Limb-girdle muscular dystrophy type 2G is caused by mutations in the gene encoding the sarcomeric protein telethonin.

Autosomal recessive limb-girdle muscular dystrophies (AR LGMDs) are a genetically heterogeneous group of disorders that affect mainly the proximal musculature. There are eight genetically distinct forms of AR LGMD, LGMD 2A-H (refs 2-10), and the genetic lesions underlying these forms, except for LGMD 2G and 2H, have been identified. LGMD 2A and LGMD 2B are caused by mutations in the genes encoding calpain 3 (ref. 11) and dysferlin, respectively, and are usually associated with a mild phenotype. Mutations in the genes encoding gamma-(ref. 14), alpha-(ref. 5), beta-(refs 6,7) and delta (ref. 15)-sarcoglycans are responsible for LGMD 2C to 2F, respectively. Sarcoglycans, together with sarcospan, dystroglycans, syntrophins and dystrobrevin, constitute the dystrophin-glycoprotein complex (DGC). Patients with LGMD 2C-F predominantly have a severe clinical course. The LGMD 2G locus maps to a 3-cM interval in 17q11-12 in two Brazilian families with a relatively mild form of AR LGMD (ref. 9). To positionally clone the LGMD 2G gene, we constructed a physical map of the 17q11-12 region and refined its localization to an interval of 1.2 Mb. The gene encoding telethonin, a sarcomeric protein, lies within this candidate region. We have found that mutations in the telethonin gene cause LGMD 2G, identifying a new molecular mechanism for AR LGMD.

Enzyme replacement therapy in two patients with classic Fabry disease from the same family tree: Two case reports.

BACKGROUND: The pathophysiology of Fabry disease (FD)-induced progressive vital organ damage is irreversible. Disease progression can be delayed using enzyme replacement therapy (ERT). In patients with classic FD, sporadic accumulation of globotriaosylceramide (GL-3) in the heart and kidney begins in utero; however, until childhood, GL-3 accumulation is mild and reversible and can be restored by ERT. The current consensus is that ERT initiation during early childhood is paramount. Nonetheless, complete recovery of organs in patients with advanced FD is challenging. CASE SUMMARY: Two related male patients, an uncle (patient 1) and nephew (patient 2), presented with classic FD. Both patients were treated by us. Patient 1 was in his 50s, and ERT was initiated following end-organ damage; this was subsequently ineffective. He developed cerebral infarction and died of sudden cardiac arrest. Patient 2 was in his mid-30s, and ERT was initiated when the patient was diagnosed with FD, during which the damage to vital organs was not overtly apparent. Although he had left ventricular hypertrophy at the beginning of this treatment, the degree of hypertrophy progression was limited to a minimal range after > 18 years of ERT. CONCLUSION: We obtained discouraging ERT outcomes for older patients but encouraging outcomes for younger adults with classic FD.

First Report of Colletotrichum boninense Causing Anthracnose on Pepper in Brazil.

Colletotrichum boninense was isolated from pepper (Capsicum annuum) fruits (cv. Amanda) with preharvest anthracnose symptoms collected in the Brazilian states of Rio Grande do Sul and São Paulo in July of 2005. In the field, the disease affected mature fruits and leaves with an incidence near 25%. Typical symptoms in fruits were circular, sunken lesions with orange spore masses in a dark center. Three single conidia isolates were obtained from infected fruits. When grown on potato dextrose agar at 25°C with a 12-h photoperiod, these isolates produced white colonies with a cream-to-orange color in the opposite side, but no sclerotia. Conidia were cylindrical, had obtuse ends and a hilum-like low protuberance at the base, and measured 13.5 to 15.5 × 4.6 to 5.1 µm. Conidial length/width ratio was 2.8 to 3.0. These morphological characteristics are consistent with the description of C. boninense (1). To confirm pathogen identity, the internal transcribed spacer rRNA region was sequenced (GenBank Accession Nos. FJ010199, FJ010200, and FJ010201) and compared with the same region of C. boninense (GenBank Accession No. DQ286160.1). Similarity between these sequences was 98 to 99%. The pathogenicity of the three isolates was determined on pepper fruits cv. Amanda. Attached as well as detached fruits from potted plants were inoculated. Inoculation was performed by depositing 40-µl droplets of a suspension (105 conidia per ml) on the surfaces of nonwounded (detached n = 5; attached n = 5) and wounded (detached n = 5; attached n = 5) fruits with a sterilized hypodermic needle. Incubation took place in a moist chamber for 12 days at 25°C with a 12-h photoperiod. Inoculation of control fruits was similar in procedure and number to that of test fruits, except sterile distilled water was used instead of the conidial suspension. Symptoms, observed in wounded and nonwounded test fruits 3 to 5 days after inoculation, were characterized by necrotic, sunken zones containing acervuli, black setae, and orange spore masses. Control fruits presented no symptoms. Pathogens reisolated from infected fruits showed the same morphological and molecular characteristics of the isolates previously inoculated. To our knowledge, this is the first report of C. boninense infecting pepper in Brazil. Reference: (1) J. Moriwaki et al. Mycoscience 44:47, 2003.

Bone regeneration by synthetic octacalcium phosphate and its role in biological mineralization.

Octacalcium phosphate (Ca8H2(PO4)6 * 5H2O; OCP) has been advocated to be a precursor of biological apatite crystals in bone and tooth. Recent studies, using physical techniques, showed that OCP is present as a transient phase during biological apatite formation in human dentin, porcine enamel and murine bone. However, there is still a controversy regarding the chemical nature of the first mineral formed in the biominerals. A number of studies have demonstrated that synthetic OCP shows bone regenerative and biodegradable characteristics, rather than other calcium phosphate bone substitute materials, such as hydroxyapatite (Ca10(PO4)6(OH)2; HA) ceramic. It seems likely that synthetic OCP may be an alternative to autogenous bone graft. It is known that OCP contains alternative layers of water molecules and an apatite structure, and that the transition of OCP to HA is likely to be spontaneous and irreversible. The conversion process induces modification of local environment adjacent to OCP surface, including the changes in adsorption of serum proteins and concentration of calcium and inorganic phosphate ions. This article reviews the possible application to bone regeneration by synthetic OCP and the mechanism to enhance bone regeneration in relation to biological mineralization in bone and tooth.

Computed tomography evaluation of regional lymph node metastases in patients with biliary cancer.

BACKGROUND: Identification of lymph node metastases in biliary cancer is important for determining prognosis and surgical planning, but the effectiveness of computed tomography (CT) in diagnosing node metastases of the hepatoduodenal ligament (peribiliary and retroportal nodes) or around the common hepatic artery is unknown. METHODS: CT scans and pathological results from 146 patients who had undergone regional lymphadenectomy for biliary carcinoma were reviewed. To evaluate the regional lymph nodes, long- and short-axis diameters of lymph nodes were measured and axial ratios calculated (short-axis diameter/long-axis diameter). Nodes were considered round if the axial ratio exceeded 0.7. Internal lymph node structures were also evaluated. RESULTS: The presence of a round node with a short-axis diameter exceeding 16 mm had a positive predictive value (PPV) of 56 per cent for the presence of metastatic foci, and node heterogeneity had a PPV of 64 per cent. The highest PPV (67 per cent) was obtained for round nodes greater than 18 mm in short-axis diameter, but nodes of this size and character were rare. CONCLUSION: CT is not useful for predicting regional lymph nodal metastases in biliary carcinoma.

Analysis of appositional bone formation using a novel rat experimental model.

AIM: To analyze the process of appositional bone formation using our original rat experimental model. MATERIALS AND METHODS: Rats were anesthetized and a ring made of polytetrafluorethylene was placed on the parietal bone surface in the surgical procedure. The time course of appositional bone formation was analyzed with histomorphometry and in situ hybridization for type I collagen and bone sialoprotein. RESULTS: The rat experimental model allowed new bone to be formed on the pre-existing bone surface and persist for 12 weeks. We demonstrated that bone is apposed actively for the first 4 weeks and less actively thereafter. CONCLUSIONS: The experimental model may contribute to biological analysis for appositional bone formation expected to occur in clinical procedures such as alveolar bone augmentation and sinus lifting.

Formation of bone-like apatite enhanced by hydrolysis of octacalcium phosphate crystals deposited in collagen matrix.

It has been shown that granules of synthetic octacalcium phosphate (OCP) or the composites with collagen are capable of enhancing bone regeneration, accompanied by a gradual conversion from OCP to apatite with time. The present study was designed to investigate whether formation of bone-like apatite can be accelerated by OCP deposited throughout collagen matrix (OCP collagen complex, OCC) immersed in simulated body fluid (SBF). The formation of bone-like apatite has been suggested to be essential to induce osteoconductivity of various substrates. The formation of OCP in collagen solution was investigated in calcium or phosphate ions in the range between 22.5 and 142.5 mM and pH 6.26 and 8.56. X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy (SEM) showed that condition to nucleate OCP was limited to that of a solution with Ca/P 0.43 around pH 7.16 in the presence of collagen. OCP was shown to be formed throughout the collagen matrix by SEM observation. The immersion of OCC in SBF up to 10 days enhanced apatite crystal deposition, probably through OCP-apatite conversion: the apatite formation in OCC took place within only 1 day. The present study indicated that the existence of OCP deposited throughout the collagen matrix promotes bone-like apatite formation under physiological condition.

A polymorphism in endostatin, an angiogenesis inhibitor, predisposes for the development of prostatic adenocarcinoma.

We have performed association studies between a novel coding single nucleotide polymorphism (D104N) in endostatin, one of the most potent inhibitors of angiogenesis, and prostate cancer. We observed that heterozygous N104 individuals have a 2.5 times increased chance of developing prostate cancer as compared with homozygous D104 subjects (odds ratio, 2.4; 95% confidence interval, 1.4-4.16). Modeling of the endostatin mutant showed that the N104 protein is stable. These results together with the observation that residue 104 is evolutionary conserved lead us to propose that: (a) the DNA segment containing this residue might contain a novel interaction site to a yet unknown receptor; and (b) the presence of N104 impairs the function of endostatin.

Metabolism of acetylpolyamines by monoamine oxidase, diamine oxidase and polyamine oxidase.

N1-Monoacetylspermine, N1,N12-diacetylspermine and N1-monoacetylspermidine were found to be good substrates for rat liver polyamine oxidase, but not for rat liver mitochondrial monoamine oxidase. N8-Monoacetylspermidine, monoacetylcadaverine, monoacetylputrescine and monoacetyl-1,3-diaminopropane were oxidized by the monoamine oxidase when the substrate concentration was 10.0 mM, but not by the polyamine oxidase. All the acetylpolyamines except N1,N12-diacetylspermine were also oxidized by hog kidney diamine oxidase although their affinities for the oxidase appeared low. The present data suggest that acetylpolyamines are not easily metabolized in vivo by either monoamine oxidase or diamine oxidase in mammalian tissues although N1-monoacetylspermine, N1,N12-diacetylspermine and N1-monoacetylspermidine are attacked by polyamine oxidase.

Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography/mass spectrometry.

A detailed procedure for the assay of free pyrroloquinoline quinone (PQQ) in human and rat samples by gas chromatography/mass spectrometry (GC/MS) has been established with stable-isotopic PQQ as internal standard. PQQ was extracted from the samples, after addition of the internal standard, with butanol under acid conditions and with Sep-Pak C18 cartridges. After derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ by selected ion monitoring, respectively. Trace amounts of free PQQ were detected in eight organs, plasma and urine of the human, and in three organs of the rat. The PQQ level was highest in the human spleen (5.9 +/- 3.4 ng/g tissue, followed by the pancreas and lung, and it was below detection limits for human brain and heart. Trace levels of PQQ were also found in rat small intestine, liver and testis. Our data are far below those measured by the redox cycling method of Gallop's group for human plasma, adrenal and urine.

Post-proline endopeptidase in human placenta.

Post-proline endopeptidase (EC 3.4.21.26) was found in human placenta, purified 3390-fold from it and briefly characterized. The post-proline endopeptidase could be completely separated from dipeptidyl peptidase IV (EC 3.4.14.5) by hydrophobic phenyl-Sepharose chromatography. The pH optimum of the enzyme was 6.7. The Km values for 7-(Succinyl-Gly-Pro)-4- methylcoumarinamide was 1.0 mM. The molecular weight of this enzyme was estimated to be 140 000 by gel filtration and 67 000 by dodecyl sulfate gel electrophoresis, indicating its dimeric structure. Human placental post-proline endopeptidase was suggested to be a thiol proteinase by inhibition studies.

Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae.

House dust mite allergen is thought to be a major cause of asthma. Characterization of these allergen molecules is therefore an important step for the development of effective diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report molecular cloning and expression of the group 6 (chymotrypsin-like) allergen from the house dust mite, Dermatophagoides farinae. Sequencing analysis indicates that cloned cDNA, designated Der f 6, encodes a 279 amino acid polypeptide which conserves a primary structure characteristic for chymotrypsin-like serine proteases found in mammals. Recombinant Der f 6 expressed in Escherichia coli bound IgE in a pool made of 20 sera, and induced histamine release from patients' peripheral blood cells.

Predicting time of parturition from changing vaginal temperature measured by data-logging apparatus in beef cows with twin fetuses.

Vaginal temperatures (VT) of crossbred (Japanese Black crossed Holstein-Friesian) beef cows (n = 31) were measured by a data-logging apparatus to obtain serial data from days 0 to 6 before parturition. For both single and twin pregnancies, no significant differences were observed in VT during days 3-6 before parturition. Maternal VT was not affected by maternal weight just after parturition, parity, fetal sex, or total fetal litter weight. Average of twin litter weights for two males (MM) and two females (FF) had the strong positive correlations (r = 0.84; P < 0.05) with maternal VT, whereas twin weights of mixed-gender twins (FM) did not correlate with maternal VT (r = -0.26; P = 0.61). Maternal temperature decreased as weights of the female fetus of FM twins became heavier (r = -0.82; P < 0.05). In contrast, maternal VT of FF and MM twins increased as twin weights increased. We defined when the VT began to decrease before parturition by two different methods. One was the "same hours method" where differences in VT between a particular time of day and the corresponding time of the preceding day were compared when the VT was consistently > or =0.3 or > or =0.5 degrees C for more than 3h. The second method was the "maximum-minimum method" where decreased in the maximum or the minimum values of the day over > or =0.3 and > or =0.5 degrees C were compared to values of preceding day. Onset of decreased VT before expulsion was not different between singletons and twins. In an attempt to define the critical condition in predicting parturition, we estimated assumable predicting probability using the 31 cows that were collected VT. When the parturition occurred within 60 h in the "same hours method" and 72 h since VT was > or =0.3 degrees C and in the "maximum-minimum method", the assumable probability was 100%. In verification experiment under these condition, the "same hours method" had a higher probability of predicting the time of parturition than the "maximum-minimum method", and it was possible to detect the onset of decreased VT at the correct time by the minutes. We concluded that "same hours method" was useful for predicting parturition time in cattle with single and twin pregnancies by the serial measurement of vaginal temperature.

T-cell epitope analysis of Mag 3, an important allergen from the house dust mite, Dermatophagoides farinae.

Here we describe the detection of T-cell epitope region on the house dust mite allergen Mag 3, which has been shown to trigger T-cell proliferation in mite-allergic asthmatic patients. We first examined murine T-cell epitope using T-cell fraction prepared from recombinant Mag 3 (r-Mag 3)-primed H-2k mice. Initial proliferation assay with truncated r-Mag 3 indicated that N-terminal 113 amino acid region was required for triggering T-cell activation. Subsequent epitope scanning with synthetic overlapping peptides revealed that T-cell reactive region was assigned within amino acid range 56-75. We also explored human T-cell determinant using specific T-cells from mite-allergic patients. Intriguingly, we found that amino acid range 56-85, a portion partially overlapping with that identified in r-Mag 3-primed mice, was exclusively recognized by T-cells from different patients. Further investigation of unique T-cell epitope region found in this study would provide insight into the development of animal therapeutic model and/or peptide vaccine for asthma.

Long-term survival in aged patients with corrected transposition of the great arteries.

Corrected transposition of the great arteries is a rare condition, and few patients with this abnormality survive past 50 years of age because of associated congenital defects or the subsequent development of atrioventricular valvular insufficiency or heart block or both. We describe four men with uncomplicated C-TGA. Our patients are of interest for the following reasons: (a) their condition is very rare; (b) the diagnosis of C-TGA traditionally has been verified through invasive cardiac catheterization procedures; however, in our latest two patients, recently developed noninvasive diagnostic techniques played the decisive role in the diagnosis of C-TGA; (c) in these modalities, they presented as a "natural experimental model" that the right ventricle submitted to a high systemic pressure load is capable of increasing muscle mass over long-term adaptation. Our four patients illustrate that patients with C-TGA, even with the associated cardiac anomalies, may live a normal life span with proper management.

A new enzymatic assay for total diamines and polyamines in urine of cancer patients.

A detailed procedure of a new photometric assay for total diamines and polyamines in human urine using soybean seedling amine oxidase (SSAO) as an enzyme reagent is described. It is based on the unique substrate specificity of SSAO that the enzyme is active toward all diamines and polyamines. The amines were purified from urine by cation-exchange chromatography and incubated with SSAO. Hydrogen peroxide formed in the oxidase reaction was measured photometrically by coupling 4-aminoantipyrine with phenol in the presence of peroxidase. For its simplicity and sensitivity, our method seems useful for routine clinical investigation. The data obtained from normal subjects and patients of various cancers are presented to validate the present method.

A simple fluorometric assay for type B monoamine oxidase activity in rat tissues.

A simple fluorometric assay for monoamine oxidase (MAO) [EC 1.4.3.4] activity towards beta-phenylethylamine (PEA) was devised. The procedure consists in measuring the disappearance of PEA fluorometrically. The disappearance of PEA was completely inhibited by pargyline, a potent inhibitor of MAO. MAO activity for PEA was linear with 10 mg to 100 mg of liver tissue in 3 ml of reaction mixture for up to 90 min of incubation. Using this method, the V max values and the apparent Km values of MAO for PEA in several rat tissues were determined, and compared with those for benzylamine and 5-hydroxytryptamine (5-HT).

Serotonin in neuroblastoma x glioma NG108-15 hybrid cells.

Mouse neuroblastoma x rat glioma NG108-15 hybrid cells contain a considerable amount of serotonin, and possess small but significant tryptophan hydroxylase activity. The results suggest that NG108-15 hybrid cells are serotonergic, in addition to the known cholinergic property.

Determination of phenothiazines in human body fluids by solid-phase microextraction and liquid chromatography/tandem mass spectrometry.

Eleven phenothiazine derivatives with heavy side-chains were found to be extractable from human whole blood and urine samples by solid-phase microextraction (SPME) with a polyacrylate-coated fiber. The fiber was then injected into the desorption chamber of an SPME-liquid chromatography (LC) interface for LC/tandem mass spectrometry (MS/MS) with positive ion electrospray (ES) ionization. All compounds formed base peaks due to [M + 1](+) ions by LC/ES-MS/MS. By use of LC/ES-MS/MS, the product ions produced from each [M + 1](+) ion showed base peaks due to side-chain liberation. Selected reaction monitoring (SRM) and selected ion monitoring (SIM) were compared for the detection of the 11 phenothiazine derivatives from human whole blood and urine. SRM showed much higher sensitivity than SIM for both types of sample. Therefore, a detailed procedure for the detection of drugs by SRM with SPME-LC/MS/MS was established and carefully validated. The extraction efficiencies of the 11 phenothiazine derivatives spiked into whole blood and urine were 0. 0002-0.12 and 2.6-39.8%, respectively. The regression equations for the 11 phenothiazine derivatives showed excellent linearity with detection limits of 0.2-200 ng ml(-1) for whole blood and 4-22 pg ml(-1) for urine. The intra- and inter-day precisions for whole blood and urine samples were not greater than 15.1%. The data obtained after oral administration of perazine or flupentixol to a male subject are presented.

Determination of pentazocine in human whole blood and urine by gas chromatography/surface ionization organic mass spectrometry.

Pentazocine has been found to be measurable with much higher sensitivity by gas chromatography (GC)/surface ionization (SI) organic mass spectrometry (OMS) than by the conventional GC/electron ionization (EI) mass spectrometry. The compound was extracted from human whole blood and urine with Sep-Pak C(18) cartridges before analysis by GC/SIOMS; recoveries were > 96.6% for both samples. The calibration curves were linear in the range 6.25-100 ng ml(-1) and the detection limits were 500 pg ml(-1) of a sample by selected ion monitoring (SIM) with GC/SIOMS. The intra- and inter-day relative standard deviations for the determination of pentazocine in whole blood and urine were not greater than 9.6%. The sensitivity for pentazocine obtained by SI-SIM was about 60 times higher than that obtained by EI-SIM. To validate the present GC/SIOMS method for pentazocine, whole blood and urine samples collected from two volunteers 1-6 h after intramuscular injection of 15 mg of pentazocine were analyzed. The concentrations were 13.5-59.3 ng ml(-1) for whole blood and 0.39-4.00 microg ml(-1) for urine.