Characterization of a 23-kDa protein associated with CD40.

CD40 is a 45-kDa glycoprotein member of the tumor necrosis factor receptor (TNFR) family expressed on B cells, thymic epithelial cells, dendritic cells, and some carcinoma cells. The unique capacity of CD40 to trigger immunoglobulin isotype switching is dependent on the activation of protein-tyrosine kinases, yet CD40 possesses no kinase domain and no known consensus sequences for binding to protein-tyrosine kinases. Recently, an intracellular protein (CD40bp/LAP-1/CRAF-1) which belongs to the family of TNFR-associated proteins was reported to associate with CD40. We describe a 23-kDa cell surface protein (p23) which is specifically associated with CD40 on B cells and on urinary bladder transitional carcinoma cells. Protein microsequencing revealed that p23 shows no homology to any known protein. A rabbit antibody raised against a peptide derived from p23 recognized a 23-kDa protein in CD40 immunoprecipitates. In contrast to CD40bp/LAP-1/CRAF-1, p23 was not associated with TNFR p80 (CD120b). These findings suggest that p23 is a novel member of the CD40 receptor complex.

Primary structure and functional expression of a cGMP-gated potassium channel.

Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcn1) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcn1 also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcn1 represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.

Indirect coupling of phosphate release to de novo tension generation during muscle contraction.

A key question in muscle contraction is how tension generation is coupled to the chemistry of the actomyosin ATPase. Biochemical and mechanochemical experiments link tension generation to a change in structure associated with phosphate release. Length-jump and temperature-jump experiments, on the other hand, implicate phase 2slow, a significantly faster, markedly strain-sensitive kinetic process in tension generation. We use a laser temperature jump to probe the kinetics and mechanism of tension generation in skinned rabbit psoas fibers--an appropriate method since both phosphate release and phase 2slow are readily perturbed by temperature. Kinetics characteristic of the structural change associated with phosphate release are observed only when phosphate is added to fibers. When present, it causes a reduction in fiber tension; otherwise, no force is generated when it is perturbed. We therefore exclude this step from tension generation. The kinetics of de novo tension generation by the temperature-jump equivalent of phase 2slow appear unaffected by phosphate binding. We therefore propose that phosphate release is indirectly coupled to de novo tension generation via a steady-state flux through an irreversible step. We conclude that tension generation occurs in the absence of chemical change as the result of an entropy-driven transition between strongly bound crossbridges in the actomyosin-ADP state. The mechanism resembles the operation of a clock, with phosphate release providing the energy to tension the spring, and the irreversible step functions as the escapement mechanism, which is followed in turn by tension generation as the movement of the hands.

An alternative pathway for signal flow from rod photoreceptors to ganglion cells in mammalian retina.

Rod signals in the mammalian retina are thought to reach ganglion cells over the circuit rod-->rod depolarizing bipolar cell-->AII amacrine cell-->cone bipolar cells-->ganglion cells. A possible alternative pathway involves gap junctions linking the rods and cones, the circuit being rod-->cone-->cone bipolar cells-->ganglion cells. It is not clear whether this second pathway indeed relays rod signals to ganglion cells. We studied signal flow in the isolated rabbit retina with a multielectrode array, which allows the activity of many identified ganglion cells to be observed simultaneously while the preparation is stimulated with light and/or exposed to drugs. When transmission between rods and rod depolarizing bipolar cells was blocked by the glutamate agonist 2-amino-4-phosphonobutyric acid (APB), rod input to all On-center and briskly responding Off-center ganglion cells was dramatically reduced as expected. Off responses persisted, however, in Off-center sluggish and On-Off direction-selective ganglion cells. Presumably these responses were generated by the alternative pathway involving rod-cone junctions. This APB-resistant pathway may carry the major rod input to Off-center sluggish and On-Off direction-selective ganglion cells.

Chimeric Na+/H+ exchangers: an epithelial membrane-bound N-terminal domain requires an epithelial cytoplasmic C-terminal domain for regulation by protein kinases.

All cloned members of the mammalian Na+/H+ exchanger gene family encode proteins that consist of two functionally distinct domains: a membrane-bound N terminus and a cytoplasmic C terminus, which are required for ion transport and regulation of transport, respectively. Despite their similarity in structure, three members of this family, designated NHE1, NHE2, and NHE3, exhibit different kinetic mechanisms in response to growth factors and protein kinases. For instance, growth factors stimulate NHE1 by a change in the affinity constant for intracellular H+, K'(Hi+), and regulate NHE2 and NHE3 by a change in Vmax. We have constructed chimeric Na+/H+ exchangers by exchanging the N and C termini among three cloned rabbit Na+/H+ exchangers (NHE1 to NHE3) to determine which domain is responsible for the above Vmax-vs.-K'(H(i)+) effect of the Na+/H+ isoforms. All of the chimeras had functional exchange activity and basal kinetic properties similar to those of wild-type exchangers. Studies with serum showed that the N terminus is responsible for the Vmax-vs.-K'(H(i)+) stimulation of the Na+/H+ exchanger isoforms. Moreover, phorbol 12-myristate 13-acetate and fibroblast growth factor altered Na+/H+ exchange only in chimeras that had an epithelial N-terminal domain matched with an epithelial C-terminal domain. Therefore, the protein kinase-induced regulation of Na+/H+ exchangers is mediated through a specific interaction between the N- and C-termini, whcih is restricted so that epithelial N- and epithelial N-and C-terminal portions of the exchangers are required for regulation.

Efficient gene transfer into human hepatocytes by baculovirus vectors.

Viral vectors are the most efficient tools for gene delivery, and the search for tissue-specific infecting viruses is important for the development of in vivo gene therapy strategies. The baculovirus Autographa californica nuclear polyhedrosis virus is widely used as a vector for expression of foreign genes in insect cells, and its host specificity is supposed to be restricted to arthropods. Here we demonstrate that recombinant A. californica nuclear polyhedrosis virus is efficiently taken up by human hepatocytes via an endosomal pathway. High-level reporter gene expression from heterologous promoters was observed in human and rabbit hepatocytes in vitro. Mouse hepatocytes and some other epithelial cell types are targeted at a considerably lower rate. The efficiency of gene transfer by baculovirus considerably exceeds that obtained by calcium phosphate or lipid transfection. These properties of baculovirus suggest a use for it as a vector for liver-directed gene transfer but highlight a potential risk in handling certain recombinant baculoviruses.

Immunophenotyping of melanomas for tyrosinase: implications for vaccine development.

Tyrosinase (EC 1.14.18.1), the key enzyme in melanin synthesis, has been shown to be one of the targets for cytotoxic T-cell recognition in melanoma patients. To develop serological reagents useful for immunophenotyping melanoma for tyrosinase, human tyrosinase cDNA was expressed in an Escherichia coli expression vector. The purified recombinant tyrosinase was used to generate mouse monoclonal and rabbit polyclonal antibodies. The prototype monoclonal antibody, T311, recognized a cluster of protein moieties ranging from 70 to 80 kDa in tyrosinase mRNA-positive melanoma cell lines and melanoma specimens as well as in L cells transfected with tyrosinase cDNA. Untransfected L cells and L cells transfected with tyrosinase-related protein 1, TRP-1(gp75), were nonreactive. Immunohistochemical analysis of melanomas with T311 showed tyrosinase in melanotic and amelanotic variants, and tyrosinase expression correlated with the presence of tyrosinase mRNA. Melanocytes in skin stained with T311, whereas other normal tissues tested were negative. The expression pattern of three melanosome-associated proteins--tyrosinase, TRP-1(gp75), and gp100--in melanoma was also compared. Tyrosinase and gp100 are expressed in a higher percentage of melanomas than TRP-1(gp75), and the expression of these three antigens was discordant. Tyrosinase expression within individual tumor specimen is usually homogenous, distinctly different from the commonly observed heterogeneous pattern of gp100 expression.

Apolipoprotein B mRNA-editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals.

Apolipoprotein (apo-) B mRNA editing is the deamination of cytidine that creates a new termination codon and produces a truncated version of apo-B (apo-B48). The cytidine deaminase catalytic subunit [apo-B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1)] of the multiprotein editing complex has been identified. We generated transgenic rabbits and mice expressing rabbit APOBEC-1 in their livers to determine whether hepatic expression would lower low density lipoprotein cholesterol concentrations. The apo-B mRNA from the livers of the transgenic mice and rabbit was extensively edited, and the transgenic animals had reduced concentrations of apo-B100 and low density lipoproteins compared with control animals. Unexpectedly, all of the transgenic mice and a transgenic rabbit had liver dysplasia, and many transgenic mice developed hepatocellular carcinomas. Many of the mouse livers were hyperplastic and filled with lipid. Other hepatic mRNAs with sequence motifs similar to apo-B mRNA were examined for this type of editing (i.e., cytidine deamination). One of these, tyrosine kinase, was edited in livers of transgenic mice but not of controls. This result demonstrates that other mRNAs can be edited by the overexpressed editing enzyme and suggests that aberrant editing of hepatic mRNAs involved in cell growth and regulation is the cause of the tumorigenesis. Finally, these findings compromise the potential use of APOBEC-1 for gene therapy to lower plasma levels of low density lipoproteins.

Reconsideration of the catalytic center and mechanism of mammalian paraoxonase/arylesterase.

For three decades, mammalian paraoxonase (A-esterase, aromatic esterase, arylesterase; PON, EC 3.1.8.1) has been thought to be a cysteine esterase demonstrating structural and mechanistic homologies with the serine esterases (cholinesterases and carboxyesterases). Human, mouse, and rabbit PONs each contain only three cysteine residues, and their positions within PON have been conserved. In purified human PON, residues Cys-41 and Cys-352 form an intramolecular disulfide bond and neither could function as an active-center cysteine. Highly purified, enzymatically active PON contains a single titratable sulfhydryl group. Thus, Cys-283 is the only probable candidate for an active-center cysteine. Through site-directed mutagenesis of the human cDNA, Cys-283 was replaced with either serine (C283S) or alanine (C283A). The expressed C283 (wild-type) enzyme was inactivated by para-hydroxymercuribenzoate, but the C283S and C283A mutant enzymes were not inactivated. C283A and C283S mutant enzymes retained both paraoxonase and arylesterase activities, and the Km values for paraoxon and phenyl acetate were similar to those of the wild type. Clearly, residue Cys-283 is free in active PON, but a free sulfhydryl group is not required for either paraoxonase or arylesterase activities. Consequently, it is necessary to examine other models for the active-site structure and catalytic mechanism of PON.

Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury.

Disruption of the renal proximal tubule (PT) brush border is a prominent early event during ischemic injury to the kidney. The molecular basis for this event is unknown. Within the brush border, ezrin may normally link the cytoskeleton to the cell plasma membrane. Anoxia causes ezrin to dissociate from the cytoskeleton and also causes many cell proteins to become dephosphorylated in renal PTs. This study examines the hypothesis that ezrin dephosphorylation accompanies and may mediate the anoxic disruption of the rabbit renal PT. During normoxia, 73 +/- 3% of the cytoskeleton-associated (Triton-insoluble) ezrin was phosphorylated, but 88 +/- 6% of dissociated (Triton-soluble) ezrin was dephosphorylated. Phosphorylation was on serine/threonine resides, since ezrin was not detectable by an antibody against phosphotyrosine. After 60 min of anoxia, phosphorylation of total intracellular ezrin significantly decreased from 72 +/- 2% to 21 +/- 9%, and ezrin associated with the cytoskeleton decreased from 91 +/- 2% to 58 +/- 2%. Calyculin A (1 microM), the serine/threonine phosphatase inhibitor, inhibited the dephosphorylation of ezrin during anoxia by 57% and also blocked the dissociation of ezrin from the cytoskeleton by 53%. Our results demonstrate that (i) the association of ezrin with the renal microvillar cytoskeleton is correlated with phosphorylation of ezrin serine/threonine residues and (ii) anoxia may cause disruption of the renal brush border by dephosphorylating ezrin and thereby dissociating the brush border membrane from the cytoskeleton.

Functional anatomy of human eyeblink conditioning determined with regional cerebral glucose metabolism and positron-emission tomography.

Relative cerebral glucose metabolism was examined with positron-emission tomography (PET) as a measure of neuronal activation during performance of the classically conditioned eyeblink response in 12 young adult subjects. Each subject received three sessions: (i) a control session with PET scan in which unpaired presentations of the tone conditioned stimulus and corneal airpuff unconditioned stimulus were administered, (ii) a paired training session to allow associative learning to occur, and (iii) a paired test session with PET scan. Brain regions exhibiting learning-related activation were identified as those areas that showed significant differences in glucose metabolism between the unpaired control condition and well-trained state in the 9 subjects who met the learning criterion. Areas showing significant activation included bilateral sites in the inferior cerebellar cortex/deep nuclei, anterior cerebellar vermis, contralateral cerebellar cortex and pontine tegmentum, ipsilateral inferior thalamus/red nucleus, ipsilateral hippocampal formation, ipsilateral lateral temporal cortex, and bilateral ventral striatum. Among all subjects, including those who did not meet the learning criterion, metabolic changes in ipsilateral cerebellar nuclei, bilateral cerebellar cortex, anterior vermis, contralateral pontine tegmentum, ipsilateral hippocampal formation, and bilateral striatum correlated with degree of learning. The localization to cerebellum and its associated brainstem circuitry is consistent with neurobiological studies in the rabbit model of eyeblink classical conditioning and neuropsychological studies in brain-damaged humans. In addition, these data support a role for the hippocampus in conditioning and suggest that the ventral striatum may also be involved.

Accumulation of plasma cells in atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits.

By screening a cDNA library constructed from aortic total RNA derived from Watanabe heritable hyperlipidemic (WHHL) rabbits by differential hybridization, we have obtained a cDNA encoding the kappa light chain of immunoglobulin. Northern blot analysis of total RNA prepared from aortas of WHHL and normal rabbits of various ages revealed that this light-chain mRNA accumulates gradually with age in aortas in WHHL rabbits. Northern blotting and in situ hybridization with an antisense oligonucleotide specific to rabbit immunoglobulin gamma heavy-chain mRNA also detected accumulation of this heavy-chain mRNA in advanced lesions of WHHL rabbit aortas. Moreover, immunohistochemical and electron microscopic analyses demonstrated the presence of plasma cells in the atherosclerotic lesions.

Genetic engineering of vein grafts resistant to atherosclerosis.

Previously, researchers have speculated that genetic engineering can improve the long-term function of vascular grafts which are prone to atherosclerosis and occlusion. In this study, we demonstrated that an intraoperative gene therapy approach using antisense oligodeoxynucleotide blockage of medial smooth muscle cell proliferation can prevent the accelerated atherosclerosis that is responsible for autologous vein graft failure. Selective blockade of the expression of genes for two cell cycle regulatory proteins, proliferating cell nuclear antigen and cell division cycle 2 kinase, was achieved in the smooth muscle cells of rabbit jugular veins grafted into the carotid arteries. This alteration of gene expression successfully redirected vein graft biology away from neointimal hyperplasia and toward medial hypertrophy, yielding conduits that more closely resembled normal arteries. More importantly, these genetically engineered grafts proved resistant to diet-induced atherosclerosis. These findings establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular disease.

Spatial organization of retinal information about the direction of image motion.

The visual stimuli that elicit neural activity differ for different retinal ganglion cells and these cells have been categorized by the visual information that they transmit. If specific visual information is conveyed exclusively or primarily by a particular set of ganglion cells, one might expect the cells to be organized spatially so that their sampling of information from the visual field is complete but not redundant. In other words, the laterally spreading dendrites of the ganglion cells should completely cover the retinal plane without gaps or significant overlap. The first evidence for this sort of arrangement, which has been called a tiling or tessellation, was for the two types of "alpha" ganglion cells in cat retina. Other reports of tiling by ganglion cells have been made subsequently. We have found evidence of a particularly rigorous tiling for the four types of ganglion cells in rabbit retina that convey information about the direction of retinal image motion (the ON-OFF direction-selective cells). Although individual cells in the four groups are morphologically indistinguishable, they are organized as four overlaid tilings, each tiling consisting of like-type cells that respond preferentially to a particular direction of retinal image motion. These observations lend support to the hypothesis that tiling is a general feature of the organization of information outflow from the retina and clearly implicate mechanisms for recognition of like-type cells and establishment of mutually acceptable territories during retinal development.

Refinement of odor molecule tuning by dendrodendritic synaptic inhibition in the olfactory bulb.

Mitral/tufted cells (M/T cells) and granule cells form reciprocal dendrodendritic synapses in the main olfactory bulb; the granule cell is excited by glutamate from the M/T cell and in turn inhibits M/T cells by gamma-aminobutyrate. The trans-synaptically excited granule cell is thought to induce lateral inhibition in neighboring M/T cells and to refine olfactory information. It remains, however, elusive how significantly and specifically this synaptic regulation contributes to the discrimination of different olfactory stimuli. This investigation concerns the mechanism of olfactory discrimination by single unit recordings of responses to a series of normal aliphatic aldehydes from individual rabbit M/T cells. This analysis revealed that inhibitory responses are evoked in a M/T cell by a defined subset of odor molecules with structures closely related to the excitatory odor molecules. Furthermore, blockade of the reciprocal synaptic transmission by the glutamate receptor antagonist or the gamma-aminobutyrate receptor antagonist markedly suppressed the odor-evoked inhibition, indicating that the inhibitory responses are evoked by lateral inhibition via the reciprocal synaptic transmission. The synaptic regulation in the olfactory bulb thus greatly enhances the tuning specificity of odor responses and would contribute to discrimination of olfactory information.