The peroxin pex3p initiates membrane assembly in peroxisome biogenesis.

Rat cDNA encoding a 372-amino-acid peroxin was isolated, primarily by functional complementation screening, using a peroxisome-deficient Chinese hamster ovary cell mutant, ZPG208, of complementation group 17. The deduced primary sequence showed approximately 25% amino acid identity with the yeast Pex3p, thereby we termed this cDNA rat PEX3 (RnPEX3). Human and Chinese hamster Pex3p showed 96 and 94% identity to rat Pex3p and had 373 amino acids. Pex3p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. A homozygous, inactivating missense mutation, G to A at position413, in a codon (GGA) for Gly(138) and resulting in a codon (GAA) for Glu was the genetic cause of peroxisome deficiency of complementation group 17 ZPG208. The peroxisome-restoring activity apparently required the full length of Pex3p, whereas its N-terminal part from residues 1 to 40 was sufficient to target a fusion protein to peroxisomes. We also demonstrated that Pex3p binds the farnesylated peroxisomal membrane protein Pex19p. Moreover, upon expression of PEX3 in ZPG208, peroxisomal membrane vesicles were assembled before the import of soluble proteins such as PTS2-tagged green fluorescent protein. Thus, Pex3p assembles membrane vesicles before the matrix proteins are translocated.

Epidemiology of influenza-associated encephalitis-encephalopathy in Hokkaido, the northernmost island of Japan.

BACKGROUND: It is well known that acute onset brain dysfunction, which usually is diagnosed as encephalitis or encephalopathy, occurs in association with influenza. However, this may have been underestimated as a rather infrequent event. Sixty-four infants and children developed encephalitis-encephalopathy during the five recent influenza seasons in Hokkaido, the northernmost island of Japan. METHODS: Inquiries were sent at the end of each season, from October 1994 to March 1999, to 94 hospitals and institutes in Hokkaido which accept pediatric age patients, asking if there were any admitted cases of encephalitis or encephalopathy. RESULTS: The patients were 42 boys and 22 girls and 47 (73.4%) were 4 years of age or younger. None of them had received an influenza vaccine nor had an oral administration of aspirin. Most of the patients became comatose with or without convulsions within a few days of the onset of fever. Twenty-eight (43.8%) patients died and 13 (20.3%) had neurological sequelae. Patients with clotting disorders, elevations of serum creatine kinase and/or aspartate aminotransferase and alanine aminotransferase, and brain CT abnormalities had a poor prognosis compared with patients without. Among these affected patients, the influenza genome (H3) was detected by polymerase chain reaction in nine cerebrospinal fluid samples, influenza virus A (H3N2) was isolated in 18 nasopharyngeal swab samples and a four-fold or greater rise in serum hemagglutinin inhibition antibody titer against H3N2 was observed in seven patients. CONCLUSIONS: It appears urgent to promote vaccination against influenza in young children to prevent these devastating disease conditions.

Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly.

At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p. Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein.

Analysis of mumps vaccine failure by means of avidity testing for mumps virus-specific immunoglobulin G.

To characterize patients with mumps vaccine failure, avidity testing was performed with the Enzygnost Anti-Parotitis Virus/IgG kit using a single-dilution-6 M urea denaturation method. Five groups of patients were tested. Group 1 consisted of 29 patients with primary mumps infections; group 2 was 20 children and adults with a definite history of natural infection; group 3 was 7 patients with a recent mumps vaccination, 1 of whom developed parotid gland swelling and aseptic meningitis; group 4 was 14 patients with mumps vaccine failure; and group 5 was 6 patients with recurrent episodes of parotitis in addition to a history of vaccination. On the basis of the results of groups 1 and 2, an avidity of /=32% was determined to be high. Avidity maturation from low to high appears to occur around 180 days after the acute illness. The results of group 3 showed that the vaccine-induced immunoglobulin G (IgG) had very low avidity. Among the 14 patients in group 4, 12 patients, including 7 with a positive IgM response, were diagnosed as having secondary vaccine failures. The results of group 5 suggested the possibility that the avidity of the mumps vaccine-induced IgG remains low or borderline. These results showed that secondary mumps vaccine failure occurs not infrequently, even among school age children under condition in which the vaccine coverage is low (i.e., 33% in our study population), and therefore, vaccinees are prone to be exposed to wild-type viruses. Avidity testing should provide information useful for the analysis of mumps virus infections.

Newly identified Chinese hamster ovary cell mutants are defective in biogenesis of peroxisomal membrane vesicles (Peroxisomal ghosts), representing a novel complementation group in mammals.

We isolated peroxisome biogenesis-defective mutants from Chinese hamster ovary cells by the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method. Seven cell mutants, ZP116, ZP119, ZP160, ZP161, ZP162, ZP164, and ZP165, of 11 P9OH/UV-resistant cell clones showed cytosolic localization of catalase, a peroxisomal matrix enzyme, apparently indicating a defect of peroxisome biogenesis. By transfection of PEX cDNAs and cell fusion analysis, mutants ZP119 and ZP165 were found to belong to a novel complementation group (CG), distinct from earlier mutants. CG analysis by cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome indicated that ZP119 and ZP165 were in the same CG as the most recently identified human CG-J. The peroxisomal matrix proteins examined, including PTS1 proteins as well as a PTS2 protein, 3-ketoacyl-CoA thiolase, were also found in the cytosol in ZP119 and ZP165. Furthermore, these mutants showed typical peroxisome assembly-defective phenotype such as severe loss of resistance to 12-(1'-pyrene)dodecanoic acid/UV treatment. Most strikingly, peroxisomal reminiscent vesicular structures, so-called peroxisomal ghosts noted in all CGs of earlier Chinese hamster ovary cell mutants as well as in eight CGs of patients' fibroblasts, were not discernible in ZP119 and ZP165, despite normal synthesis of peroxisomal membrane proteins. Accordingly, ZP119 and ZP165 are the first cell mutants defective in import of both soluble and membrane proteins, representing the 14th peroxisome-deficient CG in mammals, including humans.

Oral-facial-digital syndrome type IX in a patient with Dandy-Walker malformation.

We report a girl with oral, facial, and digital anomalies including multiple alveolar frenula, lobulated tongue with nodules, a posterior cleft palate, hypertelorism, a prominent forehead with a large anterior fontanelle, and postaxial polydactyly in both hands and the right foot, features compatible with the oral-facial-digital syndrome (OFDS). In addition, she had bilateral microphthalmia, optic disc coloboma, and retinal degeneration with partial detachment, thus establishing a diagnosis of OFDS type IX. Dandy-Walker malformation and retrobulbar cysts were observed on MRI. These additional malformations have not been reported in OFDS type IX. The frequent apnoeic spells which occurred immediately after birth were relieved after cystoperitoneal shunt implantation for hydrocephalus. Considering our case and previous reports of OFDS type IX, including two male sibs, a boy born to consanguineous parents, and three females, inheritance is probably autosomal recessive.

Analysis of mycoplasmal pleural effusion by the polymerase chain reaction.

Ten pediatric patients with mycoplasmal pleuritis were tested for the presence of Mycoplasma pneumoniae in pleural fluid by the polymerase chain reaction (PCR). Three of the four PCR positive cases left a persistent consolidation. The remaining one was an infant who required mechanical ventilation. PCR may be useful in predicting delayed resolution of roentgenographic abnormality.

Measles virus-specific immunoglobulin G subclass response in serum and cerebrospinal fluid.

BACKGROUND: While many previous studies have focused on the impairment in the cellular immunity during measles virus infection, to date, a limited amount of data is available concerning the virus-specific IgG subclass response during measles virus infection. OBJECTIVE: The purpose of this study is to analyze the measles virus infection on the basis of virus-specific IgG subclass (G 1 and G 3). STUDY DESIGN: Frozen-stored, serum and/or cerebospinal fluid samples from three groups of patients were tested retrospectively; Group 1 comprised 14 patients with measles primary infection, group 2, ten patients with reinfection/vaccine failure, and group 3, seven patients with subacute sclerosing panencephalitis. The method used was a modified ELISA method utilizing the Enzygnost IgG detection kit with mouse-monoclonal antibodies (clone HP6091 for IgG 1 and clone HP6050 for IgG 3). Avidity testing for each subclass IgG was also performed for selected samples by means of an 8 M urea-denaturation method. RESULTS: In group 1, the IgG 3 could be detected in serum within 7 days from the onset of rash more frequently than IgG 1. In the cases of group 2, both subclasses were detected in very acute phase serum samples. In these cases, the IgG 1-specific avidity was always higher than that of IgG 3. In group 3, the subclass IgGs detected in the cerebrospinal fluid had a lower avidity than those in the serum. CONCLUSIONS: Our results suggested that in measles virus infection, like other viral infections, the IgG 3 response normally occurs before the IgG 1 response, and plays a major role in the acute phase immunity during the primary infection, while the IgG 1 plays a major role in the maintenance of immunity. Continuously produced IgG 1 and IgG 3 in the central nervous system in cases of subacute sclerosing panencephalitis may be derived from cell populations different from those in the blood.

[Acute encephalitis and encephalopathy at the height of influenza in childhood].

Twenty six infants and children with acute encephalitis and encephalopathy during two influenza seasons in Hokkaido, the northernmost island of Japan, were reported. Thirteen patients died and 5 had residual neurological sequelae. Influenza virus genome was detected by PCR in 9 out of 10 cerebrospinal fluid samples from these patients. CT and MRI of the brain demonstrated symmetrical changes in the thalamus and brainstem. The prevalence of these encephalitis and encephalopathy of childhood should be surveyed by nationwide scale.

Survey of mycoplasmal bacteremia detected in children by polymerase chain reaction.

To determine whether mycoplasmal bacteremia occurs during ordinary or complicated diseases due to M. pneumoniae (and if so, how frequently), we used polymerase chain reaction (PCR) to detect M. pneumoniae in serum samples. The PCR primers used were modified for nested amplification. The genome of this organism was detected in 1 of the 25 patients with pneumonia and 10 of the 17 patients without pneumonia (P < .001, chi test). The genome was detected more frequently in patients who had encephalitis of which the neurological onset was within 7 days of the onset of fever rather than later. We hypothesize that mycoplasmal bacteremia occurs more frequently than previously appreciated, specifically in the absence of pneumonia, and that certain types of complications (e.g., encephalitis of early onset) are associated with its occurrence.

Immunoglobulin G avidity testing in serum and cerebrospinal fluid for analysis of measles virus infection.

We studied a variety of patients with measles virus infection by using avidity testing for measles virus-specific immunoglobulin G (IgG) in serum and cerebrospinal fluid samples. For the avidity testing, an Enzygnost measles IgG enzyme-linked immunosorbent assay kit was used with an 8 M urea denaturing method. With this method, low-avidity IgG (acute primary infection, avidity of < 30% within 15 days of the onset of rash) and high-avidity IgG (subacute sclerosing panencephalitis, avidity of > 75%) could be clearly distinguished by using serum samples. One patient, who developed a typical course of measles despite a previous vaccination, showed a positive IgM response with an initial low titer of measles virus-specific IgG of low avidity, but a later sample revealed a high titer of IgG of intermediate (40%) avidity, suggesting previous immunological priming. Two patients with breakthrough infection (secondary vaccine failure), both having central nervous system involvement, showed a positive IgM response with initial high titers of serum IgG of high avidity. In addition, one of the patients had a detectable level of measles-specific IgG in cerebrospinal fluid. In this patient, the avidity of both serum and cerebrospinal fluid IgG decreased during the short follow-up period. This phenomenon has never before been reported. In subacute sclerosing panencephalitis patients, the avidity of cerebrospinal fluid IgG was consistently lower than that of serum IgG. The difference in avidity between cerebrospinal fluid and serum IgG may be used as a direct indicator of intrathecal production of IgG. In conclusion, the avidity testing is simple to perform, reliable, and highly informative in the analysis of measles virus infection.

Interleukin-6 in cerebrospinal fluid of patients with central nervous system infections.

Interleukin(IL)-6 levels were measured in cerebrospinal fluid (CSF) and serum samples from pediatric patients with central nervous system (CNS) infections by means of an enzyme-linked immunosorbent assay. Mean IL-6 concentrations in CSF samples from patients with bacterial meningitis (49,017 +/- 44,730 pg/ml) were significantly higher than those in patients with aseptic meningitis (1076 +/- 1572 pg/ml) or encephalitis (409 +/- 835 pg/ml). In aseptic meningitis and encephalitis, IL-6 levels in serum were within the lower ranges (< 100 pg/ml), in contrast with the highly elevated levels found in bacterial meningitis (14,332 +/- 18,385 pg/ml). In 5 of the 15 patients with encephalitis, elevated levels of IL-6 were observed in the initial CSF samples despite normal findings of routine CSF examinations. Also, sequential CSF samples revealed that there was an increase in the CSF cell count in two of the five patients. These results validated the potential of measuring IL-6 in CSF samples for the purpose of providing additional information on routine laboratory test results.

Measles encephalomyelitis in a patient with a history of vaccination.

Secondary vaccine failure (SVF) of measles is generally believed to run a milder course of illness than an ordinary course of infection. Severe complications such as central nervous system involvement have rarely been reported. A 12 year old girl, who had received a live attenuated measles vaccine 10 years earlier, developed an encephalomyelitis in the absence of symptoms indicative of ordinary measles such as Koplik spots. Anti-measles hemagglutination inhibition (HI) titer and measles IgM and IgG antibody titers were measured in a commercial laboratory. Measles virus genomic sequence was detected by polymerase chain reaction. Both serum and cerebrospinal fluid (CSF) samples obtained at acute phase already showed extremely high titers of HI (x8192 in serum and x1024 in CSF, respectively) and IgG antibody along with the presence of IgM antibody. Polymerase chain reaction detected the measles virus genomic sequence in the acute phase CSF. The patient's definite history of measles vaccination, high titers of HI and IgG antibodies observed at the very early stage of illness and the clinical course indicated that this patient has an encephalomyelitis due to SVF of measles. It is suggested that measles virus can be a pathogen of encephalitis without symptoms indicative of ordinary measles in individuals who received live attenuated measles vaccines.

Pulmonary aspergillosis and pseudosequestration of the lung in chronic granulomatous disease.

We present a case of chronic granulomatous disease with an angiographically proven pseudosequestration of the lung. The patient was a 15-year-old boy who was admitted to the hospital with symptoms of fever, cough, hemoptysis and a subcutaneous abscess. Aspergillus fumigatus was isolated from the sputum and the abscess. During treatment, angiography demonstrated on anomalous blood supply to the right middle lobe. The therapeutic implications of pseudosequestration of the lung for the treatment of chronic granulomatous disease are discussed.

Detection of measles virus from clinical samples using the polymerase chain reaction.

OBJECTIVE: To evaluate the usefulness of the polymerase chain reaction to detect the measles virus sequence using clinical samples. DESIGN: Centers for Disease Control and Prevention case definition of measles with or without IgM serology as a standard. SETTING: A laboratory in the Department of Pediatrics of the Hokkaido University Hospital, Sapporo, Japan. PATIENTS: Thirty-two serum samples, 16 throat swab samples, and nine cerebrospinal fluid samples from 32 patients with measles, including four patients with central nervous system involvement, and one serum sample and two throat swab samples from two patients with modified courses of measles were obtained. Ten serum samples, 10 throat swab samples, and 10 cerebrospinal fluid samples were obtained from patients without apparent measles infection as negative controls. MEASUREMENTS AND MAIN RESULTS: Sensitivity and specificity were comparable with those as obtained by culture or other methods reported in the literature. The polymerase chain reaction was positive in 24 (75.0%) of 32 by serum samples and in 13 (81.3%) of 16 by throat swab samples from the patients with measles, in contrast to none within the negative control group. In three of the four patients with central nervous system involvement, the measles virus sequence was detected in cerebrospinal fluid samples obtained within 1 day following the onset of the manifestations. All three samples from the patients with modified measles yielded positive results. CONCLUSIONS: The polymerase chain reaction can be used with sufficient sensitivity and specificity to detect the measles virus sequence using clinical samples. Transient and direct invasion of the central nervous system by this virus at the initial stage of the central nervous system involvement was strongly suggested.

Nested amplification protocol for the detection of Mycobacterium tuberculosis.

Several methods for rapid diagnosis of tuberculosis have been devised through DNA amplification. However, the chemically strong cell wall of the species, the presumptively low numbers of organisms and their uneven distribution in clinical samples, and the lack of a "gold standard" for diagnosing tuberculosis, have hindered the routine clinical use of this method. In a pediatric patient group, these factors are more perplexing. To circumvent these problems, we made use of nested amplification and developed a standard protocol for extracting DNA from various forms of clinical samples which were suitable to our clinical laboratory. It is our impression that the overall sensitivity, including technical bias accompanying this method, is equal to, or at least greater than, that of culture. Most notably, the rapidity in obtaining results and the simplicity in handling, storage and transfer of samples are the principal advantages of this method.

DNA diagnosis of central nervous system infection by Mycoplasma pneumoniae.

A nested polymerase chain reaction method for the detection of Mycoplasma pneumoniae was devised and applied to clinical samples. This system could detect 5 to 50 fg of the DNA from M pneumoniae and did not amplify the DNA from Mycoplasma genitalium. With this method, the sequence of this organism was detected successfully in cerebrospinal fluid samples from four of six patients and in serum samples from three of four patients with clinically and serologically confirmed mycoplasmal central nervous system infection. This strongly suggested the direct invasion of this organism into the central nervous system and the concomitant occurrence of mycoplasmaremia. The nested amplification method is considered to be simple, rapid, and sensitive without the use of radioisotopes, thereby being highly applicable as a useful tool in routine clinical laboratories for the preliminary detection and diagnosis of mycoplasmal infections, particularly in extrapulmonary cases.

A case of traumatic intramural hematoma of the duodenum effectively treated with ultrasonically guided aspiration drainage and endoscopic balloon catheter dilation.

A 52-year-old man was admitted on February 15, 1990, with hiccups and vomiting. He had been well until 13 days before admission when he stumbled and fell when intoxicated, striking his abdomen. A diagnosis of intramural hematoma was made with computerized tomography and sonography of the abdomen after admission, revealing a mass that was intimately related to the duodenum. Treatment of the intramural duodenal hematoma is controversial. However, this case illustrates the ideal situation where conservative management could be applied with total parenteral nutrition, percutaneous aspiration drainage, and endoscopic balloon catheter dilatation of the narrowed lumen of the duodenum. The patient's subsequent course supports the concept of planned conservative management.