Biological qualification of biomarkers of chemical-induced renal toxicity in two strains of male rat.

This study reports the evaluation of four urinary biomarkers of renal toxicity, α-glutathione-S-transferase (α-GST), μ-GST, clusterin, and renal papillary antigen-1 (RPA-1), in male Sprague-Dawley and Han-Wistar rats given cisplatin, gentamicin, or N-phenylanthranilic acid (NPAA). Kidney injury was diagnosed histopathologically, according to site/nature of renal injury, and graded for severity. The area under the receiver operating characteristic (ROC) curve was used to compare the diagnostic accuracy of each exploratory renal biomarker with traditional indicators of renal function and injury (blood urea nitrogen [BUN], serum creatinine [sCr] as well as urinary N-acetyl-β-D-glucosaminidase [NAG] and protein).

Renal biomarker qualification submission: a dialog between the FDA-EMEA and Predictive Safety Testing Consortium.

The first formal qualification of safety biomarkers for regulatory decision making marks a milestone in the application of biomarkers to drug development. Following submission of drug toxicity studies and analyses of biomarker performance to the Food and Drug Administration (FDA) and European Medicines Agency (EMEA) by the Predictive Safety Testing Consortium's (PSTC) Nephrotoxicity Working Group, seven renal safety biomarkers have been qualified for limited use in nonclinical and clinical drug development to help guide safety assessments. This was a pilot process, and the experience gained will both facilitate better understanding of how the qualification process will probably evolve and clarify the minimal requirements necessary to evaluate the performance of biomarkers of organ injury within specific contexts.

Localization of the linker domain of Ca2+/calmodulin-dependent protein kinase II.

Electron micrographs of rotary shadowed replicas of alpha-Ca2+/calmodulin-dependent protein kinase II reveal a flower-shaped multimeric molecule with a central particle surrounded by 8-10 smaller peripheral particles. Peripheral particles are attached to the central particle by thin arms or "linkers." Movement of peripheral particles to contact each other for autophosphorylation is likely to involve these linkers.

Kinesin light chains are essential for axonal transport in Drosophila.

Kinesin is a heterotetramer composed of two 115-kD heavy chains and two 58-kD light chains. The microtubule motor activity of kinesin is performed by the heavy chains, but the functions of the light chains are poorly understood. Mutations were generated in the Drosophila gene Kinesin light chain (Klc), and the phenotypic consequences of loss of Klc function were analyzed at the behavioral and cellular levels.

Kinesin light chain in a eubacterium.

A eubacterial homolog of a kinesin light chain gene has been isolated and characterized from the cyanobacterium Plectonema boryanum. Although the eubacterial and eukaryotic kinesin light chains are highly similar in amino acid sequence, the eubacterial sequence differs in several distinguishing structural features, including the absence of a putative PEST domain and the presence of additional highly conserved imperfect tandem repeats. Two soluble kinesin light chain antigens have been identified from whole-cell lysates by immunoblot analysis.

Calexcitin: a signaling protein that binds calcium and GTP, inhibits potassium channels, and enhances membrane excitability.

A previously uncharacterized 22-kDa Ca(2+)-binding protein that also binds guanosine nucleotides was characterized, cloned, and analyzed by electrophysiological techniques. The cloned protein, calexcitin, contains two EF-hands and also has homology with GTP-binding proteins in the ADP ribosylation factor family. In addition to binding two molecules of Ca2+, calexcitin bound GTP and possessed GTPase activity.

Application of a membrane fusion assay for rapid drug screening.

Purpose: The purpose of this study is to develop an in vitro assay for screening drug and their effects on membrane fusion and lysis of intracellular organelles.

Methods: A 96-well microtiter-dish turbidimetric assay using membrane components of the eggs of sea urchins, a marine invertebrate, was applied to monitor granule fusion and/or lysis.

Results: Of 18 drugs screened, 16 had no effect.

Molecular characterization of a neuronal-specific protein that stimulates the activity of Cdk5.

Cyclin-dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and tau protein phosphorylation. This report describes the characterization of a 62-kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity.

cAMP-dependent phosphorylation of Aplysia twitchin may mediate modulation of muscle contractions by neuropeptide cotransmitters.

Acting through a cAMP-cAMP-dependent protein kinase (cAPK) cascade, members of two neuropeptide families, the small cardioactive peptides and myomodulins, modulate contraction amplitude and relaxation rate in the accessory radula closer (ARC) muscle of the marine mollusc Aplysia californica. An approximately 750-kDa phosphoprotein was identified in the ARC muscle as the major substrate for cAPK activated either by application of neuropeptides or by peptides released by motorneuron stimulation at physiological frequencies. Immunoblot and immunoelectron microscopy experiments revealed the widespread presence of this protein in Aplysia muscles and its colocalization with contractile filaments in the ARC muscle.

Expression of the synaptic vesicle proteins VAMPs/synaptobrevins 1 and 2 in non-neural tissues.

The VAMPs/synaptobrevins (Vp/Sybs) are small integral membrane proteins. Two isoforms, Vp/Syb 1 and Vp/Syb 2, are considered to be specific to neural tissue. They are associated with synaptic vesicles and are believed to play an important role in neurotransmitter release.

Kinesin light chains: identification and characterization of a family of proteins from the optic lobe of the squid Loligo pealii.

Multiple transcripts coding for kinesin light chain isoforms are present in the tissues of the squid Loligo pealii. Isoform diversity arises through alternative RNA splicing in the amino and carboxyl termini of the putative proteins. Comparison to rat and Drosophila proteins demonstrates a remarkable conservation of structural domains and regulatory motifs.

The myomodulin-related neuropeptides: characterization of a gene encoding a family of peptide cotransmitters in Aplysia.

The myomodulin-related peptides comprise a family of cotransmitters that modulate neuromuscular signaling in the feeding system of Aplysia. In this study, cDNA clones encoding a myomodulin precursor polypeptide were isolated and characterized. This precursor contains seven different myomodulin-related peptides, one of which, myomodulin A, is present in 10 contiguous copies.

The buccalin-related neuropeptides: isolation and characterization of an Aplysia cDNA clone encoding a family of peptide cotransmitters.

The buccalin-related peptides, buccalin A and buccalin B, are members of a family of cotransmitters that modulate neuromuscular transmission in Aplysia. In this study, a third buccalin-related peptide, buccalin C, was purified from neuronal elements in the accessory radula closer, a muscle involved in the animal's feeding behavior. Oligonucleotide probes based upon the amino acid sequence of buccalin C were used to isolate cDNA clones that encode a buccalin precursor polypeptide.

Catalytic subunits of Aplysia neuronal cAMP-dependent protein kinase with two different N termini.

Previously, two forms of cAMP-dependent protein kinase catalytic subunit generated by mutually exclusive use of two internal exon cassettes (A1 and A2) were demonstrated in Aplysia neurons. Here, it is shown that there also exist catalytic subunits with alternative N termini derived from two exons, N1 and N2, expressed in combination with either of the internal cassettes. Processed transcripts including N1 or N2 sequences are of about equal abundance in the nervous system, arise through alternative promoter use, and encode catalytically active polypeptides.

A regulatory subunit of the cAMP-dependent protein kinase down-regulated in aplysia sensory neurons during long-term sensitization.

Binding of cAMP by the five neuronal isoforms (N1-5) of the regulatory (R) subunit of the Aplysia cAMP-dependent protein kinase is diminished in sensory neurons stimulated to produce long-term presynaptic facilitation. To determine how the cAMP-binding activity of the R subunits is lost, we isolated cDNAs encoding N4, which is a homolog of mammalian RI. Immunoblots with antisera raised against the R protein overexpressed in E.

Cloning and characterization of Ca(2+)-dependent and Ca(2+)-independent PKCs expressed in Aplysia sensory cells.

We isolated cDNA clones from an Aplysia sensory-cell library encoding two isoforms of protein kinase C (PKC). Several isozyme-specific regions are conserved in the Aplysia kinases, notably the variable regions V5 in the Ca(2+)-dependent PKC (Apl I) and V1 in the Ca(2+)-independent PKC (Apl II). Neuronal proteins with the properties expected of these two isoforms can be identified with antibodies raised against peptides synthesized from the amino acid sequences deduced from the clones.

Primary structure of a molluscan egg-specific NADase, a second-messenger enzyme.

An egg-specific NADase has been purified from the ovotestis of the marine mollusk Aplysia californica. The enzyme converts NAD to cyclic ADP-ribose (cADPR), which is a potent mobilizer of Ca2+. It is likely that the NADase serves to raise Ca2+ levels in the ova at appropriate times.

A relative of the catalytic subunit of cyclic AMP-dependent protein kinase in Aplysia spermatozoa.

Transcripts encoding CAPL-B, an apparent member of the cyclic-nucleotide-regulated kinase subfamily in Aplysia californica, are found exclusively in the ovotestis and are concentrated in meiotic and postmeiotic spermatogenic cells. The CAPL-B polypeptide is present in mature spermatozoa, suggesting that the kinase plays a part in regulating events associated with fertilization.

Two catalytic subunits of cAMP-dependent protein kinase generated by alternative RNA splicing are expressed in Aplysia neurons.

The amino acid sequences of two catalytic (C) subunits of Aplysia cAMP-dependent protein kinase (cAPK) have been deduced from the nucleotide sequences of cDNAs generated from neuronal poly(A)+ RNA. Both subunits contain 352 residues and are identical except for amino acids 142-183, which differ at 10 out of 42 positions. They derive from alternatively spliced transcripts of a single gene (CAPL) containing two mutually exclusive exon cassettes.

In vivo and in vitro models of demyelinating disease: activation of the adenylate cyclase system influences JHM virus expression in explanted rat oligodendrocytes.

The specificity of JHM virus (JHMV) tropism for rat oligodendrocytes, as one of the primary host cells in the central nervous system, is maintained after explanation (S. Beushausen and S. Dales, Virology 141:89-101, 1985).

In vivo and in vitro models of demyelinating diseases. XV. Differentiation influences the regulation of coronavirus infection in primary explants of mouse CNS.

Mouse oligodendrocytes and astrocytes, in primary cerebral explant cultures, were infected with JHMV and MHV3 coronaviruses. Contrary to previous findings with neural cells from the rat (S. Beushausen and S.

In vivo and in vitro models of demyelinating disease. XI. Tropism and differentiation regulate the infectious process of coronaviruses in primary explants of the rat CNS.

The coronaviruses, ubiquitous in mammals, including man, manifest serotype-related predeliction for different tissues. This presentation deals with specificity of the murine viscerotropic MHV3 and neurotropic JHMV for explanted cells from the CNS of newborn, inbred, Wistar-Furth rats. An unambiguous tropism of MHV3 for astrocytes and JHMV for oligodendrocytes is demonstrated.