GCP5 and GCP6: two new members of the human gamma-tubulin complex.

The gamma-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human gamma-tubulin complex and the identification of its subunits. The human gamma-tubulin complex is a ring of ~25 nm, has a subunit structure similar to that reported for gamma-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human gamma-tubulin complex components confirmed the presence of four previously identified components (gamma-tubulin and gamma-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the gamma-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire gamma-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of gamma-tubulin, GCP2, GCP3, and GCP4 within the gamma-tubulin complex. Thus, the gamma-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.

Flavin-containing monooxygenase-mediated metabolism of N-deacetyl ketoconazole by rat hepatic microsomes.

Although ketoconazole is extensively metabolized by hepatic microsomal enzymes, the route of formation and toxicity of suspected metabolites are largely unknown. Reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice. DAK may be susceptible to successive oxidative attacks on the N-1 position by flavin-containing monooxygenases (FMO) producing potentially toxic metabolites. Previous laboratory findings have demonstrated that postnatal rat hepatic microsomes metabolize DAK by NADPH-dependent monooxygenases to two metabolites as determined by HPLC. Our current investigation evaluated DAK's metabolism in adult male and female rats and identified metabolites that may be responsible for ketoconazole's hepatotoxicity. DAK was extensively metabolized by rat liver microsomal monooxygenases at pH 8.8 in pyrophosphate buffer containing the glucose 6-phosphate NADPH-generating system to three metabolites as determined by HPLC. The initial metabolite of DAK was a secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole, which was confirmed by liquid chromatography/mass spectrometry and NMR spectroscopy. Extensive metabolism of DAK occurred at pH 8.8 in pyrophosphate buffer (female 29% and male 53% at 0.25 h; female 55% and male 57% at 0.5 h; and female 62% and male 66% at 1.0 h). Significantly less metabolism of DAK occurred at pH 7.4 in phosphate buffer (female 11%, male 17% at 0.25 h; female 20%, male 31% at 0.5 h; and female 27%, male 37% at 1 h). Heat inactivation of microsomal-FMO abolished the formation of these metabolites from DAK. SKF-525A did not inhibit this reaction. These results suggest that DAK appears to be extensively metabolized by adult FMO-mediated monooxygenation.

An integrated approach to proteome analysis: identification of proteins associated with cardiac hypertrophy.

Hypertrophy of cardiac myocytes is a primary response of the heart to overload, and is an independent predictor of heart failure and death. Distinct cellular phenotypes are associated with hypertrophy resulting from different causes. These phenotypes have been described by others at the molecular level by analysis of gene transcription patterns. An alternative approach is the analysis of large-scale protein expression patterns (the proteome) by two-dimensional polyacrylamide gel electrophoresis. Realization of this goal requires the ability to rigorously analyze complex 2D gel images, efficiently digest individual gel isolated proteins (especially those expressed at low levels), and analyze the resulting peptides with high sensitivity for rapid database searches. We have undertaken to improve the technology and experimental approaches to these challenges in order to effectively study a cell culture model for cardiac hypertrophy. The 2D gel patterns for cell lysates from multiple samples of cardiac myocytes with or without phenylephrine-induced hypertrophy were analyzed and spots which changed in abundance with statistical significance were located. Eleven such spots were identified using improved procedures for in-gel digestion of silver-stained proteins and high-sensitivity mass spectrometry. The incorporation of low levels of sodium dodecyl sulfate into the digestion buffer improved peptide recovery. The combination of matrix-assisted laser desorption mass spectrometry for initial measurements and capillary liquid chromatography-ion trap mass spectrometry for peptide sequence determination yielded efficient protein identification. The integration of 2D gel image analysis and routine identification of proteins present in gels at the subpicomole level represents a general model for proteome studies relating genomic sequence with protein expression patterns.

Identification of mouse liver proteins on two-dimensional electrophoresis gels by matrix-assisted laser desorption/ionization mass spectrometry of in situ enzymatic digests.

A number of proteins from a silver-stained two-dimensional (2-D) electrophoresis gel of mouse liver whole-cell lysate were identified by peptide mass mapping and sequence database searching. The excised protein spots were processed by in situ reduction and alkylation, followed by Lys-C digestion. The masses of the resulting peptide mixtures were measured with a matrix-assisted laser desorption/ionization (MALDI) reflection-time-of-flight mass spectrometer. These masses were used successfully to search a protein sequence database. Optimized silver staining and digestion protocols allowed proteins to be identified routinely at the low picomole level. The high mass accuracy and resolution provided by delayed extraction were important for high specificity in the database search. Fragment ion data obtained by MALDI post-source decay (PSD) measurements not only provided confirmation of peptide identification, but could be used to identify the protein from a single peptide without spectral interpretation.

Attention deficit hyperactivity disorder.

Attention Deficit Hyperactivity Disorder is found in a significant number of children. The symptoms of the disorder can cause problems in learning, socialization, and behavior for those individuals afflicted with it and put them at high risk for serious psychopathology in adulthood. This article describes the causes, neurobiology, symptoms, and treatment for ADHD and the pediatric nurse's role in identification, referral, and symptom management of this disorder.

Schizoaffective disorder: a case study.

1. Schizoaffective disorder includes a combination of both schizophrenia and affective disorder symptoms. 2. Diagnosis of schizoaffective disorder is based on a careful evaluation of the client's history, as well as current symptoms. Misdiagnosis may occur if only current symptoms are considered. 3. Treatment of schizoaffective disorder includes neuroleptics, antimanics and antidepressants. The client must be carefully assessed for side effects. The elderly client poses special challenges. 4. Case management by a clinical nurse specialist or psychiatric nurse practitioner with the ultimate goal of increased quality of life for the client includes: assessment of client for positive and negative effects of medication; client, family and caregiver education regarding medications; identification of significant symptoms; and assistance with coping skills and support.

The disulfide linkages and glycosylation sites of the human natriuretic peptide receptor-C homodimer.

The natriuretic peptide receptor-C (NPR-C) constitutes greater than 95% of the natriuretic peptide binding sites in vivo. This cell surface glycoprotein is a disulfide-linked homodimer with a subunit molecular weight of 68,000. Two sources and types of ANP affinity-purified human NPR-C were used to map disulfide linkages and glycosylation sites of this receptor by mass spectrometry: the extracellular domain obtained by papain cleavage of a receptor-IgG fusion protein expressed in Chinese hamster ovary cells, and a baculovirus/Sf9-expressed cytoplasmic truncation mutant in which 34 of 37 cytoplasmic domain amino acids were deleted. Two intramolecular disulfide bonded loops were found in the 435 amino acid extracellular domain (C63-C91, C168-C216). The juxtamembrane residues C428 and C431 are involved in homodimer formation, confirmed by site-directed mutagenesis of full-length NPR. Three of the four potential Asn-linked glycosylation sites are occupied: N41 (complex), N248 (high mannose), and N349 (complex; partial occupancy). These data describe the intra- and intermolecular linkages in NPR-C, providing a model for the homologous guanylyl cyclase receptors, NPR-A and NPR-B; both of the cyclase receptors likely contain the first amino-terminal 29 amino acid loop, but only NPR-A possesses the second 49 amino acid loop in common with NPR-C.

Zinc inhibition of renin and the protease from human immunodeficiency virus type 1.

We report here for the first time that Zn2+ is an effective inhibitor of renin and the protease from HIV-1, two aspartyl proteinases of considerable physiological importance. Inhibition of renin is noncompetitive and is accompanied by binding of 1 mol of Zn2+/mol of enzyme. Depending on the substrate, inhibition of the HIV protease by Zn2+ can be either competitive or noncompetitive, but in neither case is loss of activity due to disruption of the protease dimer. Inhibition of both enzymes is first order with respect to Zn2+ and is rapidly reversed by addition of EDTA. Ki values are strongly pH dependent and optimal in the range of 20 microM at or above pH 7. All of the data in hand suggest that the inhibitory effect of Zn2+ is a consequence of its binding at, or near, the active-site carboxyl groups of these aspartyl proteinases. This inhibition of the viral enzyme may help to explain some of the beneficial effects seen in AIDS patients who have received Zn2+ therapy.