bolivian population. CONCLUSION: This is the first description of a family with two SCA mutations with affected subjects having a combined SCA2 and SCA10 phenotype.

Consensus paper: pathological mechanisms underlying neurodegeneration in spinocerebellar ataxias.

Intensive scientific research devoted in the recent years to understand the molecular mechanisms or neurodegeneration in spinocerebellar ataxias (SCAs) are identifying new pathways and targets providing new insights and a better understanding of the molecular pathogenesis in these diseases. In this consensus manuscript, the authors discuss their current views on the identified molecular processes causing or modulating the neurodegenerative phenotype in spinocerebellar ataxias with the common opinion of translating the new knowledge acquired into candidate targets for therapy. The following topics are discussed: transcription dysregulation, protein aggregation, autophagy, ion channels, the role of mitochondria, RNA toxicity, modulators of neurodegeneration and current therapeutic approaches. Overall point of consensus includes the common vision of neurodegeneration in SCAs as a multifactorial, progressive and reversible process, at least in early stages. Specific points of consensus include the role of the dysregulation of protein folding, transcription, bioenergetics, calcium handling and eventual cell death with apoptotic features of neurons during SCA disease progression. Unresolved questions include how the dysregulation of these pathways triggers the onset of symptoms and mediates disease progression since this understanding may allow effective treatments of SCAs within the window of reversibility to prevent early neuronal damage. Common opinions also include the need for clinical detection of early neuronal dysfunction, for more basic research to decipher the early neurodegenerative process in SCAs in order to give rise to new concepts for treatment strategies and for the translation of the results to preclinical studies and, thereafter, in clinical practice.

Affective communication deficits associated with cerebellar degeneration.

The cerebellum has extensive connections with the frontal lobes. Cerebellar injury has been reported to induce frontal-executive cognitive dysfunction and blunting of affect. We examined a patient with idiopathic cerebellar degeneration with impaired family relationships attributed to an "emotional disconnection." Examination revealed ataxia, dysmetria, and adiadochokinesia more severe on the left and frontal-executive dysfunction; memory and cognitive functions were otherwise normal. Testing of emotional communication included assessments of emotional semantic knowledge, emotional prosody, and emotional facial expressions. Comprehension was normal but expression was severely impaired. Cerebellar dysfunction can cause a defect in facial and prosodic emotional communication.

Divalent cations enhance fluoride binding to Streptococcus mutans and Streptococcus sanguinis cells and subsequently inhibit bacterial acid production.

One preventive effect of topical fluoride application is derived from the fact that fluoride can inhibit bacterial acid production. Furthermore, divalent cations such as Ca(2+) and Mg(2+) increase the binding of fluoride to bacterial cells. These findings suggest that exposure of oral bacteria to fluoride in the presence of divalent cations increases fluoride binding to bacterial cells and subsequently enhances fluoride-induced inhibition of bacterial acid production. This study investigated the effects of fluoride exposure (0-20,000 ppm F) in the presence of Ca(2+) or Mg(2+) prior to glucose challenge on pH fall ability by bacterial sugar fermentation, as well as fluoride binding to bacterial cells by exposure to fluoride, and fluoride release from bacterial cells during bacterial sugar fermentation, using caries-related bacteria, Streptococcus mutans and Streptococcus sanguinis. The pH fall by both streptococci was inhibited by exposure to over 250 ppm F in the presence of Ca(2+) (p < 0.01), whereas in the presence of Mg(2+), the pH fall by S. mutans and S. sanguinis was inhibited after exposure to over 250 and 950 ppm F, respectively (p < 0.05). The amounts of fluoride binding to and released from streptococcal cells increased with the concentration of fluoride the cells were exposed to in the presence of Mg(2+), but were high enough even after 250 ppm F exposure in the presence of Ca(2+). The enhanced inhibition of acid production in the presence of divalent cations is probably due to the improved efficiency of fluoride binding to bacterial cells being improved via these divalent cations.

Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel.

A polymorphic CAG repeat was identified in the human alpha 1A voltage-dependent calcium channel subunit. To test the hypothesis that expansion of this CAG repeat could be the cause of an inherited progressive ataxia, we genotyped a large number of unrelated controls and ataxia patients. Eight unrelated patients with late onset ataxia had alleles with larger repeat numbers (21-27) compared to the number of repeats (4-16) in 475 non-ataxia individuals. Analysis of the repeat length in families of the affected individuals revealed that the expansion segregated with the phenotype in every patient. We identified six isoforms of the human alpha 1A calcium channel subunit. The CAG repeat is within the open reading frame and is predicted to encode glutamine in three of the isoforms. We conclude that a small polyglutamine expansion in the human alpha 1A calcium channel is most likely the cause of a newly classified autosomal dominant spinocerebellar ataxia, SCA6.

Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.

Spinocerebellar ataxia type 10 (SCA10; MIM 603516; refs 1,2) is an autosomal dominant disorder characterized by cerebellar ataxia and seizures. The gene SCA10 maps to a 3.8-cM interval on human chromosome 22q13-qter (refs 1,2). Because several other SCA subtypes show trinucleotide repeat expansions, we examined microsatellites in this region. We found an expansion of a pentanucleotide (ATTCT) repeat in intron 9 of SCA10 in all patients in five Mexican SCA10 families. There was an inverse correlation between the expansion size, up to 22.5 kb larger than the normal allele, and the age of onset (r2=0.34, P=0.018). Analysis of 562 chromosomes from unaffected individuals of various ethnic origins (including 242 chromosomes from Mexican persons) showed a range of 10 to 22 ATTCT repeats with no evidence of expansions. Our data indicate that the new SCA10 intronic ATTCT pentanucleotide repeat in SCA10 patients is unstable and represents the largest microsatellite expansion found so far in the human genome.

An unstable triplet repeat in a gene related to myotonic muscular dystrophy.

Synthetic oligonucleotides containing GC-rich triplet sequences were used in a scanning strategy to identify unstable genetic sequences at the myotonic dystrophy (DM) locus. A highly polymorphic GCT repeat was identified and found to be unstable, with an increased number of repeats occurring in DM patients. In the case of severe congenital DM, the paternal triplet allele was inherited unaltered while the maternal, DM-associated allele was unstable. These studies suggest that the mutational mechanism leading to DM is triplet amplification, similar to that occurring in the fragile X syndrome. The triplet repeat sequence is within a gene (to be referred to as myotonin-protein kinase), which has a sequence similar to protein kinases.

Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy.

The myotonic dystrophy mutation has recently been identified; however, the molecular mechanism of the disease is still unknown. The sequence of the myotonin-protein kinase gene was determined, and messenger RNA spliced forms were identified in various tissues. Antisera were developed for analytical studies. Quantitative reverse transcription-polymerase chain reaction and radioimmunoassay were used to demonstrate that decreased levels of the messenger RNA and protein expression are associated with the adult form of myotonic dystrophy.

Functioning adrenocortical oncocytoma: the first documented case producing interleukin-6 and review of the literature.

Adrenocortical oncocytoma is an extremely rare and predominantly non-functioning tumor. We herein report the first case of an adrenocortical oncocytoma that produces interleukin (IL)-6. A 38-yr-old woman was referred for treatment of a 4-cm adrenal mass. Laboratory test results showed elevated inflammatory parameters. Intriguingly, IL-6 serum level was also high at 30 pg/ml (normal 0-4 pg/ml). The patient underwent laparoscopic right adrenalectomy. Microscopic examination showed that the tumor was an adrenocortical oncocytoma with a unique peripheral lymphoid cuff with germinal centers. Electron microscopy demonstrated that the cytoplasm of the neoplastic cells was packed with numerous abnormal mitochondria. Three observations lead us to consider that this tumor was the primary source of serum IL-6. First, the IL-6 level in blood collected from the right adrenal vein was highest (527 pg/ml) among intra-operative blood samples. Second, neoplastic cells stained positively for IL-6. Third, the serum IL-6 returned to normal levels immediately after surgery.

ABO-incompatible adult living donor liver transplantation for hepatocellular carcinoma.

Living donor liver transplantation (LDLT) offers timely transplantation for patients with hepatocellular carcinoma (HCC). If ABO-incompatible LDLT is feasible, the need for pretransplantation treatment may be eliminated, which may reduce overall morbidity. In this article, we have described 8 adult HCC patients who successfully underwent LDLT from ABO-incompatible donors. Antirejection therapy included multiple preoperative plasmaphereses, splenectomy, and an immunosuppressive regimen with tacrolimus, methylprednisolone, and mycophenolate mofetil. The maintenance dose of immunosuppression did not differ from that of the ABO-identical cases. In addition, we also performed intrahepatic arterial infusion of prostaglandin E1. In 5 patients, we administered a single dose of rituximab, a chimeric CD20 monoclonal antibody. As a result of this treatment, 6/8 patients are still alive. Our experience has shown that it is possible to control antibody-mediated humoral rejection and other complications in adult ABO-incompatible LDLT.

Biliary complications after 52 adult living donor liver transplantations: a single-center experience.

OBJECTIVE: The incidence of biliary complications after adult living donor liver transplantation (ALDLT) are still high even though various devices have been reported to overcome them. METHOD: From October 2000 to April 2007, we performed 52 ALDLTs which included 15 ABO-incompatible grafts. Median follow-up was 565 days. In 49 procedures, we used duct-to-duct anastmosis with a stent inserted in the recipient duct and out through the common bile duct wall as an external stent, and in 3 procedures, we used duct-to-jejunostomy anastomosis. We investigated postoperative biliary complications and their management. RESULTS: Forty-four patients received right lobe grafts and 8 received left lobe grafts. Among patients in whom duct-to-duct anastomosis was used, nine (20.5%) developed biliary complications including bile leakage in five and biliary strictures in four. All bile leakage was treated with reoperation. Three biliary strictures were treated with stent placement, and one biliary stricture was treated with magnetic compression anastomosis. Among the three patients in whom duct-to-jejunostomy was used, two (66.7%) had bile leakage and stricture, respectively. Two of four ABO-incompatible patients (50%) had hepatic artery thrombosis with biliary complications, a high incidence. CONCLUSION: In our series of ABO-incompatible patients undergoing ALDLT, those who developed hepatic artery thrombosis exhibited a high incidence of biliary complications.

How can we increase living related donor renal transplantations?

BACKGROUND: In Japan, living donor renal transplantation has gained momentum due to an increased number of patients with end-stage renal disease. Living donation not only provides better outcomes, but also the recipients usually need less medications, thereby increasing the quality of life and reducing the potential side effects of immunosuppression. MATERIALS AND METHODS: For the past 25 years, our center had performed 140 open donor nephrectomy (OPNx) renal transplantations. Since July 2003, we changed our procurement operation to living hand-assisted laparoscopic donor nephrectomy (HALNx) in 49 cases. Our operative technique consisted of two 12-mm ports placed in the midaxillary line at the superior and inferior levels of the umbilicus. Next, a 5-cm incision was made in the midline periumbilicus and the hand port system fitted through a midline abdominal incision. RESULTS: In 49 cases, HALNx was completed successfully; no patient required conversion to laparotomy. The estimated blood loss was 33.0 +/- 43.4 g and no patient required blood transfusion. In comparison, in OPNx the blood loss was 426.5 +/- 247.6 g (P < .001). The mean operative times were 167.4 +/- 39.7 minutes for HALNx and 228.4 +/- 35.7 minutes for OPNx (P < .001). The postoperative hospital stays were 9.1 +/- 3.8 days for HALNx and 13.0 +/- 1.9 days for OPNx (P < .001). For 3 years prior to introduction of HALNx, we had performed only 10 living donor renal transplantations. Since the introduction of HALNx in 2003, the number of living donors has tripled during the following 3 years. CONCLUSIONS: Herein we have reported that HALNx was superior in terms of less operative time and blood loss, postoperative pain and recovery, and shorter hospital stay. Overall donor patient satisfaction was also better in the HALNx group. HALNx is a safe procedure that makes kidney donation more appealing to potential live donors and has increased the living donor pool at our center.

Disruption of the purine nucleotide cycle. A potential explanation for muscle dysfunction in myoadenylate deaminase deficiency.

A patient with symptoms of easy fatigability, postexercise myalgias, and delayed recovery of muscle strength after activity is described. Skeletal muscle from this patient had <1.0% normal myoadenylate deaminase activity and NH(3) was not released from muscle after ischemic exercise. In association with this enzyme deficiency, exercise led to a >90% reduction in muscle content of adenine nucleotides. No inosine monophosphate accumulated after exercise and total purine content of the muscle fell to 21% of control. Repletion of the adenine nucleotide pool in this patient was delayed compared to controls, and ATP content had only returned to 68% of control at 165 min after exercise. These studies demonstrate that disruption of the purine nucleotide cycle as a consequence of myoadenylate deaminase deficiency results in marked alterations in ATP content of muscle, and potentially, these changes in ATP content could account for muscle dysfunction in this patient.

Genetic and physical mapping of the partial resistance gene, pi34, to blast in rice.

ABSTRACT Partial resistance to rice blast in the Oryza sativa japonica group cv. Chubu 32 is controlled by Pi34, a major quantitative trait locus (QTL) on chromosome 11, and several uncharacterized QTLs. The objectives of the study were (i) high-resolution genetic and physical mapping of Pi34 and (ii) identification of new QTL imparting resistance to rice blast. Chubu 32 was crossed with a susceptible chromosomal segment substitution line (CSSL) of cv. Koshihikari. From 4,012 of segregating individuals, 213 recombinants in the Pi34 region were screened by using polymerase chain reaction-based markers and tested resistance in the field and greenhouse. The Pi34 locus is located in the 54.1-kb region on the genomic sequence of cv. Nipponbare. We constructed a bacterial artificial chromosome (BAC) library of Chubu 32, selected the clone containing Pi34, and sequenced it. The Pi34 locus consequently was located on an interval of 65.3 kb containing 10 predicted open reading frames (ORFs). Two of these ORFs were predicted only in Chubu 32 and encoded transposable elements. The other eight ORFs were found in both Chubu 32 and Nipponbare and one of them, which encoded an unknown protein, showed significantly different amino acid sequences between two cultivars. The new QTL, Piq6(t), was detected on the short arm of chromosome 6 and the genetic distance of flanking markers was 16.9 centimorgans in Nipponbare. Pi34 and Piq6(t) acted additively on resistance to rice blast but the effect of Piq6(t) was relatively small compared with Pi34.

Length-dependent structure formation in Friedreich ataxia (GAA)n*(TTC)n repeats at neutral pH.

More than 15 human genetic diseases have been associated with the expansion of trinucleotide DNA repeats, which may involve the formation of non-duplex DNA structures. The slipped-strand nucleation of duplex DNA within GC-rich trinucleotide repeats may result in the changes of repeat length; however, such a mechanism seems less likely for the AT-rich (GAA)n*(TTC)n repeats. Using two-dimensional agarose gels, chemical probing and atomic force microscopy, we characterized the formation of non-B-DNA structures in the Friedreich ataxia-associated (GAA)n*(TTC)n repeats from the FRDA gene that were cloned with flanking genomic sequences into plasmids. For the normal genomic repeat length (n = 9) our data are consistent with the formation of a very stable protonated intramolecular triplex (H-DNA). Its stability at pH 7.4 is likely due to the high proportion of the T.A.T triads which form within the repeats as well as in the immediately adjacent AT-rich sequences with a homopurine. homopyrimidine bias. At the long normal repeat length (n = 23), a family of H-DNAs of slightly different sizes has been detected. At the premutation repeat length (n = 42) and higher negative supercoiling, the formation of a single H-DNA structure becomes less favorable and the data are consistent with the formation of a bi-triplex structure.

The role of plasmapheresis therapy for perioperative management in ABO-incompatible adult living donor liver transplantation.

BACKGROUND: Although living donor liver transplantation (LDLT) was established as a treatment for end-stage liver disease in Japan, the indication for LDLT across an ABO-incompatible barrier remains controversial. The purpose of this study was to elucidate the role of plasmapheresis in incompatible LDLT. METHODS: Eleven adult patients (seven men and four women) who underwent incompatible LDLT were enrolled in this study. Of these three patients had hepatocellular carcinoma, three chronic hepatitis C, one Wilson's disease, one autoimmune hepatitis, one chronic hepatitis B, one hemochromatosis, and one fulminant hepatic failure. The immunosuppressive regimen consisted of tacrolimus, prednisolone, mycophenolate mofetil (or cyclophosphamide), and prostaglandin E1 in all patients. Multiple plasmapheresis was performed perioperatively to reduce the recipient's antibody titers against the donor's blood type. RESULTS: Plasmapheresis was useful for the reduction of the recipient's antibody titers to x 16 or lower before and after transplantation. There was no difference in transplant outcome between the 11 patients with incompatible blood group and 30 patients with identical or compatible blood groups. DISCUSSION: Major postoperative complications such as intrahepatic biliary complications and hepatic necrosis may occur in incompatible transplantation. Several investigators suggested that anti-immunoglobulin (Ig) M and anti-IgG antibody titers sustained these complications. The antibody titers must be decreased sufficiently with plasmapheresis. An elevation of anti-ABO titers after transplantation may be a predictive risk factor for increased mortality and morbidity. In order to perform LDLT in a safer manner, plasmapheresis is an indispensable treatment to improve the outcome of ABO-incompatible cases.

Antitumor quinolones with mammalian topoisomerase II mediated DNA cleavage activity.

Ninety quinolones were evaluated to determine whether their ability to induce mammalian topoisomerase II mediated DNA cleavage in vitro correlated with their antitumor activity in vivo. Ten quinolones generated linear DNA at a yield of more than 10% of substrate supercoiled DNA in the mammalian topoisomerase II mediated DNA cleavage assay. All of these compounds showed a significant increase in life span (greater than 20%) in the murine leukemia P388 model. These antitumor quinolones have closely related structures: two halogens at C-6 and C-8; and cyclopropyl at N-1 of quinolone skeleton. In contrast, many analogues of the above quinolones, as well as new quinolones used clinically as an antibacterial drug, did not induce the cleavable complex in vitro or show antitumor activity in vivo. These findings indicate that quinolone derivatives can be a promising new class of antitumor agent targeting mammalian topoisomerase II.

Antitumor activity of 7-N-[[2-[[2-(gamma-L-glutamylamino)ethyl]dithio]ethyl]]-mitomycin C.

Through the extensive investigation of new mitomycin C (MMC) derivatives, several compounds with disulfide at N-7 were found to show activities superior to MMC against murine Sarcoma 180 solid tumor. Among them, 7-N-[[2-[[2-(gamma-L-glutamylamino)ethyl]dithio]ethyl]]- mitomycin C (KW-2149) was selected for further evaluation of antitumor activity and toxicity in mice. KW-2149 exhibited activity superior to MMC in increasing survival of i.p. inoculated P388 leukemia-, M5076 sarcoma-, and B16 melanoma-bearing mice. KW-2149 administered i.v. also exhibited superior activity in inhibiting the growth of s.c. inoculated P388 leukemia, M5076 sarcoma, and colon 26 adenocarcinoma and in increasing survival of i.v. inoculated P388 leukemia- and M5076 sarcoma-bearing mice. Furthermore, KW-2149 remarkably increased the life span of MMC-resistant P388 leukemia- and L1210 leukemia-bearing mice. KW-2149 and MMC inhibited the growth of human tumors inoculated into nude mice. The activity of KW-2149 was prominent in human lung carcinoma Lu-65 and Lu-99, bladder carcinoma T24, and epidermoid carcinoma A431. KW-2149 was comparable to MMC in decreasing the number of WBC in the peripheral blood, and the thrombopenia induced by KW-2149 was mild and recovery was rapid. The in vitro anticellular spectrum of KW-2149 against 23 human tumor cell lines was similar to that of MMC. However, KW-2149 inhibited the growth of the cell lines at concentrations of 10- to 100-fold lower than MMC and showed efficient cytotoxicity against MMC-insensitive tumor cell lines. These included lung epidermoid carcinoma Calu-1, stomach carcinoma MKN-28, colon adenocarcinoma DLD-1, colon adenocarcinoma LoVo, bladder carcinoma HT-1197, sarcoma G-292, and melanoma SK-MEL-28 cells. These results indicate that KW-2149 bears interesting characteristics as a new anticancer drug and warrants further development.

Radial flow bioreactor for the creation of bioartificial liver and kidney.

A radial flow bioreactor (RFB) is used for a three-dimensional perfusion culture of hepatocellular carcinoma (HCC) cells and renal cells, to create a bioartificial liver and kidney. The cylindrical reactor is filled with porous cellulose microcarrier. RFB can be characterized as a system in which the medium flows from the periphery toward the center of the reactor, thereby delivering an adequate supply of oxygen and nutrients to cells at the center as well as at the periphery. HCC cells incubated in the RFB system at high density maintained viability for long periods of time. Proximal tubular cells (LLC-PK1) as well as HCC cells, but not human immortalized mesangial cells (HMC) were cultured in the RFB for more than 14 days. The mRNA expression of some enzymes involved in the urea cycle, cytochrome P450s in HCC cells, and the 1-alpha-hydroxylase (CYP27B1) in LLC-PK1 cells was higher than that in monolayer cultures. These results suggest that the RFB system composed of HCC cells or renal cells may be useful for a bioartificial liver and kidney.

Evaluation of appropriate blood level in continuous intravenous infusion from trough concentrations after oral administration based on area under trough level in tacrolimus and cyclosporine therapy.

The target blood concentrations of tacrolimus (TAC) and cyclosporine (CYA) during continuous intravenous infusion (C(ss)) have been determined based on clinical experience. However, it is desirable that C(ss) should be set so that the AUC after intravenous infusion is equal to the AUC after oral administration (AUC(po)). Accordingly, we performed 12-hour monitoring of blood concentrations to calculate C(ss) from the blood trough levels (C(TL)) on 15 kidney recipients administered TAC and 12 recipients administered CYA (Neoral). We used an area under the trough level (AUTL) as a new pharmacokinetic parameter. The C(ss) was evaluated from C(TL), AUC(po), and AUTL was calculated to be C(ss) = C(TL) x (AUC(po)/AUTL). In addition, AUTL/AUC(po) ratio and blood peak/trough level ratio (C(max)/C(min)) were examined to compare pharmacokinetics of TAC and CYA. The formula for TAC was C(ss) = C(TL) x 1.40 and that for CYA, C(ss) = C(TL) x 2.55. The calculated target C(ss) of TAC was 1.40 times that of C(TL), which was similar to the present clinical C(TL). In contrast, the calculated target C(ss) of CYA was 2.55 times the C(TL), and therefore an extremely high C(ss) was necessary to obtain a sufficient AUC that will be available after oral administration. Consequently, intravenous administration of CYA twice a day was considered to be more appropriate to obtain sufficient CYA pharmacokinetics, rather than a continuous intravenous administration. We conclude that the formula, C(ss) = C(TL) x (AUC(po)/AUTL) was useful to calculate the target blood concentration of calcineurin inhibitors when changing from continuous intravenous infusion to oral administration of these drugs.