Clinical and molecular characterization of a novel PLIN1 frameshift mutation identified in patients with familial partial lipodystrophy.

Perilipin 1 is a lipid droplet coat protein predominantly expressed in adipocytes, where it inhibits basal and facilitates stimulated lipolysis. Loss-of-function mutations in the PLIN1 gene were recently reported in patients with a novel subtype of familial partial lipodystrophy, designated as FPLD4. We now report the identification and characterization of a novel heterozygous frameshift mutation affecting the carboxy-terminus (439fs) of perilipin 1 in two unrelated families. The mutation cosegregated with a similar phenotype including partial lipodystrophy, severe insulin resistance and type 2 diabetes, extreme hypertriglyceridemia, and nonalcoholic fatty liver disease in both families. Poor metabolic control despite maximal medical therapy prompted two patients to undergo bariatric surgery, with remarkably beneficial consequences. Functional studies indicated that expression levels of the mutant protein were lower than wild-type protein, and in stably transfected preadipocytes the mutant protein was associated with smaller lipid droplets. Interestingly, unlike the previously reported 398 and 404 frameshift mutants, this variant binds and stabilizes ABHD5 expression but still fails to inhibit basal lipolysis as effectively as wild-type perilipin 1. Collectively, these findings highlight the physiological need for exquisite regulation of neutral lipid storage within adipocyte lipid droplets, as well as the possible metabolic benefits of bariatric surgery in this serious disease.

Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative stress.

The etiology and pathophysiology of schizophrenia remain unknown. A parallel transcriptomics, proteomics and metabolomics approach was employed on human brain tissue to explore the molecular disease signatures. Almost half the altered proteins identified by proteomics were associated with mitochondrial function and oxidative stress responses. This was mirrored by transcriptional and metabolite perturbations. Cluster analysis of transcriptional alterations showed that genes related to energy metabolism and oxidative stress differentiated almost 90% of schizophrenia patients from controls, while confounding drug effects could be ruled out. We propose that oxidative stress and the ensuing cellular adaptations are linked to the schizophrenia disease process and hope that this new disease concept may advance the approach to treatment, diagnosis and disease prevention of schizophrenia and related syndromes.

Low molecular weight constituents of male mouse urine mediate the pregnancy block effect and convey information about the identity of the mating male.

Mouse urine contains a complex mixture of chemosignals including a variety of small volatile molecules that are bound to major urinary proteins. In addition to signalling maleness, male urine also conveys information about individuality, which allows recently mated female mice to distinguish the urinary chemosignals of the mating male from those of an unfamiliar male. The highly polymorphic nature of the major urinary proteins makes them a likely candidate for conveying individuality information in the context of the pregnancy block effect. This was investigated by comparing the pregnancy-blocking effectiveness of a high molecular weight urinary fraction, containing major urinary proteins, with that of a low molecular weight fraction containing volatile ligands. Not only was the high molecular weight fraction ineffective in blocking pregnancy, but it also appeared to be less important in signalling individuality than the low molecular fraction. The high molecular weight fraction was ineffective in inducing expression of the immediate early gene product egr-1 in the accessory olfactory bulb. In contrast, the low molecular weight fraction induced egr-1 expression in the mitral/tufted neurons in the anterior subregion of the accessory olfactory bulb, suggesting that they activate the V1R class of vomeronasal receptor neuron.

Chronic dexamethasone-treatment alters mineralocorticoid receptor, truncated trkB and selected glutamate receptor subunit mRNA expression in the porcine hippocampus.

Prepubertal boars (n = 4/treatment) were killed 24 h after a 5 day course of intravenous injections of dexamethasone (Dex, 1 and 5 mg kg(-1)), or saline vehicle. Gene expression was quantified in brain sections following in situ hybridisation histochemistry. The objective was to determine whether chronic glucocorticoid treatment would alter the expression of mRNAs for gluco- and mineralocorticoid receptors (GR and MR), brain-derived neurotrophic factor (BDNF), its receptor, trkB, and selected ionotropic glutamate receptor (iGluR) subunits in the hippocampus. Although Dex did not alter GR message, the higher dose reduced MR mRNA in all hippocampal subfields studied. There was no effect of Dex on the expression of BDNF, or the full-length form of its receptor but there was evidence to suggest that mRNA for the truncated form of trkB was increased. Expression of mRNA for glutamate receptor subunits was either unaffected (NR1) or decreased (GluR2 and GluR3). These findings indicate that acute and chronic glucocorticoid treatment has differential effects on hippocampal gene expression in the porcine brain.

Down-regulation of BDNF mRNA, with no effect on trkB or glucocorticoid receptor m RNAs, in the porcine hippocampus after acute dexamethasone treatment.

Glucocorticoids bind to hippocampal mineralo-(MR) and gluco-(GR) corticoid receptors and, at high concentrations (e.g. as seen following treatment with pharmacological doses of corticosteroids or during stress), may affect hippocampal neuronal function. Such actions could involve brain-derived neurotrophic factor (BDNF), its receptor, trkB, and the excitatory neurotransmitter, glutamate. This experiment investigated the effect of a single intravenous (i.v.) injection of the synthetic glucocorticoid, dexamethasone (Dex, 5 mg kg(-1)) on gene expression for MR s, GR s, BDNF, trkB, and selected ionotropic glutamate receptor subunits (iGluRs), in the porcine hippocampus. Quantification of m RNA s in the brains of pigs (n = 4/treatment) killed 24 hours after saline or Dex administration indicated a significant Dex-induced decrease in BDNF m RNA in all hippocampal regions. However, gene expression for MR s, GR s, trkB and iGluRs was unaffected at this time-point.

Distribution of the neurotensin receptor NTS1 in the rat CNS studied using an amino-terminal directed antibody.

The distribution of neurotensin receptor 1 immunoreactivity in the rat brain was studied using an antibody against the amino-terminal of the receptor expressed as a fusion protein with glutathione-S transferase. Affinity purified antibodies detected the fusion protein and the complete neurotensin receptor sequence expressed in Escherichia coli. The immunostaining was abolished by preabsorption with the amino-terminal fusion protein. Immunoreactive neurotensin receptor 1 immunoreactivity was detected on cell bodies and their processes in a number of CNS regions. In agreement with previous binding studies neurotensin receptor 1 immunoreactivity was particularly localised in cell bodies in the basal forebrain, nucleus basalis and substantia nigra. At the electron microscope level immunoreactivity was found both in axonal bouton and dendrites and spines in the basal forebrain indicating that neurotensin may act both pre- and post-synaptically. There were several regions such as the substantia gelatinosa, ventral caudate-putamen and the lateral reticular nucleus where the neurotensin receptor 1 positive cells had not previously been reported, indicating that distribution of this receptor is widespread.

Is right hemisphere specialization for face discrimination specific to humans?

Patterns of neural activation during face recognition were investigated in sheep by quantifying altered c-fos mRNA expression in situations where faces (sheep vs. human) can (faces upright) and cannot (faces inverted) be discriminated. Exposure to upright faces selectively increased expression significantly more in the right inferior temporal cortex than in the left, and active choice between upright faces additionally increased expression bilaterally in basal amygdala and hippocampus (CA1-4). Exposure to inverted faces did not lead to enhanced activation in the right inferior temporal cortex, amygdala or hippocampus but instead increased expression levels in the diagonal band of Broca, parietal and cingulate cortices. These results show that discrimination of upright faces in sheep preferentially engages the right temporal cortex, as it does in humans, and that performance of active choices between such faces may additionally involve the basal amygdala and hippocampus.

Isolation of mouse vomeronasal receptor genes and their co-localization with specific G-protein messenger RNAs.

Four mouse vomeronasal receptors (mV1Rs) have been isolated by similarity to rat vomeronasal receptor (V1R) motifs. The four mV1Rs identified in this study are members of two distinct subfamilies. Specific in situ hybridization probes (ISH) derived from the 3' non-coding regions of the mV1R genes, were used to detect expression of a single receptor and probes from the homologous coding regions were used to detect expression of subfamily members. The ISH results showed that the mV1Rs expressing neurons were scattered in the middle/upper layer of the vomeronasal organ (VNO) sensory epithelium in serial VNO sections but were excluded from the deeper layers of the VNO sensory epithelium and these neurons were found to co-express the mRNA for the G-protein Galphai2, and were distinct from the deeper layers of the VNO sensory epithelium where the mRNA for Galphao positive neurons was located.

Expression of olfactory receptors, G-proteins and AxCAMs during the development and maturation of olfactory sensory neurons in the mouse.

Three mouse olfactory receptors have been cloned and sequenced and were found to be expressed in different zones of the olfactory epithelium. In situ hybridisation (ISH) results showed that each olfactory receptor was expressed at an early stage in development (E12), was not dependent on the maturation of the receptor neurons, and was present long before the onset of odour detection. Cells positive for these same olfactory receptors and the G-protein (Gbeta) were also found in non-neural regions of the nasal epithelium in the earlier stages of development (E12-16). Ncam, and Big-2 expression were, however, restricted to the region of developing olfactory neurons. Ncam expression appeared in advance of the olfactory receptor expression, while Big-2 appeared after olfactory receptor expression and neither were expressed in cells outside the olfactory epithelium. Both showed the highest number of positive cells in the early post-partum period when olfactory detection is functional. Ncam is known to be involved in guidance of the developing olfactory axons and was expressed earlier than any of the olfactory receptors, while Big-2 appears somewhat later (E14) at a time when developing axons reach the olfactory bulb. Moreover the highest periods of expression occur at post-natal day 7 when a proliferation of bulbar glomeruli are observed, suggesting the role of Big-2 to be primarily concerned with synaptogenesis.

Immunohistochemical localisation of mGluR7 protein in the rodent and human cerebellar cortex using subtype specific antibodies.

Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein coupled receptors that are linked to multiple second messenger systems to regulate neuronal excitability and synaptic transmission. To characterise the protein expression of the two mGluR7 receptor splice variants in human and rat cerebellar cortex, antibodies specific to mGluR7 were generated. Antibodies were raised against a glutathione-S-transferase fusion protein containing amino acid residues located in the extracellular domain common to both the human and rat mGluR7 splice variants. These antibodies specifically detected human mGluR7a in mammalian cells transfected with this receptor. In agreement with mGluR7 in situ hybridisation studies, immunohistochemistry performed at the light microscope level revealed that mGluR7 protein expression occurred most prominently in a particular population of nerve cells common to both the human and rat, located within the cerebellar cortex of gray matter contained within transverse folia. Moreover, strong mGluR7-like immunoreactivity was seen in Purkinje cell body cytoplasm of the Purkinje cell layer. In the most superficial cerebellar cortical layer, the molecular layer, immunostaining was observed in Purkinje cell associated proximal and distal dendritic trees. No detectable labelling was evident in intrinsic deep cerebellar nuclei known to contain GABAergic terminals of projecting Purkinje cell axons. These data are suggestive of a post-synaptic location of mGluR7 in this central nervous system structure. In the rodent, additional non-Purkinje cells thought to represent inhibitory interneurones were labelled at all levels in the molecular layer. mGluR7-like immunoreactivity was not associated with glial cells.

Formation of olfactory memories mediated by nitric oxide.

Sheep learn to recognize the odours of their lambs within two hours of giving birth, and this learning involves synaptic changes within the olfactory bulb. Specifically, mitral cells become increasingly responsive to the learned odour, which stimulates release of both glutamate and GABA (gamma-aminobutyric acid) neurotransmitters from the reciprocal synapses between the excitatory mitral cells and inhibitory granule cells. Nitric oxide (NO) has been implicated in synaptic plasticity in other regions of the brain as a result of its modulation of cyclic GMP levels. Here we investigate the possible role of NO in olfactory learning. We find that the neuronal enzyme nitric oxide synthase (nNOS) is expressed in both mitral and granule cells, whereas the guanylyl cyclase subunits that are required for NO stimulation of cGMP formation are expressed only in mitral cells. Immediately after birth, glutamate levels rise, inducing formation of NO and cGMP, which potentiate glutamate release at the mitral-to-granule cell synapses. Inhibition of nNOS or guanylyl cyclase activity prevents both the potentiation of glutamate release and formation of the olfactory memory. The effects of nNOS inhibition can be reversed by infusion of NO into the olfactory bulb. Once memory has formed, however, inhibition of nNOS or guanylyl cyclase activity cannot impair either its recall or the neurochemical release evoked by the learned lamb odour. Nitric oxide therefore seems to act as a retrograde and/or intracellular messenger, being released from both mitral and granule cells to potentiate glutamate release from mitral cells by modulating cGMP concentrations. We propose that the resulting changes in the functional circuitry of the olfactory bulb underlie the formation of olfactory memories.

A novel splice variant of the cell adhesion molecule BIG-2 is expressed in the olfactory and vomeronasal neuroepithelia.

We have cloned a mouse cDNA encoding a novel truncated form of the gene BIG-2 from the vomeronasal organ. The related proteins BIG-2 and BIG-1 possess a C-terminal glycosylphosphatidylinositol anchor, six immunoglobulin domains and four fibronectin type III repeats. They are related to certain axonal-associated cell adhesion molecules (AxCAMs) exhibiting most similarity to the TAG-1/F3 subgroup of neural cell adhesion molecules. The cDNA we have identified, termed BIG-2A, appears to represent a novel splice variant of BIG-2 possessing six Ig-like domains, a single fibronectin repeat and lacking the glycosylphosphatidylinositol-anchoring domain (GPI). To determine the expression of this gene, in situ hybridization analysis was performed in adult and developing mice using a riboprobe specific for BIG-2A. Maximum expression was observed in mature sensory cells of the vomeronasal neuroepithelium and a less intense signal was also evident in the olfactory neuroepithelium. These results suggest that alternative splicing of the BIG-2 gene transcript may play an important role in the organization of the vomeronasal and olfactory neuroepithelia.

A region of a Sym plasmid of Rhizobium leguminosarum biovar phaseoli has similarity to prokaryotic insertion sequences and to eukaryotic integrases.

Near the nod and nif genes of the Sym plasmid pRP2JI of Rhizobium leguminosarum biovar phaseoli are three open reading frames whose deduced polypeptide products have similarities to those of genes in bacterial insertion sequences. The similarity of one of these ORFs was significantly greater to that of the integrase region of pol proteins of eukaryotic retroviruses and transposable elements in animals and plants than it was to the transposases of prokaryotic insertion sequences. In the noncoding region of the IS-like element, there was a sequence similar to that which had been identified close to nod genes in Azorhizobium caulinodans.

The psi operon of Rhizobium leguminosarum biovar phaseoli: identification of two genes whose products are located at the bacterial cell surface.

We have delineated three short open reading frames, psiA, ORF-P and psiB within the psi operon of Rhizobium leguminosarum biovar phaseoli. psiA, in a multi-copy plasmid, causes inhibition of exopolysaccharide synthesis in R. leguminosarum. In addition, the suppression of exopolysaccharide synthesis due to the multi-copy psiA caused R. leguminosarum strains to stain with the dye calcofluor, a response that does not occur with wild-type strains of this species. Insertions of a defective phoA gene (lacking its promoter, ribosomal binding site and leader sequence) into psiA and psiB were isolated and the precise locations of the insertions were established. PsiA-PhoA and PsiB-PhoA protein fusions were found to express alkaline phosphatase activity indicating that PsiA and PsiB span the inner membrane or are translocated across it.

Sequence and regulation of psrA, a gene on the Sym plasmid of Rhizobium leguminosarum biover phaseoli which inhibits transcription of the psi genes.

The psr region of Rhizobium leguminosarum biovar phaseoli had originally been recognized on the basis of its ability to repress the transcription of the psi genes, one of which, psiA, inhibits exopolysaccharide synthesis when cloned in multi-copy plasmids. Both psr and psi are located on the symbiotic plasmid pRP2JI. The psrA gene was localized and sequenced. The deduced amino acid sequence of PsrA was shown to have similarity to the DNA-binding region of a family of other transcriptional regulators, consistent with its known effects on the expression of psi. The transcription of psrA itself appears to be constitutive in free-living Rhizobium, but is regulated by another gene on the Sym plasmid pRP2JI.

Membrane topology of the integral membrane components, OppB and OppC, of the oligopeptide permease of Salmonella typhimurium.

The oligopeptide permease of Salmonella typhimurium is a periplasmic binding protein-dependent transport system. Five gene products, OppABCDF, are required for the functioning of this transporter, two of which (OppB and OppC) are highly hydrophobic, integral membrane proteins and are responsible for mediating passage of peptides across the cytoplasmic membrane. OppB and OppC are each predicted, from their sequences, to span the membrane many times. In this paper we describe experimental evidence confirming these predictions using a combination of biochemical, immunological and genetic procedures. Each of these two proteins is shown to span the membrane six times, with the N- and C-termini both being located at the cytoplasmic face of the membrane. Opp is apparently a typical member of the ABC (ATP-binding cassette) superfamily of transporters. These findings, therefore, have general implications for the organization and function of other ABC transporters, including the human multidrug resistance protein and the product of the cystic fibrosis gene.

Binding protein-dependent transport systems.

Bacterial binding protein-dependent transport systems are the best characterized members of a superfamily of transporters which are structurally, functionally, and evolutionary related to each other. These transporters are not only found in bacteria but also in yeasts, plants, and animals including man, and include both import and export systems. Although any single system is relatively specific, different systems handle very different substrates which can be inorganic ions, amino acids, sugars, large polysaccharides, or even proteins. Some are of considerable medical importance, including Mdr, the protein responsible for multidrug resistance in human tumors, and the product of the cystic fibrosis locus. In this article we review the current state of knowledge on the structure and function of the protein components of these transporters, the mechanism by which transport is mediated, and the role of ATP in the transport process.

Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport.

The ATP-binding cassette (ABC) superfamily of transport systems now includes over thirty proteins that share extensive sequence similarity and domain organization. This superfamily includes the well characterized periplasmic binding protein-dependent uptake systems of prokaryotes, bacterial exporters, and eukaryotic proteins including the P-glycoprotein associated with multidrug resistance in tumours (MDR), the STE6 gene product that mediates export of yeast a-factor mating pheromone, pfMDR that is implicated in chloroquine resistance of the malarial parasite, and the product of the cystic fibrosis gene (CFTR). Here we present a tertiary structure model of the ATP-binding cassettes characteristic of this class of transport system, based on similarities between the predicted secondary structures of members of this family and the previously determined structure of adenylate kinase. This model has implications for both the molecular basis of transport and cystic fibrosis and provides a framework for further experimentation.

Periplasmic binding protein-dependent transport systems: the membrane-associated components.

Periplasmic binding protein-dependent transport systems are multicomponent, consisting of several inner membrane-associated proteins and a periplasmic component. The membrane-associated components of different systems are related in organization and function suggesting that, despite different substrate specificities, each transport system functions by a common mechanism. Current understanding of these components is reviewed. The nature of energy coupling to periplasmic transport systems has long been debated. Recent data now demonstrate that ATP hydrolysis is the primary source of energy for transport. The ATP-binding transport components are the best characterized of a family of closely related ATP-binding proteins believed to couple ATP hydrolysis to a variety of different biological processes. Intriguingly, systems closely related to periplasmic binding protein-dependent transport systems have recently been identified in several Gram-positive organisms (which lack a periplasm) and in eukaryotic cells. This class of transport system appears to be widespread in nature, serving a variety of important and diverse functions.