Effect of 'attenuated' mutations in mucopolysaccharidosis IVA on molecular phenotypes of N-acetylgalactosamine-6-sulfate sulfatase.

Mucopolysaccharidosis IVA is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS gene was performed for seven MPS IVA patients with attenuated phenotypes from three unrelated families. Four of 5 missense mutations identified in this study (p.F167V, p.R253W, p.R380S, p.P484S) and two reported (p.F97V, p.N204K), associated with attenuated phenotypes, were characterized using in vitro stable expression experiments, enzyme kinetic study, protein processing and structural analysis. The stably expressed mutant enzymes defining the attenuated phenotype exhibited a considerable residual activity (1.2-36.7% of the wild-type GALNS activity) except for p.R380S. Enzyme kinetic studies showed that p.F97V, p.F167V and p.N204K have lower affinity to the substrate compared with other mutants. The p.F97V enzyme was the most thermolabile at 55 degrees C. Immunoblot analyses indicated a rapid degradation and/or an insufficiency in processing in the mutant proteins. Tertiary structure analysis revealed that although there was a tendency for 'attenuated' mutant residues to be located on the surface of GALNS, they have a different effect on the protein including modification of the hydrophobic core and salt-bridge formation and different potential energy. This study demonstrates that 'attenuated' mutant enzymes are heterogeneous in molecular phenotypes, including biochemical properties and tertiary structure.

[Left ventriculoplasty for ischemic cardiomyopathy with a left ventricular aneurysm].

We report 3 cases of left ventriculoplasty (LVP). They were chosen according to classification of the preoperative left venticle (LV) shape; an apex type and anteroseptal type. We think that an apex type has an indication for a Dor operation and the treatment of an anteroseptal type should be chosen between the following 2 methods. One is an overlapping method. It has the advantage of having to use no intracardiac patch which would remain akinetic area. It is therefore suitable for relatively small LV aneurysms without involvement of the proximal diagonal branches. However, it has the disadvantage of having to cut some distal diagonal branches in order to perform the volume reduction. The other method is a septal anterior ventricular exclusion (SAVE) operation. It is suitable for larger LV aneurysms which involve the proximal diagonal branches due to its advantage of being able to perform the LVP without cutting the diagonal branches. However, it has the disadvantage of leaving an akinetic area that corresponds to the intracardiac patch. We believe that choice of the LVP method according to the preoperative LV shape will bring about a better postoperative LV function and shape.

The mouse N-acetylgalactosamine-6-sulfate sulfatase (Galns) gene: cDNA isolation, genomic characterization, chromosomal assignment and analysis of the 5'-flanking region.

Deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) leads to mucopolysaccharidosis IV A (MPS IV A), for which there is no definitive treatment so far. Although a number of mutations of the GALNS gene of MPS IV A patients have been described, pathogenesis of the disorder still remains elusive. In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kb cDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84% similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was isolated from S129vJ genomic library and its genomic organization was characterized. The mouse Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron-exon splice junctions conformed to the GT/AG consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between -44 and -75 although major transcription initiation site was observed at -90 bp from the ATG codon. The 5'-flanking region lacks canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.

Mitochondrial trifunctional protein deficiency associated with recurrent myoglobinuria in adolescence.

A 23-year-old man with recurrent myoglobinuria had low muscle-free carnitine levels and deficient fasting ketogenesis. Urinary organic acid analysis showed large amounts of C6-C14 3-hydroxydicarboxylic acids. Mitochondrial trifunctional protein (TP), harboring long-chain enoyl-coenzyme A (CoA) hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase showed markedly decreased activity in fibroblasts. On immunoblot analysis, the TP content of his fibroblasts was less than 2% that of the control cells. TP deficiency can be a life-threatening disorder with early infantile onset, but it can also present in adolescence with recurrent myoglobinuria.

Acyl-CoA thioesterases belong to a novel gene family of peroxisome proliferator-regulated enzymes involved in lipid metabolism.

Acyl-CoA thioesterases hydrolyze acyl-CoAs to the corresponding free fatty acid plus coenzyme A. The activity is strongly induced in rat and mouse liver after feeding the animals peroxisome proliferators (PPs). To elucidate the role of these enzymes in lipid metabolism, the authors have cloned the cDNAs corresponding to the inducible cytosolic and mitochondrial type I enzymes (CTE-I and MTE-I), and studied tissue expression and nutritional regulation of expression of the mRNAs in mice. The constitutive expression of both mRNAs was low in liver, with CTE-I expressed mainly in kidney and brown adipose tissue, and MTE-I expressed in brown adipose tissue and heart. As expected, the expression in liver of both the CTE-I and MTE-I mRNAs were strongly induced (> 50-fold) by treatment with clofibrate. A similar level of induction was observed by fasting and a time-course study showed that the CTE-I and MTE-I mRNAs were increased already at 6 h after removal of the diet. Refeeding normal chow diet to mice fasted for 24 h normalized the mRNA levels with a T1/2 of about 3-4 h. Feeding mice a fat-free diet further decreased the expression, possibly indicating repression of expression. The strong expression of MTE-I and CTE-I in the heart was increased about 10-fold by fasting. To further characterize these highly regulated enzymes, the authors have cloned the corresponding genes and promoter regions. The structures of the two genes were found to be very similar, consisting of three exons and two introns. Exon-intron borders conform to general consensus sequences, and, especially, the first exon appears to be highly conserved. The promoter regions of both the CTE-I and MTE-I genes contain putative PP response elements, suggesting an involvement of PP-activated receptors in the regulation of these genes.

Injection sclerotherapy of esophageal varices for patients undergoing emergency and elective surgery.

From October 1977 to September 1981, 68 patients with esophageal varices (30 emergency cases of bleeding and 38 elective cases) were treated by injecting 5% ethanolamine oleate into varices, using an esophagofiberscope. Esophageal bleeding was successfully controlled in 29 of 30 patients who had emergency surgery. None of the 38 patients who underwent elective operation had bleeding after treatment. When recurrence occurred 1 or 2 years after treatment, the same procedure was repeated. Pleuritis occurred in one of the patients who had emergency surgery, and bleeding (300 to 400 ml) from the esophagocardial junction occurred in two patients who underwent elective operation. These patients were treatment conservatively.

Choledocholithotomy by Yag laser with a choledochofiberscope: case reports of two patients.

The Neodymium-Yag laser was successfully used with a choledochofiberscope for choledocholithotomy in two patients. The bile duct stones were crushed with the Yag laser and removed or pushed into the duodenum without causing any injury. This procedure is highly recommended for treatment of some patients with bile duct stones.

Two patients with acute hepatitis B with suspected sexual transmission of hepatitis G virus.

Two patients with acute hepatitis B with suggested sexual transmission of hepatitis G virus (HGV) are reported. A total of 18 patients with community acquired acute hepatitis B were analyzed in this study. Two of the 18 patients (patients 1 and 2) were positive for serum HGV RNA at the initial consultation. Both patients had had sexual contact with prostitutes several weeks before the onset of acute hepatitis, and hepatitis B virus (HBV) was suggested to be infected through the sexual contacts. These patients showed no other history of exposure to possible transmission routes for blood-borne hepatitis viruses. Patient 1 was diagnosed as with acute HGV infection because the antibody to HGV envelope-2 protein seroconverted to positive during the course of acute hepatitis. HGV RNA was negative in a serum sample collected from patient 2 before the onset of acute hepatitis, also suggesting acute HGV infection. These results indicate that in patients 1 and 2 HGV was infected along with HBV through sexual contact. The clinical manifestations of acute hepatitis in the two patients with HGV co-infection did not differ from those in the 16 patients with HBV infection alone.

Transmission of and liver injury by TT virus in patients on maintenance hemodialysis.

To study the prevalence and clinical significance of TT virus (TTV) infection in hemodialysis patients, we tested for TTV DNA in serum, using the nested polymerase chain reaction. The prevalence of TTV DNA in 352 hemodialysis patients was 32%, significantly higher than that in 50 healthy blood donors (12%). The prevalence increased with age (P = 0.0098): it was 20% (22/110) in patients aged less than 49 years, 37% (69/188) in those aged 50-69 years, and 41% (22/ 54) in those aged over 70 years. Other clinical features and the prevalence of other hepatitis viral markers tested did not differ between patients with TTV DNA and those without it. The detection rate of hepatitis C virus (HCV) and hepatitis G virus (HGV) viremias increased with duration of hemodialysis and with the number of blood transfusion units, but the prevalence of TTV viremia did not. Twenty-nine of 91 patients followed for 5 years were initially positive for TTV DNA. Of these 29 patients, 17 (59%) carried this viremia for at least 5 years. Fourteen of the 62 patients (23%) who were initially negative for TTV DNA acquired TTV viremia. Serum alanine aminotransferase (ALT) levels were elevated in patients with HCV viremia but not in patients with HGV or TTV viremia. However, the mean ALT level in patients with all three viremias (HCV, HGV, and TTV) was significantly higher than that in patients with one or two of the viremias. More than 30% of the hemodialysis patients had TTV viremia and the carrier state was maintained for years. The hemodialysis procedures, including blood transfusion, did not seem to be crucial for the transmission of TTV. The pathogenic effects of TTV on hepatitis appear to be limited.

Relationship between hepatitis C virus infection and schistosomal liver disease: not simply an additive effect.

BACKGROUND: To study the association, clinical significance, and impact of hepatitis C virus (HCV) co-infection in patients with schistosomal liver disease (SLD). METHODS: A total of 240 patients with chronic liver diseases encountered consecutively were enrolled in the study. Fifty volunteer blood donors were enrolled as controls. HCV antibody determination (enzyme-linked immunosorbent assay), qualitative and quantitative HCV RNA assay (reverse transcriptase polymerase chain reaction), and HCV genotyping (line probe assay) were performed. RESULTS: Twenty-eight patients had SLD alone, 60 had both SLD and chronic hepatitis C (CH-C), 120 had CH-C alone, and 32 had other liver diseases. The positivity rates for HCV antibody (76% vs 20%; P < 0.001) and HCV RNA (59% vs 10%; P < 0.001) were significantly higher in the patients with SLD (n = 88) than in the volunteer blood donors (n = 50). Complications of liver cirrhosis were more common in patients with concomitant SLD and CH-C than in those with either SLD or CH-C alone. The mean levels of alanine aminotransferase (77 +/- 42 vs 93 +/- 55 IU/l; P = 0.049) and HCV RNA concentrations (3.5 +/- 1.0 vs 4.2 +/- 1.0 log copy/ml; P < 0.001) were significantly lower in patients with concomitant SLD and CH-C than in those with CH-C alone. HCV genotype 4 predominated in both these groups (93% and 98%). CONCLUSIONS: SLD in Egypt is significantly associated with HCV infection, with the predominance of genotype 4. Concurrent HCV infection and SLD result in much more severe liver disease than that seen with either disease alone. However, the activity of HCV infection seems to be partially suppressed in patients with SLD.

Seroepidemiological study of hepatitis E virus infection in Japan using a newly developed antibody assay.

PURPOSE: A seroepidemiological study of hepatitis E virus (HEV) infection was conducted in Japan, where HEV infection is not considered endemic. METHODS: IgG and IgM class antibodies to HEV were measured with a newly developed enzyme-linked immunosorbent assay in which recombinant virus-like particles were used as an antigen. A total of 1253 individuals (401 males and 852 females; age range, 6-89 years) were enrolled from two different areas: area 1 (n = 478), in which hepatitis C was endemic; and area 2 (n = 775), in which it was not endemic. RESULTS: The HEV antibody (IgG class) positive rate was 6.7% in area 1 and 4.6% in area 2. Similarly, the HAV antibody (IgG class) positive rates were 65.3% and 72.3%. The age- and sex-specific prevalence of both HAV and HEV antibodies was quite similar in the two areas, and the HAV antibody positive rate clearly increased with age in both males and females. On the other hand, the HEV antibody positive rate showed a slight tendency to increase with age in males, but not in females. None of the 32 individuals with the HEV antibody who were interviewed had a history of visiting countries in which hepatitis E was endemic. In both areas, the mean age, percentage of males, and HAV antibody positive rate were significantly higher in the group of individuals with the HEV antibody than in the group of those without it, according to conventional statistical analyses. Of the three factors age, male sex, presence of HAV antibody, and the area factor, only male sex was statistically significant (P < 0.001) on multivariate logistic regression analysis. Two (0.2%) of the total of 1253 individuals were positive for the IgM class antibody to HEV. CONCLUSIONS: Our results suggest the possibility that HEV infection is circulating in Japan at a low level. HEV infection was associated with male sex, but not with HAV infection.

Long-term duration of reduced serum complement level following burn injury.

We report the case of a boy whose serum CH50 was below the detection limit following burn injury and skin transplantation. The APCH50 level was slightly decreased, although C3, C4, and the other complements were within the normal range. Cold activation was not detected in his plasma. His peripheral blood monocyte ratio slightly elevated to 19% and then decreased to 5.9%. In this case, burn injury caused the depletion of the complement, particularly in the alternative pathway, and resulted in the reduced CH50 level, although C3 did not show the typical pattern of alternative pathway depletion. In previously reported cases of burn injury, the CH50 level returned to the normal range within 2 weeks. In this patient the reduced level of CH50 continued for 4 months. We should consider burn injury one of the causes of complement deficiency even in cases with a duration of more than 1 month.

Metastatic small cell lung cancer causing biliary obstruction.

We report a case with metastatic small cell lung cancer which first manifested with biliary obstruction due to metastasis. Prognosis of patients presenting with jaundice due to hepatic parenchyma involvement is thought to be poor. However, the patient was successfully treated with percutaneous transhepatic biliary drainage and combination chemotherapy with reduced dosage. We believe this to be the first such case report, despite the frequency of metastasis to the liver from small cell lung cancer.

Intense accumulation of indium-111 leukocytes in peritonitis carcinomatosa.

In order to detect the infectious foci in a case of terminal recurrent cancer of the sigmoid colon with intense inflammation, In-111 oxine leukocyte scintigraphy was performed. Leukocytes labeled with In-111 oxine quickly localized within the region of peritonitis carcinomatosa and could be imaged after 4 hours. With time, high activity appeared in this area. And 48 hours after injection, the large intestine was clearly seen. However, no activity was seen in the main recurrent tumor. This suggested that the labeled leukocytes had accumulated in regions of inflammation rather than in malignant tissue. When performing In-111 leukocyte scintigraphy for diseases in which tumor cells and inflammation are mixed, distinguishing the two components is particularly important, and time-sequential scanning is very useful.

Definitive proton beam radiation therapy for inoperable gastric cancer: a report of two cases.

Proton beam radiation therapy using 250 MeV protons was carried out on two patients with early gastric cancer (T1, N0, M0). One patient was an 85-year-old man with early gastric cancer of type IIa + IIc. The other one was a 70 year old man with early gastric cancer of type IIc. In both cases histological examination of biopsy specimens showed differentiated adenocarcinoma; distant metastasis was not found by other examinations. Both patients were considered inoperable due to their poor cardiac and/or respiratory functions. Therefore, it was decided to treat them by definitive proton irradiation, delivering total doses of 86 Gy and 83 Gy, respectively. In both patients, skin erythema that did not require any special treatment was found in the irradiation field. Hematobiological examinations did not show any abnormality. Although endoscopic examination at two years after irradiation in the former case and at seven months in the latter case showed persistent gastric ulcer at the site of the cancerous lesions, cancer cells were not found histologically. Therefore, we concluded that proton irradiation therapy was useful for inoperable early gastric cancers.

Prevention of postoperative pericardial adhesions with a defibrinogenating agent.

Pericardial adhesions pose a major problem during re-operative cardiac surgical procedures. The purpose of this study was to determine the effect of Batroxobin, a fibrinogen depleting agent, on the incidence of experimental pericardial adhesions. Twenty-four rabbits were divided into 3 groups of 8 rabbits each and pericardial mesothelial injury was induced by abrasion. Group A included rabbits receiving no further treatment, Group B included rabbits receiving intrapericardial injection of Ringer's solution, and Group C included rabbits receiving intrapericardial injection of Batroxobin. Three weeks after surgery, the incidence of adhesions in Groups B and C was compared with that in Group A. Pericardial adhesions found in 80% of the rabbits in Groups A and B. However, only 14% of the rabbits in Group C had pericardial adhesions (p < 0.05). Our findings demonstrated that intrapericardial Batroxobin reduced the incidence of pericardial adhesions in an animal model.

Schmidt syndrome due to idiopathic accessory nerve paralysis.

A patient with Schmidt syndrome due to idiopathic accessory neuropathy has had paralysis of soft palate and larynx as well as trapezius and sternocleidomastoid muscles, reflecting involvement of cranial and spinal roots, respectively. In addition to vocal cord paralysis on the laryngoscopy, videofluoroscopy confirmed diminished mobility of the soft palate. Electrodiagnosis showed accessory neuropathy at the proximal segment. Furthermore, the accessory nerve innervated not only upper but also middle and lower portions of the trapezius muscles.

Aneurysm of the left sinus of Valsalva causing aortic, mitral regurgitation and myocardial ischemia.

An aneurysm of the left sinus of Valsalva producing aortic and mitral regurgitation with myocardial ischemia was treated successfully by reconstructing the left coronary sinus while preserving the aortic cusp combined with coronary artery bypass grafting. Aortic and mitral regurgitation occurred due to distortion of the left aortic cusp by a huge aneurysm that also compressed and obstructed the main trunk of the left coronary artery. The postoperative course was uneventful and follow-up showed aortic and mitral regurgitation to be absent and the coronary graft to be patent. Aortic valve-sparing surgery thus proved to be an appropriate procedure for this case.

[Electrophysiological monitoring of the brainstem function in impending brain death--serial changes of blink reflex and brainstem auditory evoked potential].

Serial changes of EEG, BR and BAEP recordings were obtained over a period of two days on two patients who had suffered massive cerebral hemorrhage while their clinical condition evolved from coma with evidence of preserved cerebral and brainstem functions to a state meeting the criteria of brain death. As clinical evidence of deteriorating brainstem function became apparent in case 1, first wave IV and V components of BAEP disappeared while waves I to III were normal. Finally, when clinical criteria of brain death were fulfilled, the BAEP response was restricted to wave I with small amplitude to stimulation of left ear only. These serial changes were likely consistent with gradual dissolution of brainstem function in a rostrocaudal direction. By contrast, in case 2, the BAEP response was restricted to waves I and II and was stationary in the whole process of impending brain death in no association with some preservation of cerebral and brainstem function. The changes of BAEP was not parallel to the progressive deterioration of EEG and BR. After meeting clinical criteria of brain death, complete abolition of waves II and I was sequential in that order, and then Babinski sign besides withdrawal and deep tendon reflexes may revive in the extremities. Monitoring of serial electrophysiological changes is helpful in the course of impending brain death to determine whether revival of Babinski sign is due to recovery of cerebral-brainstem dysfunction or due to establishment of spinal autonomy.