Experimental diet-induced atherosclerosis in Quaker parrots (Myiopsitta monachus).

Spontaneous atherosclerosis is common in psittaciformes, and clinical signs associated with flow-limiting stenosis are encountered in pet birds. Nevertheless, a psittacine model of atherosclerosis has not been developed for research investigations. Sixteen captive-bred Quaker parrots (Myiopsitta monachus) were used in this study. While 4 control birds were fed a maintenance diet, 12 other birds were fed an atherogenic diet composed of 1% cholesterol controlling for a calorie-to-protein ratio for periods ranging from 2 to 8 months. The birds were euthanized at the end of their respective food trial period. Histopathology, transmission electron microscopy, and cholesterol measurement were performed on the ascending aorta and brachiocephalic and pulmonary arteries. Plasma lipoproteins, cholesterol, and triglycerides were also measured on a monthly basis. Significant atherosclerotic lesions were induced within 2 months and advanced atherosclerotic lesions within 4 to 6 months. The advanced lesions were histologically similar to naturally occurring lesions identified in the same parrot species with a lipid core and a fibrous cap. Ultrastructurally, there were extracellular lipid, foam cell, and endothelial changes. Arterial cholesterol content increased linearly over time. Plasma cholesterol and low-density lipoprotein (LDL) significantly increased over time by an average of 5- and 15-fold, respectively, with a shift from high-density lipoprotein to LDL as the main plasma lipoprotein. Quaker parrots also exhibited high plasma cholesteryl ester transfer protein activity that increased, although not significantly, over time. This experiment demonstrates that in Quaker parrots fed 1% cholesterol, advanced atherosclerosis can be induced relatively quickly, and lesions resemble those found in other avian models and humans.

Mutations in SIP1, encoding Smad interacting protein-1, cause a form of Hirschsprung disease.

Hirschsprung disease (HSCR) is sometimes associated with a set of characteristics including mental retardation, microcephaly, and distinct facial features, but the gene mutated in this condition has not yet been identified. Here we report that mutations in SIP1, encoding Smad interacting protein-1, cause disease in a series of cases. SIP1 is located in the deleted segment at 2q22 from a patient with a de novo t(2;13)(q22;q22) translocation. SIP1 seems to have crucial roles in normal embryonic neural and neural crest development.

[DLys(6)]-luteinizing hormone releasing hormone-curcumin conjugate inhibits pancreatic cancer cell growth in vitro and in vivo.

Pancreatic ductal adenocarcinomas are invariably lethal, and developing effective treatments that have minimal side effects is a challenge. Previous studies from our laboratory have shown that conjugates of cell membrane disrupting lytic peptides and luteinizing hormone releasing hormone (LHRH) target and destroy human prostate and breast cancer cells in xenografts in the nude mouse model (Hansel et al., Mol Cell Endocrinol 2007;260-262:183-9; Hansel et al., Mol Cell Endocrinol 2007;269:26-33), which express LHRH receptors. The objectives of our study were to synthesize a bioconjugate of LHRH analog ([DLys(6)]-LHRH) and a dietary microchemical (curcumin) and test the hypothesis that [DLys(6)]-LHRH-curcumin targets and inhibits pancreatic cancer cell growth in vitro and in vivo. In in vitro studies, we determined by confocal microscopy, flow cytometry analysis and reverse transcriptase-polymerase chain reaction that MIAPaCa-2, Panc-1 and BxPC-3 pancreatic cancer cell lines express LHRH receptors. [DLys(6)]-LHRH-curcumin inhibited cell proliferation of pancreatic cancer cell lines and induced apoptotic cell death (p < 0.05). Apoptosis was induced by cleavage of polyadenosine-5'-diphosphate-ribose-polymerase and caspase-3. The activity of [DLys(6)]-LHRH-curcumin was equal to free curcumin at equimolar concentrations in vitro. Unlike curcumin itself, the [DLys(6)]-LHRH-curcumin conjugate is water soluble which allows its intravenous administration. In two in vivo studies, [DLys(6)]-LHRH-curcumin given intravenously caused a significant (p < 0.01) reduction in tumor weights and volumes, and free curcumin given by gavage at an equal dose failed to cause a significant reduction in tumor weights and volumes in the nude mouse pancreatic cancer model. [DLys(6)]-LHRH-curcumin treatment enhanced apoptosis compared to [DLys(6)]-LHRH and vehicle-treated controls in tumor tissue. In conclusion, [DLys(6)]-LHRH-curcumin may be useful in treating pancreatic cancer.

Diagnostic Exercise: oral tumor in an aged mare.

A 17-year-old, Thoroughbred mare was presented for necropsy with a large, invasive oral mass determined via immunohistochemistry to be a soft tissue sarcoma of neural origin. Oral sarcomas are rare in veterinary medicine, and to the authors' knowledge, this is the first oral sarcoma of neural tissue origin reported in a horse.

Mowat-Wilson syndrome: facial phenotype changing with age: study of 19 Italian patients and review of the literature.

Mowat-Wilson syndrome (MWS; OMIM #235730) is a genetic condition caused by heterozygous mutations or deletions of the ZEB2 gene, and characterized by typical face, moderate-to-severe mental retardation, epilepsy, Hirschsprung disease, and multiple congenital anomalies, including genital anomalies (particularly hypospadias in males), congenital heart defects, agenesis of the corpus callosum, and eye defects. Since the first delineation by Mowat et al. [Mowat et al. (1998); J Med Genet 35:617-623], approximately 179 patients with ZEB2 mutations, deletions or cytogenetic abnormalities have been reported primarily from Europe, Australia and the United States. Genetic defects include chromosome 2q21-q23 microdeletions (or different chromosome rearrangements) in few patients, and ZEB2 mutations in most. We report on clinical and genetic data from 19 Italian patients, diagnosed within the last 5 years, including six previously published, and compare them with patients already reported. The main purpose of this review is to underline a highly consistent phenotype and to highlight the phenotypic evolution occurring with age, particularly of the facial characteristics. The prevalence of MWS is likely to be underestimated. Knowledge of the phenotypic spectrum of MWS and of its changing phenotype with age can improve the detection rate of this condition.

An unusual splicing mutation in the HEXB gene is associated with dramatically different phenotypes in patients from different racial backgrounds.

Sandhoff disease is caused by mutations affecting the beta subunit of lysosomal beta-hexosaminidase (EC 3.2.1.52) and displays a wide spectrum of clinical phenotypes. We report a 57-year-old patient with a very mild phenotype, although residual hexosaminidase A activity in his cultured fibroblasts was less than 3% of normal activity, a level observed in juvenile onset patients. Northern and Western blot analyses confirmed a similar low level of beta subunit-mRNA and mature beta-protein, respectively. Two mutations of the HEXB gene were identified in this patient, a partial 5' gene deletion (a null allele), and a C----T transition 8 nucleotides downstream from the intron 10/exon 11 junction affecting the splicing of the beta subunit-mRNA. In their homozygous forms, the 5' deletion has been previously shown to result in a severe infantile phenotype, and the C----T transition in a juvenile phenotype. The genotype and the low level of residual hexosaminidase A activity would be expected to produce a juvenile Sandhoff phenotype in this patient, as well as in four of his six clinically normal siblings. The biochemical basis of his mild phenotype is uncertain, but may result from genetic variations in the RNA splicing machinery.

A rare case of complete human erythrocyte AMP deaminase deficiency due to two novel missense mutations in AMPD3.

Human erythrocyte AMP deaminase (AMPD3) deficiency is a clinically asymptomatic condition characterized by a 50% increase in steady-state levels of ATP in affected cells. The deficiency in Japanese is associated 75% of the time with the same mutation of R573C, and 25% of the time with other heterogeneous mutations of the AMPD3 gene. The heterozygote frequency was estimated at about 1/30. We previously reported five Japanese individuals who had a complete deficiency of AMPD3. Four were homozygotes for the major mutation of R573C; however, one female did not have the R573C allele. To clarify the mutations in her AMPD3 gene, we analyzed the AMPD3 gene and detected a minor mutation, W450R, derived from the mother and a novel mutation,Q712P, derived from the father. The expression study using AMPD3 cDNA containing both mutations showed that each mutation completely reduced the enzyme activity of AMPD3. As the frequency of carriers heterozygous for these mutations seems to be very low, identifying them may lead to a better understanding of the genetic background of populations in Japan.

Molecular analysis of Japanese patients with Rett syndrome: Identification of five novel mutations and genotype-phenotype correlation.

Rett syndrome is an X-linked dominant neurodevelopmental disorder that affects females almost exclusively. The recent identification of mutations of the methyl-CpG-binding protein 2 gene (MECP2) in patients with RTT, encouraged us to analyze the gene in 37 Japanese patients divided into classical RTT (14 cases), variant RTT (13 cases), and mentally retarded patients with Rett-like features (10 cases). Mutations in MECP2 were identified from most of the patients with classical and variant RTT (25 of 27 cases). Six reported common mutations were detected in 17 cases, and rare single nucleotide substitutions were found in 3 patients. In addition, one insertion mutation (1189insA) and four deletion mutations including one double deletion mutant (451delG, 100del4, 1124del53 and 881del289 plus 1187del8) were newly identified. In the 10 mentally retarded patients with Rett-like features, however, no mutation was detected in the coding region of MECP2. The finding of MECP2 mutations in 92.5% of patients with RTT indicates that RTT fulfilling the diagnostic criteria are due to genetic alteration.

Characterization of the human MANB gene encoding lysosomal alpha-D-mannosidase.

Genomic clones of human MANB gene encoding the lysosomal enzyme, alpha-mannosidase, have been isolated, sequenced and analyzed. The human MANB gene spans approximately 22 kb and consists of 24 exons. The 5' flanking region of the gene shows a high G+C content and has two Sp1 and three AP-2 sites. Promoter analysis using deletion constructs of the 5' flanking region fused to the bacterial CAT gene showed that 150 bp of 5' sequence could drive the expression of MANB in COS 7 cells. Determination of the sequence of the 5' end of the alpha-mannosidase mRNA by 5' RACE protocol showed that transcription is initiated from a cluster of sites centered -28 and -20 bp from the first in-frame ATG. These data demonstrate that, like other lysosomal enzyme genes such as those for beta-glucuronidase or beta-hexosaminidase, the human MANB gene is controlled by a short 5' flanking sequence located near the initiation codon.

Late infantile Hirschsprung disease-mental retardation syndrome with a 3-bp deletion in ZFHX1B.

A 48-year-old woman with late infantile onset mental retardation developed megacolon. Although the patient had no typical clinical features of Hirschsprung disease-mental retardation syndrome, a new 3-base pair deletion, eliminating an Asn, was identified in the responsible gene ZFHX1B. This suggests that screening for ZFHX1B mutations is warranted even in the absence of typical clinical features of the syndrome.

Different tissue distributions of two types of thiol proteinase inhibitors from rat liver and epidermis.

The concentrations of two types of endogenous inhibitors of thiol proteinases were determined in soluble extracts of various rat tissues by means of a sensitive enzyme immunoassay method, which consisted of solid-phase immobilized anti-rat liver inhibitor or anti-rat epidermal inhibitor and antibodies labeled with horseradish peroxidase. The minimum detectable amounts of inhibitors from liver and epidermis were 30 pg and 3 pg/assay, respectively. The tissue distributions of the epidermal and liver-type inhibitors were found to differ. The liver-type inhibitor was found to be widely distributed in various tissues at levels of 76-420 ng/mg protein, whereas the epidermal-type inhibitor was found at high levels in the skin, tongue, esophagus, stomach, intestine, and vagina, but at quite low levels in other tissues tested.

Modification of rat liver fructose biphosphate aldolase by lysosomal proteinases.

In vivo proteolytic modification of liver aldolase on administration of leupeptin, a thiol proteinase inhibitor of microbial origin, is reported. When leupeptin was injected into rats, the activity of aldolase in the liver decreased to 40% of that in control rats. Molecular properties of aldolase isolated from the livers of control rats and leupeptin-treated rats indicated that a decrease of aldolase activity is attributable to hydrolysis of a peptide linkage(s) near the carboxyterminal of the enzyme. Injection of leupeptin also caused marked increase in the activities of free lysosomal proteinases, such as cathepsin A and cathepsin D and moderate increase of cathepsin B and cathepsin L. Increase in free activity of cathepsin A returned to the level of control rats by 12 hr after injection of leupeptin, whereas 36 hr was required for recovery of decreased aldolase activity. When insulin was coinjected with leupeptin, increase in the activity of free cathepsin A and decrease of activity of aldolase produced by the injection of leupeptin was prevented. These findings indicate that modification of aldolase may be due to action of a lysosomal protease(s). Incubation of the purified aldolase with the lysosomal fraction produced the same changes in properties of aldolase as those observed in vivo on injection of leupeptin. The aldolase inactivating proteinase in the lysosomal fraction was inhibited by PMSF and leupeptin and not by pepstatin. Purified cathepsin A (a serine proteinase), cathepsin B and cathepsin L (thiol proteinase) are potent inactivators of aldolase but cathepsin H and cathepsin D are not. Cathepsin A, B and L are involved in inactivation of aldolase in lysosomes. Endogenous thiol proteinase inhibitor which inhibits lysosomal thiol proteinases (cathepsin B, L and H) is found in the cytosol fraction of liver. The level of thiol proteinase inhibitor actually decreased to 60% of that in control rats in leupeptin-treated rats, suggesting that non-thiol proteinase cathepsin A is a major factor in inactivation of aldolase in lysosomes. Not only leupeptin but also other proteinase inhibitors (antipain, E-64-D, chloroquine) caused increase of labilization of the lysosomes and decrease in aldolase activity. Physiological stimuli which are known to induce the labilization of the lysosomal membrane, such as starvation and glucagon, caused slight or no significant increase of activities of free cathepsin A and D and resulted in no apparent change in aldolase activity.

Influence of carbonate on sintering of apatites.

Sintering of carbonate apatite, prepared at 100 degrees C and pH 9.0 for 3 days, was studied by thermal analysis, x-ray diffraction, and infrared spectroscopy. The sintering temperature, at which the linear thermal shrinkage of isostatically compacted specimens increased sharply, decreased in proportion to the amount of carbonate initially present in the apatite. For example, specimens with over 8 wt% carbonate could be sintered at a temperature (650 degrees C) which was nearly 400 degrees C lower than that needed for sintering a specimen with no carbonate. Amounts of carbonate lost at the end of sintering, estimated chemically and by infra-red spectroscopy, were approximately equal to sample weight losses estimated thermogravimetrically.

Memory for subject performed tasks in patients with Korsakoff syndrome.

We examined the ability of alcoholic Korsakoff patients to remember verbal and action-related information. Eight Korsakoff patients and eight alcoholic control subjects learned action phrases in either subject-performed tasks (SPTs) or verbal tasks (VTs). Free recall and recognition tests were then administered. Despite the severe anterograde amnesia observed in Korsakoff patients for VTs, their memory performance for SPTs was similarly facilitated over VTs as was the case with alcoholic controls. Domains preserved in this amnesic syndrome may account for the benefit seen when using SPTs in Korsakoff patients. The therapeutic utilization of action events for memory rehabilitation is discussed.

Effects of non-collagenous proteins on the formation of apatite in calcium beta-glycerophosphate solutions.

The effects of the non-collagenous proteins; osteonectin, bone Gla protein and dentine phosphoprotein, on the formation of apatite were studied in calcium beta-glycerophosphate solutions containing catalytic amounts of alkaline phosphatase under physiological conditions. In the system used, calcium phosphate precipitates de novo at levels of supersaturation precisely determined through the enzymatic hydrolysis of beta-glycerophosphate. At 1.7 mM of calcium beta-glycerophosphate, calcium phosphate precipitated when inorganic phosphate accumulated to about 1.4 mM. In the presence of the proteins, however, a greater accumulation of inorganic phosphate was needed for calcium phosphate to precipitate, suggesting that a higher degree of supersaturation, though still a slight undersaturation with respect to dicalcium phosphate dihydrate, is required for calcium phosphate to precipitate in the presence of the proteins. At the same protein (micrograms/ml) concentration, dentine phosphoprotein was approximately four times as effective as bone Gla protein, which was about twice as effective as osteonectin in delaying precipitation. The proteins also retarded subsequent crystal growth, with apatite formed in the presence of the more inhibitory proteins having the smallest crystals, especially in width.

Mutations in the hypoxanthine guanine phosphoribosyltransferase gene (HPRT1) in Asian HPRT deficient families.

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified 34 mutations in 28 Japanese, 7 Korean, and 1 Indian families with the patients manifesting different clinical phenotypes, including two rare cases in female subjects, by the analysis of all nine exons of HPRT from the genomic DNA and reverse transcribed mRNA using PCR technique coupled with direct sequencing.

[Juvenile Sandhoff disease with local panatrophy--a case report].

A case of juvenile Sandhoff disease with Gowers' local panatrophy was reported. A mentally retarded 39 year-old man had been noticed to have localized skin atrophy on the right leg since 7 years of age, and was also aware of the atrophy of his right leg since his junior high school age. His parents were first cousins. He had multiple localized atrophic skin lesions on the right side of his nape, the right side of lower back, and the right thigh. These skin lesions did not show any signs of sclerosis. Although he was mentally retarded (IQ=44), the neurological examination was normal including funduscopy. Histological examination of the atrophic skin le led that the epidermal basal layer was hyperpigmented and that the dermis and subcutaneous fatty tissue were extremely atrophic. The nerves meandered across the dermis and subcutaneous tissue, and had membranous lipid storage cytosomes in the axon and Schwann cells. Biochemical studies revealed marked deficiency fo leukocyte hexosaminidase activity. Total hexosaminidase activity was decreased to 18% of normal controls, and hexosaminidase B activity was completely deficient. Other lysosomal enzymes in leukocytes were normal in activities. Both parent showed intermediate levels of leukocyte hexosaminidase A and B activities. Electron microscopy analysis of a rectal biopsy specimen showed neuronal accumulation of dense osmophilic deposits, as well as membranous cytoplasmic bodies in Meissner's plexus. On the basis of the patient's age at onset and the above findings, the patient was diagnosed to have juvenile Sandhoff disease. The local panatrophy observed in the present case could be the result of the accumulation of GM2-ganglioside in autonomic nervous system, and may well be a new clinical feature of GM2-gangliosidosis.

[A case of amyotrophic lateral sclerosis associated with clinical features of progressive supranuclear palsy].

A 64-year-old male patient of amyotrophic lateral sclerosis (ALS) with frozen gait, axial rigidity and supranuclear upper gaze palsy was reported. We have followed this patient more than four years. He was well until November 1982, when he noticed weakness of left arm. In March 1983, he noticed hypogeusia and in July, he developed dysarthria and frozen gait. On admission, he was alert and oriented. Neurological examination revealed dysarthria, dysphagia and muscular weakness and atrophy in bilateral upper extremities, dominantly in left side. He showed remarkable frozen gait, retropulsion and could not walk. Brain CT showed mild dilatation of the third ventricle. In August 1988, he showed tongue atrophy, and weakness and atrophy of the extremities progressed during these four years. He also showed axial rigidity and frozen gait. Brain CT showed severe third ventricular dilatation and atrophy of tegmentum of the midbrain and cerebellum that were compatible with progressive supranuclear palsy (PSP). Six months later, he developed upper gaze palsy. From these findings, we concluded that this patient had a quite unique clinical features of both ALS and PSP.

[A case of cerebrotendinous xanthomatosis with spastic paraparesis, epilepsy, and bradykinesia].

A 26-year-old female developed mental deterioration, general convulsion, cataract and spastic gait in order since her entrance into elementary school. A diagnosis of cerebrotendinous xanthomatosis (CTX) was made because of hypercholestanolemia. At the time of admission, cataract, a mild thickening of Achilles tendons, mental deterioration, spastic paraparesis, truncal ataxia, and bradykinesia were noted. Bilateral slowing of 2 to 7 Hz was recorded in EEG, and brain CT and MRI revealed mild cerebellar atrophy. HVA and 5-HIAA levels in CSF were low. Oral administration of chenodeoxycholic acid, 300 mg per day, resulted in improvement of bradykinesia and EEG abnormality, increase of HVA and 5-HIAA levels in CSF, and decrease of serum cholesterol level in two weeks. Bradykinesia observed in the present case is a rare clinical finding of CTX, and the improvement of bradykinesia soon after the treatment with chenodeoxycholic acid has not been reported yet. This case is important for elucidating the mechanism of neurological disorders in CTX.

[Recent advances in molecular genetics of GM2 gangliosidosis].

Recent advances in molecular genetics of GM2 gangliosidosis are reviewed. GM2 gangliosidosis is an autosomal recessive, neurodegenerative disease caused by a deficiency of beta-hexosaminidase (Hex, EC 3.2.1.52) A activity, resulting in accumulation of GM2 ganglioside in the lysosomes of neuronal cells. There are two catalytically active forms of this enzyme: Hex A, composed of one alpha and one beta subunits. Three forms of this disease, Tay-Sachs disease, Sandhoff disease, and GM2 activator deficiency, have been recognized according to whether the defect involves the alpha subunit, beta subunit, or GM2 activator protein, respectively. A number of gene abnormalities responsible for the disease have been identified and mutations specific for phenotypes and racial backgrounds are summarized. Recently, the murine models of human Tay-Sachs disease and Sandhoff disease have been produced. With the finding of dramatically clinical phenotypes in these mice, these models could be useful for research on the pathogenesis or therapy of these diseases.