Identification of bdm-1, a gene involved in G protein β-subunit function and α-subunit accumulation

Targeted disruption of Gα and Gβ genes has established the requirement of an intact G protein signaling pathway for optimal execution of several important physiological processes, including pathogenesis, in the chestnut blight fungus Cryphonectria parasitica. We now report the identification of a G protein signal transduction component, beta disruption mimic factor-1, BDM-1. Disruption of the corresponding gene, bdm-1, resulted in a phenotype indistinguishable from that previously observed after disruption of the Gβ subunit gene, cpgb-1. The BDM-1 deduced amino acid sequence contained several significant clusters of identity with mammalian phosducin, including a domain corresponding to a highly conserved 11-amino acid stretch that has been implicated in binding to the Gβγ dimer and two regions of defined Gβ/phosducin contact points. Unlike the negative regulatory function proposed for mammalian phosducin, the genetic data presented in this report suggest that BDM-1 is required for or facilitates Gβ function. Moreover, disruption of either bdm-1 or cpgb-1 resulted in a significant, posttranscriptional reduction in the accumulation of CPG-1, a key Gα subunit required for a range of vital physiological processes.

Diversity and phylogeny of gephyrin: Tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing and cytoskeleton-associated proteins

Gephyrin is essential for both the postsynaptic localization of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (Moco) in different peripheral organs. Several alternatively spliced gephyrin transcripts have been identified in rat brain that differ in their 5′ coding regions. Here, we describe gephyrin splice variants that are differentially expressed in non-neuronal tissues and different regions of the adult mouse brain. Analysis of the murine gephyrin gene indicates a highly mosaic organization, with eight of its 29 exons corresponding to the alternatively spliced regions identified by cDNA sequencing. The N- and C-terminal domains of gephyrin encoded by exons 3–7 and 16–29, respectively, display sequence similarities to bacterial, invertebrate, and plant proteins involved in Moco biosynthesis, whereas the central exons 8, 13, and 14 encode motifs that may mediate oligomerization and tubulin binding. Our data are consistent with gephyrin having evolved from a Moco biosynthetic protein by insertion of protein interaction sequences.

The APC variants I1307K and E1317Q are associated with colorectal tumors, but not always with a family history

Classical familial adenomatous polyposis (FAP) is a high-penetrance autosomal dominant disease that predisposes to hundreds or thousands of colorectal adenomas and carcinoma and that results from truncating mutations in the APC gene. A variant of FAP is attenuated adenomatous polyposis coli, which results from germ-line mutations in the 5′ and 3′ regions of the APC gene. Attenuated adenomatous polyposis coli patients have “multiple” colorectal adenomas (typically fewer than 100) without the florid phenotype of classical FAP. Another group of patients with multiple adenomas has no mutations in the APC gene, and their phenotype probably results from variation at a locus, or loci, elsewhere in the genome. Recently, however, a missense variant of APC (I1307K) was described that confers an increased risk of colorectal tumors, including multiple adenomas, in Ashkenazim. We have studied a set of 164 patients with multiple colorectal adenomas and/or carcinoma and analyzed codons 1263–1377 (exon 15G) of the APC gene for germ-line variants. Three patients with the I1307K allele were detected, each of Ashkenazi descent. Four patients had a germ-line E1317Q missense variant of APC that was not present in controls; one of these individuals had an unusually large number of metaplastic polyps of the colorectum. There is increasing evidence that there exist germ-line variants of the APC gene that predispose to the development of multiple colorectal adenomas and carcinoma, but without the florid phenotype of classical FAP, and possibly with importance for colorectal cancer risk in the general population.

Mouse Deformed epidermal autoregulatory factor 1 recruits a LIM domain factor, LMO-4, and CLIM coregulators

Nuclear LIM domains interact with a family of coregulators referred to as Clim/Ldb/Nli. Although one family member, Clim-2/Ldb-1/Nli, is highly expressed in epidermal keratinocytes, no nuclear LIM domain factor is known to be expressed in epidermis. Therefore, we used the conserved LIM-interaction domain of Clim coregulators to screen for LIM domain factors in adult and embryonic mouse skin expression libraries and isolated a factor that is highly homologous to the previously described LIM-only proteins LMO-1, -2, and -3. This factor, referred to as LMO-4, is expressed in overlapping manner with Clim-2 in epidermis and in several other regions, including epithelial cells of the gastrointestinal, respiratory and genitourinary tracts, developing cartilage, pituitary gland, and discrete regions of the central and peripheral nervous system. Like LMO-2, LMO-4 interacts strongly with Clim factors via its LIM domain. Because LMO/Clim complexes are thought to regulate gene expression by associating with DNA-binding proteins, we used LMO-4 as a bait to screen for such DNA-binding proteins in epidermis and isolated the mouse homologue of Drosophila Deformed epidermal autoregulatory factor 1 (DEAF-1), a DNA-binding protein that interacts with regulatory sequences first described in the Deformed epidermal autoregulatory element. The interaction between LMO-4 and mouse DEAF-1 maps to a proline-rich C-terminal domain of mouse DEAF-1, distinct from the helix–loop–helix and GATA domains previously shown to interact with LMOs, thus defining an additional LIM-interacting domain.

The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1.302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.

Neuroimaging of cerebral activations and deactivations associated with hypercapnia and hunger for air

There are defined medullary, mesencephalic, hypothalamic, and thalamic functions in regulation of respiration, but knowledge of cortical control and the elements subserving the consciousness of breathlessness and air hunger is limited. In nine young adults, air hunger was produced acutely by CO2 inhalation. Comparisons were made with inhalation of a N2/O2 gas mixture with the same apparatus, and also with paced breathing, and with eyes closed rest. A network of activations in pons, midbrain (mesencephalic tegmentum, parabrachial nucleus, and periaqueductal gray), hypothalamus, limbic and paralimbic areas (amygdala and periamygdalar region) cingulate, parahippocampal and fusiform gyrus, and anterior insula were seen along with caudate nuclei and pulvinar activations. Strong deactivations were seen in dorsal cingulate, posterior cingulate, and prefrontal cortex. The striking response of limbic and paralimbic regions points to these structures having a singular role in the affective sequelae entrained by disturbance of basic respiratory control whereby a process of which we are normally unaware becomes a salient element of consciousness. These activations and deactivations include phylogenetically ancient areas of allocortex and transitional cortex that together with the amygdalar/periamygdalar region may subserve functions of emotional representation and regulation of breathing.

Neuroimaging evidence implicating cerebellum in the experience of hypercapnia and hunger for air

Recent neuroimaging and neurological data implicate cerebellum in nonmotor sensory, cognitive, vegetative, and affective functions. The present study assessed cerebellar responses when the urge to breathe is stimulated by inhaled CO2. Ventilation changes follow arterial blood partial pressure CO2 changes sensed by the medullary ventral respiratory group (VRG) and hypothalamus, entraining changes in midbrain, pons, thalamus, limbic, paralimbic, and insular regions. Nearly all these areas are known to connect anatomically with the cerebellum. Using positron emission tomography, we measured regional brain blood flow during acute CO2-induced breathlessness in humans. Separable physiological and subjective effects (air hunger) were assessed by comparisons with various respiratory control conditions. The conjoint physiological effects of hypercapnia and the consequent air hunger produced strong bilateral, near-midline activations of the cerebellum in anterior quadrangular, central, and lingula lobules, and in many areas of posterior quadrangular, tonsil, biventer, declive, and inferior semilunar lobules. The primal emotion of air hunger, dissociated from hypercapnia, activated midline regions of the central lobule. The distributed activity across the cerebellum is similar to that for thirst, hunger, and their satiation. Four possible interpretations of cerebellar function(s) here are that: it subserves implicit intentions to access air; it provides predictive internal models about the consequences of CO2 inhalation; it modulates emotional responses; and that while some cerebellar regions monitor sensory acquisition in the VRG (CO2 concentration), others influence VRG to adjust respiratory rate to optimize partial pressure CO2, and others still monitor and optimize the acquisition of other sensory data in service of air hunger aroused vigilance.

Mantle dynamics and seismic tomography

Three-dimensional imaging of the Earth's interior, called seismic tomography, has achieved breakthrough advances in the last two decades, revealing fundamental geodynamical processes throughout the Earth's mantle and core. Convective circulation of the entire mantle is taking place, with subducted oceanic lithosphere sinking into the lower mantle, overcoming the resistance to penetration provided by the phase boundary near 650-km depth that separates the upper and lower mantle. The boundary layer at the base of the mantle has been revealed to have complex structure, involving local stratification, extensive structural anisotropy, and massive regions of partial melt. The Earth's high Rayleigh number convective regime now is recognized to be much more interesting and complex than suggested by textbook cartoons, and continued advances in seismic tomography, geodynamical modeling, and high-pressure–high-temperature mineral physics will be needed to fully quantify the complex dynamics of our planet's interior.

The spatial and temporal representation of a tone on the guinea pig basilar membrane

In the mammalian cochlea, the basilar membrane's (BM) mechanical responses are amplified, and frequency tuning is sharpened through active feedback from the electromotile outer hair cells (OHCs). To be effective, OHC feedback must be delivered to the correct region of the BM and introduced at the appropriate time in each cycle of BM displacement. To investigate when OHCs contribute to cochlear amplification, a laser-diode interferometer was used to measure tone-evoked BM displacements in the basal turn of the guinea pig cochlea. Measurements were made at multiple sites across the width of the BM, which are tuned to the same characteristic frequency (CF). In response to CF tones, the largest displacements occur in the OHC region and phase lead those measured beneath the outer pillar cells and adjacent to the spiral ligament by about 90°. Postmortem, responses beneath the OHCs are reduced by up to 65 dB, and all regions across the width of the BM move in unison. We suggest that OHCs amplify BM responses to CF tones when the BM is moving at maximum velocity. In regions of the BM where OHCs contribute to its motion, the responses are compressive and nonlinear. We measured the distribution of nonlinear compressive vibrations along the length of the BM in response to a single frequency tone and estimated that OHC amplification is restricted to a 1.25- to 1.40-mm length of BM centered on the CF place.

Subdivisions of auditory cortex and processing streams in primates

The auditory system of monkeys includes a large number of interconnected subcortical nuclei and cortical areas. At subcortical levels, the structural components of the auditory system of monkeys resemble those of nonprimates, but the organization at cortical levels is different. In monkeys, the ventral nucleus of the medial geniculate complex projects in parallel to a core of three primary-like auditory areas, AI, R, and RT, constituting the first stage of cortical processing. These areas interconnect and project to the homotopic and other locations in the opposite cerebral hemisphere and to a surrounding array of eight proposed belt areas as a second stage of cortical processing. The belt areas in turn project in overlapping patterns to a lateral parabelt region with at least rostral and caudal subdivisions as a third stage of cortical processing. The divisions of the parabelt distribute to adjoining auditory and multimodal regions of the temporal lobe and to four functionally distinct regions of the frontal lobe. Histochemically, chimpanzees and humans have an auditory core that closely resembles that of monkeys. The challenge for future researchers is to understand how this complex system in monkeys analyzes and utilizes auditory information.

Genomic analysis of orthologous mouse and human olfactory receptor loci

Olfactory receptor (OR) genes represent ≈1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced ≈350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence ≈111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.

Molecular Cloning of Vacuolar H+-Pyrophosphatase and Its Developmental Expression in Growing Hypocotyl of Mung Bean1

Vacuolar proton-translocating inorganic pyrophosphatase and H+-ATPase acidify the vacuoles and power the vacuolar secondary active transport systems in plants. Developmental changes in the transcription of the pyrophosphatase in growing hypocotyls of mung bean (Vigna radiata) were investigated. The cDNA clone for the mung bean enzyme contains an uninterrupted open reading frame of 2298 bp, coding for a polypeptide of 766 amino acids. Hypocotyls were divided into elongating and mature regions. RNA analysis revealed that the transcript level of the pyrophosphatase was high in the elongating region of the 3-d-old hypocotyl but was extremely low in the mature region of the 5-d-old hypocotyl. The level of transcript of the 68-kD subunit of H+-ATPase also decreased after cell maturation. In the elongating region, the proton-pumping activity of pyrophosphatase on the basis of membrane protein was 3 times higher than that of H+-ATPase. After cell maturation, the pyrophosphatase activity decreased to 30% of that in the elongating region. The decline in the pyrophosphatase activity was in parallel with a decrease in the enzyme protein content. These findings indicate that the level of the pyrophosphatase, a main vacuolar proton pump in growing cells, is negatively regulated after cell maturation at the transcriptional level.

Long-lasting reduction of inhibitory function and gamma-aminobutyric acid type A receptor subunit mRNA expression in a model of temporal lobe epilepsy.

This study evaluated hippocampal inhibitory function and the level of expression of gamma-aminobutyric acid type A (GABAA) receptor mRNA in an in vivo model of epilepsy. Chronic recurrent limbic seizures were induced in rats using injections of pilocarpine. Electrophysiological studies performed on hippocampal slices prepared from control and epileptic animals 1 to 2 months after pilocarpine injections demonstrated a significant hyperexcitability in the epileptic animals. Reduced levels of mRNA expression for the alpha 2 and alpha 5 subunits of the GABAA receptors were evident in the CA1, CA2, and CA3 regions of the hippocampus of epileptic animals. No decrease in mRNA encoding alpha 1, beta 2, or gamma 2 GABAA receptor subunits was observed. In addition, no change in the mRNA levels of alpha CaM kinase II was seen. Selective decreases in mRNA expression did not correlate with neuronal cell loss. The results indicate that selective, long-lasting reduction of GABAA subunit mRNA expression and increased excitability, possibly reflecting loss of GABAergic inhibition, occur in an in vivo model of partial complex epilepsy.

Memory for object features versus memory for object location: a positron-emission tomography study of encoding and retrieval processes.

Regional cerebral blood flow was measured with positron-emission tomography during two encoding and two retrieval tasks that were designed to compare memory for object features with memory for object locations. Bilateral increases in regional cerebral blood flow were observed in both anterior and posterior regions of inferior temporal cortex and in ventral regions of prestriate cortex, when the condition that required retrieval of object locations was subtracted from the condition that required retrieval of object features. During encoding, these changes were less pronounced and were restricted to the left inferior temporal cortex and right ventral prestriate cortex. In contrast, both encoding and retrieval of object location were associated with bilateral changes in dorsal prestriate and posterior parietal cortex. Finally, the two encoding conditions activated left frontal lobe regions preferentially, whereas the two retrieval conditions activated right frontal lobe regions. These findings confirm that, in human subjects, memory for object features is mediated by a distributed system that includes ventral prestriate cortex and both anterior and posterior regions of the inferior temporal gyrus. In contrast, memory for the locations of objects appears to be mediated by an anatomically distinct system that includes more dorsal regions of prestriate cortex and posterior regions of the parietal lobe.

Cloning and characterization of cDNAs for matrix metalloproteinases of regenerating newt limbs.

Matrix metalloproteinases (MMPs) of regenerating urodele limbs have been suggested to play crucial roles in the process of the dedifferentiation of cells in the damaged tissues and the ensuing blastema formation because the activation of MMPs is an early and conspicuous event occurring in the amputated limb. MMP cDNAs were cloned as products of the reverse transcription-PCR from cDNA libraries of newt limbs, and their structures were characterized. Three cDNAs encoding newt MMPs (2D-1, 2D-19, and 2D-24) have been cloned from second day postamputation regenerating limbs, and a cDNA (EB-1) was cloned from early bud-stage regenerating limbs. These cDNAs included the full-length coding regions. The deduced amino acid sequences of 2D-1, 2D-19, 2D-24, and EB-1 had a homology with mammalian MMP9, MMP3/10, MMP3/10, and MMP13, respectively. The basic motif of these newt MMP genes was similar to mammalian counterparts and contained regions encoding a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. However, some unique molecular evolutionary features were also found in the newt MMPs. cDNAs of 2D-19 and 2D-24 contained a specific insertion and deletion, respectively. The insertion of 2D-19 is threonine-rich, similar to the threonine cluster found in the collagenase-like sea urchin hatching enzyme. Northern blot analysis showed that the expression levels of the newt MMPs were dramatically increased after amputation, suggesting that they play an important role(s) in tissue remodeling of the regenerating limb.