An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein

The human endogenous retrovirus K (HERV-K) family of endogenous retroviruses consists of ≈50 proviral copies per haploid human genome. Herein, the HERV-Ks are shown to encode a sequence-specific nuclear RNA export factor, termed K-Rev, that is functionally analogous to the HIV-1 Rev protein. Like HIV-1 Rev, K-Rev binds to both the Crm1 nuclear export factor and to a cis-acting viral RNA target to activate nuclear export of unspliced RNAs. Surprisingly, this HERV-K RNA sequence, which is encoded within the HERV-K long terminal repeat, is also recognized by HIV-1 Rev. These data provide surprising evidence for an evolutionary link between HIV-1 and a group of endogenous retroviruses that first entered the human genome ≈30 million years ago.

Neurosteroids: Deficient cognitive performance in aged rats depends on low pregnenolone sulfate levels in the hippocampus

Pregnenolone sulfate (PREG S) is synthesized in the nervous system and is a major neurosteroid in the rat brain. Its concentrations were measured in the hippocampus and other brain areas of single adult and aged (22–24 month-old) male Sprague–Dawley rats. Significantly lower levels were found in aged rats, although the values were widely scattered and reached, in about half the animals, the same range as those of young ones. The spatial memory performances of aged rats were investigated in two different spatial memory tasks, the Morris water maze and Y-maze. Performances in both tests were significantly correlated and, accompanied by appropriate controls, likely evaluated genuine memory function. Importantly, individual hippocampal PREG S and distance to reach the platform in the water maze were linked by a significant correlation, i.e., those rats with lower memory deficit had the highest PREG S levels, whereas no relationship was found with the PREG S content in other brain areas (amygdala, prefrontal cortex, parietal cortex, striatum). Moreover, the memory deficit of cognitively impaired aged rats was transiently corrected after either intraperitoneal or bilateral intrahippocampal injection of PREG S. PREG S is both a γ-aminobutyric acid antagonist and a positive allosteric modulator at the N-methyl-d-aspartate receptor, and may reinforce neurotransmitter system(s) that decline with age. Indeed, intracerebroventricular injection of PREG S was shown to stimulate acetylcholine release in the adult rat hippocampus. In conclusion, it is proposed that the hippocampal content of PREG S plays a physiological role in preserving and/or enhancing cognitive abilities in old animals, possibly via an interaction with central cholinergic systems. Thus, neurosteroids should be further studied in the context of prevention and/or treatment of age-related memory disorders.

Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Phosphorylation of the α-subunit of Na+,K+-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K+-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive 86Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K+-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

The Three-dimensional Study of Chromosomes and Upstream Binding Factor-immunolabeled Nucleolar Organizer Regions Demonstrates Their Nonrandom Spatial Arrangement during Mitosis

The volumic rearrangement of both chromosomes and immunolabeled upstream binding factor in entire well-preserved mitotic cells was studied by confocal microscopy. By using high-quality three-dimensional visualization and tomography, it was possible to investigate interactively the volumic organization of chromosome sets and to focus on their internal characteristics. More particularly, this study demonstrates the nonrandom positioning of metaphase chromosomes bearing nucleolar organizer regions as revealed by their positive upstream binding factor immunolabeling. During the complex morphogenesis of the progeny nuclei from anaphase to late telophase, the equal partitioning of the nucleolar organizer regions is demonstrated by quantification, and their typical nonrandom central positioning within the chromosome sets is revealed.

Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60–80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis suggests a very early mitochondrial endosymbiosis in eukaryotes

Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryote Entamoeba histolytica. We have now isolated from Trichomonas vaginalis a gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of α-purple bacteria. In addition, the T. vaginalis protein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria.

Human trophoblast and choriocarcinoma expression of the growth factor pleiotrophin attributable to germ-line insertion of an endogenous retrovirus

Retroviral elements are found in abundance throughout the human genome but only rarely have alterations of endogenous genes by retroviral insertions been described. Herein we report that a human endogenous retrovirus (HERV) type C is inserted in the human growth factor gene pleiotrophin (PTN) between the 5′ untranslated and the coding region. This insert in the human genome expands the region relative to the murine gene. Studies with promoter-reporter constructs show that the HERV insert in the human PTN gene generates an additional promoter with trophoblast-specific activity. Due to this promoter function, fusion transcripts between HERV and the open reading frame of PTN (HERV-PTN) were detected in all normal human trophoblast cell cultures as early as 9 weeks after gestation (n = 7) and in all term placenta tissues (n = 5) but not in other normal adult tissues. Furthermore, only trophoblast-derived choriocarcinoma cell lines expressed HERV-PTN mRNA whereas tumor cell lines derived from the embryoblast (teratocarcinoma) or from other lineages failed to do so. We investigated the significance of HERV-PTN mRNA in a choriocarcinoma model by targeting this transcript with ribozymes and found that the depletion of HERV-PTN mRNA prevents human choriocarcinoma growth, invasion, and angiogenesis in mice. This suggests that the tissue-specific expression of PTN due to the HERV insertion in the human genome supports the highly aggressive growth of human choriocarcinoma and possibly of the human trophoblast.

Suppression of glucocorticoid secretion and antipsychotic drugs have similar effects on the mesolimbic dopaminergic transmission

Specific antagonists of central dopaminergic receptors constitute the major class of antipsychotic drugs (APD). Two principal effects of APD are used as criteria for the pre-clinical screening of their antipsychotic action: (i) inhibition of basal and depolarization-induced activity of mesolimbic dopaminergic neurons; (ii) antagonism of the locomotor effects of dopaminergic agonists. Given that glucocorticoid hormones in animals increase dopamine release and dopamine-mediated behaviors and that high levels of glucocorticoids can induce psychotic symptoms in humans, these experiments examined whether inhibition of endogenous glucocorticoids might have APD-like effects on mesolimbic dopaminergic transmission in rats. It is shown that suppression of glucocorticoid secretion by adrenalectomy profoundly decreased (by greater than 50%): (i) basal dopaminergic release and the release of dopamine induced by a depolarizing stimulus such as morphine (2 mg/kg, s.c.), as measured in the nucleus accumbens of freely moving animals by microdialysis; (ii) the locomotor activity induced by the direct dopaminergic agonist apomorphine. The effects of adrenalectomy were glucocorticoid specific given that they were reversed by the administration of glucocorticoids at doses within the physiological range. Despite its profound diminution of dopaminergic neurotransmission, adrenalectomy neither modified the number of mesencephalic dopaminergic neurons nor induced gliosis in the mesencephalon or in the nucleus accumbens, as shown by tyrosine hydroxylase and glial fibrillary acidic protein immunostaining. In conclusion, these findings suggest that blockade of central effects of glucocorticoids might open new therapeutic strategies of behavioral disturbances.

Mutators and sex in bacteria: Conflict between adaptive strategies

Bacterial mutation rates can increase and produce genetic novelty, as shown by in vitro and in silico experiments. Despite the cost due to a heavy deleterious mutation load, mutator alleles, which increase the mutation rate, can spread in asexual populations during adaptation because they remain associated with the rare favorable mutations they generate. This indirect selection for a genetic system generating diversity (second-order selection) is expected to be highly sensitive to changes in the dynamics of adaptation. Here we show by a simulation approach that even rare genetic exchanges, such as bacterial conjugation or transformation, can dramatically reduce the selection of mutators. Moreover, drift or competition between the processes of mutation and recombination in the course of adaptation reveal how second-order selection is unable to optimize the rate of generation of novelty.

Development of a sensitive multi-well colorimetric assay for active NFκB

The transcription factor nuclear factor κB (NFκB) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NFκB activation. Indeed, NFκB DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NFκB. The presence of the DNA-bound transcription factor is then detected by anti-NFκB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFκB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.

Retroviral RNA identified in the cerebrospinal fluids and brains of individuals with schizophrenia

Schizophrenia is a serious brain disease of uncertain etiology. A role for retroviruses in the etiopathogenesis of some cases of schizophrenia has been postulated on the basis of clinical and epidemiological observations. We found sequences homologous to retroviral pol genes in the cell-free cerebrospinal fluids (CSFs) of 10 of 35 (29%) individuals with recent-onset schizophrenia or schizoaffective disorder. Retroviral sequences also were identified in the CSFs of 1 of 20 individuals with chronic schizophrenia. However, retroviral sequences were not identified in any of the CSFs obtained from 22 individuals with noninflammatory neurological diseases or from 30 individuals without evidence of neurological or psychiatric diseases (χ2 = 19.25, P < 0.001). The nucleotide sequences identified in the CSFs of the individuals with schizophrenia or schizoaffective disorder were related to those of the human endogenous retroviral (HERV)-W family of endogenous retroviruses and to other retroviruses in the murine leukemia virus genus. Transcription of RNA homologous to members of the HERV-W family of retroviruses also was found to be up-regulated differentially in the frontal cortex regions of brains obtained postmortem from individuals with schizophrenia, as compared with corresponding tissue from individuals without psychiatric diseases. The transcriptional activation of certain retroviral elements within the central nervous system may be associated with the development of schizophrenia in at least some individuals. The further characterization of retroviral elements within the central nervous system of individuals with schizophrenia might lead to improved methods for the diagnosis and management of this disorder.

The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences.

Human endogenous retroviruses (HERVs) are very likely footprints of ancient germ-cell infections. HERV sequences encompass about 1% of the human genome. HERVs have retained the potential of other retroelements to retrotranspose and thus to change genomic structure and function. The genomes of almost all HERV families are highly defective. Recent progress has allowed the identification of the biologically most active family, HTDV/HERV-K, which codes for viral proteins and particles and is highly expressed in germ-cell tumors. The demonstrable and potential roles of HTDV/HERV-K as well as of other human elements in disease and in maintaining genome plasticity are illustrated.

Inhibitory influence of frontal cortex on locus coeruleus neurons.

The functional influence of the frontal cortex (FC) on the noradrenergic nucleus locus coeruleus (LC) was studied in the rat under ketamine anesthesia. The FC was inactivated by local infusion of lidocaine or ice-cold Ringer's solution while recording neuronal activity simultaneously in FC and LC. Lidocaine produced a transient increase in activity in FC, accompanied by a decrease in LC unit and multiunit activity. This was followed by a total inactivation of FC and a sustained increase in firing rate of LC neurons. Subsequent experiments revealed antidromic responses in the FC when stimulation was applied to the LC region. The antidromic responses in FC were found in a population of neurons (about 8%) restricted to the dorsomedial area, FR2. The results indicate that there is a strong inhibitory influence of FC on the tonic activity of LC neurons. The antidromic responses in FC to stimulation of the LC region suggest that this influence is locally mediated, perhaps through interneurons within the nucleus or neighboring the LC.