Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members

In the intracellular death program, hetero- and homodimerization of different anti- and pro-apoptotic Bcl-2-related proteins are critical in the determination of cell fate. From a rat ovarian fusion cDNA library, we isolated a new pro-apoptotic Bcl-2 gene, Bcl-2-related ovarian killer (Bok). Bok had conserved Bcl-2 homology (BH) domains 1, 2, and 3 and a C-terminal transmembrane region present in other Bcl-2 proteins, but lacked the BH4 domain found only in anti-apoptotic Bcl-2 proteins. In the yeast two-hybrid system, Bok interacted strongly with some (Mcl-1, BHRF1, and Bfl-1) but not other (Bcl-2, Bcl-xL, and Bcl-w) anti-apoptotic members. This finding is in direct contrast to the ability of other pro-apoptotic members (Bax, Bak, and Bik) to interact with all of the anti-apoptotic proteins. In addition, negligible interaction was found between Bok and different pro-apoptotic members. In mammalian cells, overexpression of Bok induced apoptosis that was blocked by the baculoviral-derived cysteine protease inhibitor P35. Cell killing induced by Bok was also suppressed following coexpression with Mcl-1 and BHRF1 but not with Bcl-2, further indicating that Bok heterodimerized only with selective anti-apoptotic Bcl-2 proteins. Northern blot analysis indicated that Bok was highly expressed in the ovary, testis and uterus. In situ hybridization analysis localized Bok mRNA in granulosa cells, the cell type that underwent apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic Bcl-2 protein with restricted tissue distribution and heterodimerization properties could facilitate elucidation of apoptosis mechanisms in reproductive tissues undergoing hormone-regulated cyclic cell turnover.

The Thrombospondin Receptor CD47 (IAP) Modulates and Associates with α2β1 Integrin in Vascular Smooth Muscle Cells

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a β1 integrin, rather than αvβ3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to α2 and β1 integrin subunits and to IAP. A combination of antiα2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antiαvβ3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between α2β1 and IAP was detected by the coimmunoprecipitation of both α2 and β1 integrin subunits with IAP. These data suggest that IAP can associate with α2β1 integrin and modulate its function.

A missense mutation of the Na+ channel αII subunit gene Nav1.2 in a patient with febrile and afebrile seizures causes channel dysfunction

Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in β1 and αI-subunit genes of voltage-gated Na+ channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R187W) in the gene encoding the α-subunit of neuronal voltage-gated Na+ channel type II (Nav1.2) in a patient with FS associated with afebrile seizures. The mutation R187W occurring on Arg187, a highly conserved residue among voltage-gated Na+ channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNav1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na+ channel conductance was not affected. Prolonged residence in the open state of the R187W mutant channel may augment Na+ influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Nav1.2 in a human disease and propose the R187W mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.

COX-2, a synaptically induced enzyme, is expressed by excitatory neurons at postsynaptic sites in rat cerebral cortex.

Postnatal development and adult function of the central nervous system are dependent on the capacity of neurons to effect long-term changes of specific properties in response to neural activity. This neuronal response has been demonstrated to be tightly correlated with the expression of a set of regulatory genes which include transcription factors as well as molecules that can directly modify cellular signaling. It is hypothesized that these proteins play a role in activity-dependent response. Previously, we described the expression and regulation in brain of an inducible form of prostaglandin synthase/cyclooxygenase, termed COX-2. COX-2 is a rate-limiting enzyme in prostanoid synthesis and its expression is rapidly regulated in developing and adult forebrain by physiological synaptic activity. Here we demonstrate that COX-2 immunoreactivity is selectively expressed in a subpopulation of excitatory neurons in neo-and allocortices, hippocampus, and amygdala and is compartmentalized to dendritic arborizations. Moreover, COX-2 immunoreactivity is present in dendritic spines, which are specialized structures involved in synaptic signaling. The developmental profile of COX-2 expression in dendrites follows well known histogenetic gradients and coincides with the critical period for activity-dependent synaptic remodeling. These results suggest that COX-2, and its diffusible prostanoid products, may play a role in postsynaptic signaling of excitatory neurons in cortex and associated structures.

Intravenous cocaine, morphine, and amphetamine preferentially increase extracellular dopamine in the "shell" as compared with the "core" of the rat nucleus accumbens.

The nucleus accumbens is considered a critical target of the action of drugs of abuse. In this nucleus a "shell" and a "core" have been distinguished on the basis of anatomical and histochemical criteria. The present study investigated the effect in freely moving rats of intravenous cocaine, amphetamine, and morphine on extracellular dopamine concentrations in the nucleus accumbens shell and core by means of microdialysis with vertically implanted concentric probes. Doses selected were in the range of those known to sustain drug self-administration in rats. Morphine, at 0.2 and 0.4 mg/kg, and cocaine, at 0.5 mg/kg, increased extracellular dopamine selectivity in the shell. Higher doses of cocaine (1.0 mg/kg) and the lowest dose of amphetamine tested (0.125 mg/kg) increased extracellular dopamine both in the shell and in the core, but the effect was significantly more pronounced in the shell compared with the core. Only the highest dose of amphetamine (0.250 mg/kg) increased extracellular dopamine in the shell and in the core to a similar extent. The present results provide in vivo neurochemical evidence for a functional compartmentation within the nucleus accumbens and for a preferential effect of psychostimulants and morphine in the shell of the nucleus accumbens at doses known to sustain intravenous drug self-administration.