A GNAS1 imprinting defect in pseudohypoparathyroidism type IB.

Pseudohypoparathyroidism type IB (PHPIB) is characterized by renal resistance to parathyroid hormone (PTH) and the absence of other endocrine or physical abnormalities. Familial PHPIB has been mapped to 20q13, near GNAS1, which encodes G(s)alpha, the G protein alpha-subunit required for receptor-stimulated cAMP generation. However, G(s)alpha function is normal in blood cells from PHPIB patients, ruling out mutations within the G(s)alpha coding region. In mice G(s)alpha is expressed only from the maternal allele in renal proximal tubules (the site of PTH action) but is biallelically expressed in most other tissues. Studies in patients with Albright hereditary osteodystrophy suggest a similar G(s)alpha imprinting pattern in humans. Here we identify a region upstream of the G(s)alpha promoter that is normally methylated on the maternal allele and unmethylated on the paternal allele, but that is unmethylated on both alleles in all 13 PHPIB patients studied. Within this region is an alternative promoter and first exon (exon 1A), generating transcripts that are normally expressed only from the paternal allele, but that are biallelically expressed in PHPIB patients. Therefore, PHPIB is associated with a paternal-specific imprinting pattern of the exon 1A region on both alleles, which may lead to decreased G(s)alpha expression in renal proximal tubules. We propose that loss of exon 1A imprinting is the cause of PHPIB.

Isolated endobronchial atypical mycobacterium in a child: a case report and review of the literature.

Isolated endobronchial lesions caused by Mycobacterium avium are rare, especially in the pediatric population. We share the case of a 10-month-old boy who, after 1 week of cough and low-grade fever, had a radiographic examination showing endobronchial obstruction. At bronchoscopy, a granuloma of the left bronchus intermedius was found. Histopathologic examination revealed necrotizing granulomatous inflammation. Kinyoun Acid Fast stain revealed acid fast bacilli. Cultures were positive for M. avium. Current treatment options and controversies are presented. The roles of antibiotics and steroids in preventing progressive disease are discussed. The need for serial bronchoscopy and the potential benefits of surgical resection are discussed. Isolated endobronchial M. avium infection remains a rare and challenging problem. The paucity of clinical experience, and variation in patient presentation, obligates a high index of suspicion, and frequent follow-up with bronchoscopic examination and pulmonary assessment, for the child diagnosed with isolated endobronchial atypical mycobacterial infection.

Identification of a methylation imprint mark within the mouse Gnas locus.

The imprinted mouse gene Gnas produces the G protein alpha-subunit G(S)alpha and several other gene products by using alternative promoters and first exons. G(S)alpha is maternally expressed in some tissues and biallelically expressed in most other tissues, while the gene products NESP55 and XLalphas are maternally and paternally expressed, respectively. We investigated the mechanisms of Gnas imprinting. The G(S)alpha promoter and first exon are not methylated on either allele. A further upstream region (approximately from positions -3400 to -939 relative to the G(S)alpha translational start site) is methylated only on the maternal allele in all adult somatic tissues and in early postimplantation development. Within this region lies a fourth promoter and first exon (exon 1A) that generates paternal-specific mRNAs of unknown function. Exon 1A and G(S)alpha mRNAs have similar expression patterns, making competition between their promoters unlikely. Differential methylation in this region is established during gametogenesis, being present in oocytes and absent in spermatozoa, and is maintained in preimplantation E3. 5d blastocysts. Therefore, this region is a methylation imprint mark. In contrast, differential methylation of the NESP55 and XLalphas promoter regions (Nesp and Gnasxl) is not established during gametogenesis. The methylation imprint mark that we identified may be important for the tissue-specific imprinting of G(S)alpha.

Synchronous occurrence of malignant rhabdoid tumor two decades after Wilms' tumor irradiation.

We describe the first case of synchronous malignant rhabdoid tumor arising in the pelvis and the lung two decades after both sites were irradiated for Wilms' tumor. Although the malignant rhabdoid tumor phenotype is controversial as a specific clinicopathological entity, this case exhibited classic clinicopathological features of malignant rhabdoid tumor, including tissue features of a trabecular to alveolar growth pattern; cellular features of characteristic eosinophilic cytoplasmic inclusions exhibiting intermediate filament clusters, large nuclei with prominent central nucleoli, and a dual mesenchymal and epithelial immunocytochemistry profile; and clinical features of a rapidly deteriorating course leading to death 2 months after diagnosis. The occurrence of synchronous malignant rhabdoid tumors in sites irradiated for Wilms' tumor raise interesting questions concerning the relationship of radiation-induced malignancies to putative tumor suppressor gene defects, the distinction of synchronous secondaries from primary recurrences and metastases, and finally the quintessential relationship of malignant rhabdoid tumor to Wilms' tumor.

Phenobarbital monitoring in whole blood with a quantitative noninstrumented test.

The AccuLevel phenobarbital test is based on enzyme channeling and immunochromatography. AccuLevel is a noninstrumented test for the quantitative determination of phenobarbital concentration in whole blood. Within-run precision data, with 20 replicates at each of five concentrations, has coefficients of variation (CVs) of 4.7-9.2%. Between-run precision (n = 40) results in a CV of 5.9%. The AccuLevel phenobarbital test is very specific and is unaffected by endogenous substances and blood collection tube anticoagulants. Compared to the Emit method, this test has excellent linear correlation for the quantitation of 104 phenobarbital positive patient samples. Reagents stored at 4-8 degrees C are stable for 15 months with no effect on the assay quantitation. This accurate, precise, and specific test is easily performed in 20 min.

One-step enzyme immunochromatographic assay for theophylline.

The convenience of the previously described enzyme immunochromatography method for visually quantifying theophylline in whole blood has been improved with the development of a one-step protocol. The capillary migration and color generation in the two-step enzyme immunochromatographic assay have been combined into a single step. Ascorbic acid is used as a signal inhibitor to delay enzymatic color product formation until the inhibitor itself is consumed. The concept of internal delay reaction is presented and the mechanism of ascorbate's action as an inhibitor to temporarily delay color generation is described. The internal delay reaction has been applied to a practical one-step quantitative visual enzyme immunochromatographic assay for theophylline in whole blood.

An internal clock reaction used in a one-step enzyme immunochromatographic assay of theophylline in whole blood.

We describe the development and performance of a second-generation enzyme immunochromatography method for visually quantifying theophylline in whole blood without the use of instrumentation. We have developed the novel concept of an internal chemical clock reaction to combine the capillary-migration and color-generation protocol of the two-step immunochromatographic assay into a single-step, simultaneous protocol. The two assay components are (a) chromatographic paper to which glucose oxidase (EC 1.1.3.4) and monoclonal antibody to theophylline have been immobilized, and (b) an enzyme reagent consisting of glucose, dicarboxidine, ascorbate, and theophylline-labeled horseradish peroxidase (EC 1.11.1.7). The ascorbate acts as an internal clock by inhibiting premature color formation until the ascorbate has been completely consumed in the peroxidase-mediated reaction. Color is then generated rapidly, producing a clearly visible front on the paper. Performance evaluations of the 20-min one-step assay show very good precision, analytical recovery, specificity, and accuracy. This simplified protocol is reliable and convenient for therapeutic drug monitoring in the physician's office.

Calibration, quality control, and stability of a quantitative enzyme immunochromatographic method for therapeutic drug monitoring.

We describe a noninstrumented quantitative method for therapeutic drug monitoring (AccuLevel test) that uses a factory-calibrated unit test format and a novel single-level approach to quality control. The AccuLevel method is based on the principles of immunochromatography, which provides a number of convenient protocol advantages without sacrificing assay performance or quality assurance. Most of the benefits of the immunochromatographic method derive by virtue of the fact that quantification is dependent on enzyme migration rather than enzyme activity. Since migration height is almost solely a function of a highly stable, immobilized, dry antibody reagent, the AccuLevel test is extremely insensitive to environmental factors. The predictable and uniform dependence of quantification on antibody site concentration allows complete reliability with a single-level control. These features of stability, factory calibration, and unitized test components make the AccuLevel immunochromatography method amenable to new quality control schemes.

Drug-induced modulations of hepatic heme metabolism. Neurological consequences.

Administration of the porphyrinogenic agent DDEP to PB-pretreated rats results in acute hepatic heme depletion, which is a characteristic of acute hepatic porphyria. Such acute heme depletion is associated with impaired hepatic tryptophan degradation and enhanced serotonergic tone in the CNS. We showed that intestinal motility in these rats is also significantly decreased, indicating that the serotonergic tone of the enteric nervous system may also be enhanced. In addition, the marked hepatic accumulation of glucogenic precursors, observed in parallel, indicates that the elevated tryptophan levels may also block hepatic glucogenesis. The clinical implications of these findings to acute heme-deficient states, such as the acute hepatic porphyrias, was discussed.

The influence of side chain modifications of the heme moiety on prosthetic acceptance and function of rat hepatic cytochrome P-450 and tryptophan pyrrolase.

The relative potential of various structural isomers (III, XIII) and various 2,4-side chain modified analogs of heme (iron-protoporphyrin IX) to incorporate into rat liver hemoproteins, cytochrome P-450(s), and tryptophan pyrrolase was examined. Such assessments for hepatic cytochrome P-450 relied on generation of reconstitutible apocytochrome(s) P-450 by suicidal alkylation of the existing prosthetic heme moiety by allylisopropylacetamide (AIA) in vivo. Subsequent replacement of the prosthetic heme was brought about by incubating the apocytochrome(s) P-450-enriched preparations with a particular heme isomer or analog. Structure-function relationships of the reconstituted isozymes were assessed in microsomal preparations by monitoring cytochrome P-450 content (structure) and its mixed function oxidase activity (function). In parallel, the relative ability of these heme isomers and analogs to functionally constitute hepatic tryptophan pyrrolase was also assessed by monitoring the relative increase in holoenzyme activity when preparations deliberately enriched in constitutible apoenzyme were incubated with each of these compounds. The findings reveal that 2,4-side chain modifications on the heme IX skeleton markedly influence the function of the constituted hemoproteins possibly by affecting their structural assembly through steric, electronic, and/or hydrophobic interactions with the corresponding apoproteins. Furthermore, these studies not only reveal that the structural specifications of the active prosthetic site of rat liver cytochrome P-450(s) differ from those of tryptophan pyrrolase, but also that the structural specifications of these mammalian hemoproteins for their prosthetic heme differ considerably from those reported for their bacterial counterparts.

Enzyme immunochromatography--a quantitative immunoassay requiring no instrumentation.

We describe a novel test-strip immunoassay for quantifying drugs in biological fluids. This enzyme immunochromatographic ("immunograph") method combines many features of the enzyme-channeling homogeneous immunoassay with immunochromatography and capillary migration to provide a non-separation, non-instrumental assay for theophylline in which quantification is based on the spatial distribution of enzyme label rather than on the modulation of enzyme activity. Sample antigen and hapten-enzyme conjugate are combined and moved by capillary action up a paper strip on which specific antibody has been immobilized. After color development, the assay result is evaluated by measuring the height of the colored zone on the test strip. Quantification is not a function of enzyme activity, so the method is relatively insensitive to sample matrix effects, enzyme instability, temperature, and incubation timing. Either whole blood or plasma can be used as sample. Results correlate well with those by established instrumental methods. The simple, rapid (15 min), two-incubation protocol is well suited for on-site testing in non-laboratory environments.

Elevated brain tryptophan and enhanced 5-hydroxytryptamine turnover in acute hepatic heme deficiency: clinical implications.

Administration of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine, a suicide inhibitor of hepatic cytochrome P-450, to phenobarbital-pretreated rats rapidly causes a marked and sustained hepatic heme depletion and results in porphyria. We have shown that this event results in marked impairment of hepatic tryptophan pyrrolase activity and consequently in elevated tryptophan content and enhanced 5-hydroxytryptamine (5-HT) turnover in the brain of such porphyric rats. All these effects were reversed by administration of exogenous heme. Using an indirect assay of 5-HT-dependent function, we now show that this elevated 5-HT turnover in porphyric animals is associated with enhanced serotonergic tone. We also show that it can be potentiated by tryptophan administration, reversed by administration of exogenous heme, alleviated by treatment with p-chlorophenylalanine, an inhibitor of 5-HT synthesis, and attenuated by administration of valine, an amino acid that is known to compete with tryptophan uptake in the brain. In patients with hepatic porphyria, acute hepatic heme depletion results in severe, often life-threatening attacks. These attacks are hallmarked by neuropsychiatric symptoms of unknown etiology, but that can often be successfully treated by i.v. administration of heme. Because acute hepatic heme depletion may also be expected to compromise hepatic tryptophan metabolism in such individuals, our findings raise the possibility that elevated tryptophan content and 5-HT turnover in the brain may play a role in the neurological dysfunction associated with acute attacks of hepatic porphyria.

Ultrasound-accelerated immunoassay, as exemplified by enzyme immunoassay of choriogonadotropin.

The rate-limiting step in many solid-phase immunoassays is associated with the slow kinetics of binding of macro-molecular antigen and conjugate to the immobilized phase. We demonstrate that the use of ultrasonic energy to enhance mass transport across liquid/solid interfaces can dramatically accelerate antigen binding to immobilized antibodies. We use an ultrasound-accelerated procedure with an enzyme-channelling test strip containing glucose oxidase and specific antibody to the alpha-subunit of human choriogonadotropin (HCG) co-immobilized onto a cellulose support. A horseradish peroxidase conjugate of monospecific antibody to the beta-subunit of HCG is used in the liquid phase to complete the immune "sandwich." Use of ultrasound to accelerate binding and of enzyme channelling to eliminate wash steps result in a simple two-incubation protocol by which 25 int. units of urinary HCG per liter can be detected visually in less than 20 min of assay time.

L-tryptophan: a common denominator of biochemical and neurological events of acute hepatic porphyria?

Hepatic porphyrias are disorders of heme synthesis characterized by genetically determined lesions of one of the key enzymes of heme synthesis. In carriers of such lesions, several factors (drugs, environmental chemicals, or diet) precipitate acute and often fatal attacks of neurologic dysfunction, which are promptly relieved by intravenous infusion of heme. However, the mechanism of such heme-induced amelioration remains elusive. To probe this mechanism, the biochemical events triggered by acute hepatic heme deficiency were examined in an animal model of chemically induced porphyria. Acute hepatic heme depletion in porphyric rats was found to impair hepatic tryptophan pyrrolase activity which, in turn, elevated tryptophan and 5-hydroxytryptamine turnover in the brain. These alterations in porphyric rats were dramatically reversed by parenteral heme administration. These findings suggest that increased tryptophan and 5-hydroxytryptamine in the nervous system may be responsible for the neurologic dysfunctions observed in humans with acute attacks of hepatic porphyria.

An internally referenced test strip immunoassay for morphine.

We describe an internally referenced immunochemical test-strip for use in the rapid detection of morphine. The method is based on the "enzyme-channelling" immunoassay technique, and a glucose oxidase-horseradish peroxidase enzyme pair is used to immunospecifically generate an insoluble, colored reaction product on the test-strip surface. Test strips are composed of two active surfaces, each of which contain co-immobilized glucose oxidase and antibody. The indicator pad contains antibody directed against the drug, and the color that develops on its surface is inhibited by the presence of drug in the sample. The reference pad contains anti-peroxidase and is used to set the assay detection limit and normalize for variations in temperature, timing, and sample interference. The 10-min assay protocol involves incubating the strip in sample, then incubating it in a developer solution containing glucose, a peroxidase chromogenic substrate, and a peroxidase conjugate of the analyte. The ratio of the color formed on the indicator pad to that formed on the reference pad is used to score the test as positive or negative for drug at a predetermined concentration.

Evidence that spectrin binds to macromolecular complexes on the inner surface of the red cell membrane.

Spectrin binds to a population of high-affinity sites on the exposed surface of inverted vesicles prepared from human red blood cell ghost membranes. Optimal spectrin binding requires the presence of monovalent salts but does not require calcium or magnesium. The band 2 subunit of spectrin, prepared in SDS, can also bind to vesicles, but isolated band 1 is inactive. Pre-incubation of inverted vesicles with antibodies directed against the cytoplasmic segment of band 3 or against bands 4.1-4.2 inhibits the binding of spectrin to the same vesicles. Antibodies against the cytoplasmic portion of glycophorin A have no effect. These results suggest that spectrin binds to a protein acceptor on the cytoplasmic surface of the red cell membrane which is close to the cytoplasmic segments of bands 3 and 4.1 and/or 4.2.