A common polymorphism in serotonin receptor 1B mRNA moderates regulation by miR-96 and associates with aggressive human behaviors.

Non-coding regulatory elements can transduce the human genome's response to environmental stimuli. Thus, there is a possibility that variation in non-coding regulatory elements may underlie some of the diversity in human behavior. However, this idea has remained largely untested due to the difficulty in accurately identifying regulatory elements in the 98% of the human genome that does not encode protein. The recent recognition that small trans-acting RNAs anneal to mRNA and regulate gene expression provides a means to identify and test such variants. Here, we show that microRNA-directed silencing of mRNA can be attenuated by a common human polymorphism. We have identified an element (A-element) within serotonin receptor 1B (HTR1B) mRNA that confers repression by miR-96. The repressive activity of this element is attenuated by a common human variant (G-element) that disrupts a nucleotide critical for its interaction with miR-96. Because deletion of the HTR1B gene leads to an aggressive phenotype in mice, we hypothesized an association between the A/G polymorphism and aggressive phenotypes in a sample of 359 college students. As predicted, individuals homozygous for the ancestral A-element reported more conduct-disorder behaviors than individuals with the G-element. Our studies suggest that such functional variants may be common and may help to refine the search for genes involved in complex behavioral disorders.

The identification of an endonuclease that cleaves within an HuR binding site in mRNA.

Messenger RNAs (mRNAs) that contain U-rich elements are targeted for rapid decay. Selective inhibition of this decay results in a rapid increase in steady state level. Thus, this is an important regulatory step in gene expression. Previously, we have found that these mRNAs are selectively stabilized by a specific mRNA binding protein called HuR. The mechanism of action of HuR is not well understood. It has been postulated that HuR stabilizes mRNA by the displacement or inhibition of factors that specifically cleave or deadenyl-ate these mRNAs. In this paper, we report the identification and characterization of a novel endo-nuclease that cleaves within an HuR binding site in p27kip1 mRNA. The specificity of this endonuclease and its inhibition by HuR argue for it playing a role in the postranscriptional regulation of gene expression.

Expression of transforming growth factor alpha in human gliomas.

Expression of transforming growth factor alpha (TGF alpha) is frequently associated with the development of human and animal tumors. Using a sensitive immunohistochemical assay, which can be applied on formalin-fixed, paraffin-embedded tissue, we have examined the expression of TGF alpha in 71 human gliomas (63 untreated and 8 recurrent tumors). Tumors were graded by a 3-grade-system: grade I = low grade gliomas, grade II = anaplastic gliomas and grade III = glioblastomas. A strong positive correlation between tumor grade and extent of TGF alpha expression was found (P less than 0.0001). Polymerase chain reaction (PCR) was used to amplify the fourth exon of the TGF alpha gene of 8 glioma DNA specimens and increasing amounts of normal human DNA, which served as a standard. No amplification of the TGF alpha gene copy number in tumors could be detected.

Detection of the anti-Hu antibody in specific regions of the nervous system and tumor from patients with paraneoplastic encephalomyelitis/sensory neuronopathy.

We studied the nervous systems and tumors of five patients with anti-Hu-positive paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/PSN) to determine if the autoantibody found in the serum and CSF was also present in those tissues. Immunohistochemical studies of the nervous system revealed the presence of IgG bound predominantly to the nuclei of most of the neurons and the cytoplasm of some glial cells. IgG was also present to a lesser degree in the neuropil. In brains of patients who died of cancer without the paraneoplastic syndrome, IgG was present in the immediate perivascular areas and to a very limited degree in the neuropil. There was no IgG in neurons, and in only some of the controls a few glial cells showed IgG immunoreactivity in the cytoplasm. The amount of anti-Hu IgG relative to total IgG in various brain regions and tumor was determined by quantitative Western blot analysis. The proportion of anti-Hu IgG was greater in some areas of the brain and tumor than in serum and CSF. Control brains did not contain anti-Hu IgG. There was a limited correlation among (1) the principal clinical symptoms, (2) regions of major tissue injury, and (3) the quantitative anti-Hu IgG distribution. We conclude that although the role of the antibody in the pathogenesis of the disease is still uncertain, its specific localization in the nervous system and tumor suggests an immunologic etiology of this paraneoplastic syndrome.

HuR, a novel target of anti-Hu antibodies, is expressed in non-neural tissues.

Paraneoplastic encephalomyelitis (PEM) is characterized by a diverse set of clinical signs that are limited to the nervous system. The serologic hallmark of PEM is the presence of circulating autoantibodies, collectively referred to as 'anti-Hu,' which immunoreact specifically with members of the Elav protein family. Until recently, the ELAV antigens were only detected in neurons, thus strongly supporting a role for anti-Hu antibodies in the selective neural tissue injury in PEM. The identification of HuR, however, a new member with a broad, non-neural pattern of RNA expression, raises several fundamental questions regarding PEM. First, why are non-neural tissues spared in PEM? Second, why is PEM predominantly associated with neuroendocrine tumors? To begin addressing these questions, we sought to determine whether the antibody response to HuR differs from the neural-specific counterparts in patients with PEM, and to characterize the protein expression pattern of this novel antigen in peripheral tissues and tumors. Using sera from 11 patients with Hu-positive PEM, we found that the majority of samples (73%) were weakly or non-reactive for recombinant HuR on Western blot, in contrast to consistently strong immunoreactivity with the neural-specific members HuD and Hel-N1. We also demonstrate that HuR is expressed at the protein level in both non-neural tissues and non-neuroendocrine tumors. These findings suggest that immunoreactive differences among Elav family members may contribute to the neural-restrictive pattern of tissue injury in patients with PEM.

Intracellular NAD+ content and ADP-ribose polymerase activity of serum-stimulated baby hamster kidney fibroblasts.

We have examined a number of events relating to ADP-ribose metabolism during serum-stimulated growth of BHK-21/C13 fibroblasts. Both the intracellular NAD+ content and the ADP-ribose polymerase activity were found to increase after serum stimulation of cells that were previously arrested by growth in low-serum medium. NAD+ content increased about two-fold, reaching a maximum of 4.2 nmol/microgram of DNA 8 hr after serum steK-21/C13 fibroblasts. Both the intracellular NAD+ content and the ADP-ribose polymerase activity were found to increase after serum stimulation of cells that were previously arrested by growth in low-serum medium. NAD+ content inreased about two-fold, reaching a maximum of 4.2 nmol/microgram of DNA 8 hr after serum step-up. The polymerase exhibited a sharp rise in activity, reaching a peak at about 5 hr after step-up; the activity declined below initial values by 10 hr, and then increased again to reach a plateau at 20 hr. We also report evidence which suggests a possible effect of ADP-ribosylation on the activity of DNA-dependent RNA polymerase I. The activity of this enzyme is diminished in isolated nuclei, and in a subsequent (NH4)2SO4 extract, when the nuclei are incubated with NAD+, the substrate for poly(ADP-ribose) polymerase. This inhibitory effect on the RNA polymerase is abolished when nuclei are incubated also with nicotinamide, a powerful inhibitor of the poly(ADP-ribose) polymerase.

Adenosine diphosphate ribose transferase from baby-hamster kidney cells (BHK-21/C13). Characterization of the reaction and product.

Some properties of ADP-ribose transferase, and its reaction product, from BHK-21/C13 cells are described. Enzyme activity was found almost exclusively in nuclei (90%), with the remaining 10% located in the cytosolic fraction. The nuclear enzyme is chromatin-bound and requires bivalent cations, preferably Mg2+, a pH of 8.0 and a temperature of 25 degrees C for optimal activity. Chromatin preparations incorporated radioactivity from [14C]NAD+ into acid-insoluble material for about 60 min. Kinetics for substrate NAD+ utilization were not of Michaelis--Menten type; biphasic kinetics were shown from a double-reciprocal plot (1/reaction velocity against 1/[NAD+]) and from a 'Hofstee' plot (reaction velocity/[NAD+] against reaction velocity). The transferase is unstable in the absence of Mg2+ ions. It is inhibited by thymidine, nicotinamide and nicotinamide analogues, but not by ATP, which stimulates it at concentrations of 5 mM and above. The enzyme requires thiol groups for activity; it is readily inhibited by N-ethylmaleimide at 0.5 mM. The product of the reaction is stable under acid conditions at temperatures up to 25 degrees C, but it is hydrolysed by HClO4 at 70 degrees C. It is resistant to NaOH, but is cleaved from its attachment to protein with alkali into trichloroacetic acid-insoluble and -soluble components. On the basis of Cs2SO4- density-gradient analysis under denaturing conditions (gradients included urea and guanidinium hydrochloride), and analysis of the reaction product directly on hydroxyapatite, we conclude that most of the radioactive ADP-ribose residues are firmly bound to protein, presumably in covalent linkage. Hydroxyapatite-chromatographic analysis of ADP-ribose residues released from protein by alkaline digestion showed a spectrum of molecular sizes including mono-, oligo- and poly-(ADP-ribose), when chromatin was incubated initially with [14C]NAD+ for 10 min and then for a further 30 min after addition of excess non-radioactive NAD+, only about 10% of the radioactive mono-(ADP-ribose) could be 'chased' into longer-chain molecules. Hydroxyapatite analysis was also used to show that, whereas all ADP-ribose residues were released from protein with NaOH, only 50% of them were susceptible to hydroxylamine. These hydroxylamine-sensitive residues included all size classes, although mono-(ADP-ribose) predominated. Finally, there was an approximately equal distribution of ADP-ribose incorporated into HCl-soluble proteins (including the histones) and HCl-insoluble proteins (including the non-histone proteins) when chromatin was incubated with NAD+ up to 0.5 mM, but at higher NAD+ concentrations more ADP-ribose was incorporated into the HCl-soluble fraction (82% at 4.0 mM-NAD+).

Antiserum from a patient with cerebellar degeneration identifies a novel protein in Purkinje cells, cortical neurons, and neuroectodermal tumors.

The serum and cerebrospinal fluid of a patient (NB) with subacute cerebellar degeneration were found to contain a novel antineuronal autoantibody (anti-Nb). Using this antibody, we have identified and characterized antigens present in a subset of neurons in the CNS and in some neuroectodermal tumor lines. Anti-Nb antibody bound to antigens of Mr 150, 120, and 65 kDa in Western blots using extracts of human cerebellar Purkinje cells or human cerebral cortical neurons. Immunohistochemistry demonstrated relatively specific binding of anti-Nb IgG to Purkinje cells in sections of human cerebellum and to some neocortical neurons, especially those in layer VI. Because of the association of cerebellar degeneration with occult malignancies, we screened a number of tumor cell lines for immunoreactivity to anti-Nb antibody; only tumor lines of neuroectodermal origin (melanoma, small-cell lung cancer, and neuroblastoma) expressed the Nb antigen. Anti-Nb antibody thus identifies neuronal and tumor cell antigens that appear to be unique in size and distribution of expression.

RNA splicing products formed with isolated fractions from HeLa cells are associated with fast-sedimenting complexes.

Three fractions (designated Ia, Ib, and II) have been isolated from HeLa cell nuclear extracts that are required for splicing of adenovirus and human beta-globin RNA transcripts in vitro. The incubation of two of the fractions (Ib and II) in the presence of ATP resulted in cleavage of precursor mRNA at the 5' splice site and formation of the intron-exon lariat. Addition of fraction Ia to the combination of Ib and II resulted in the formation of spliced RNA and the intron lariat. When fraction II was incubated with precursor RNA in the presence of ATP and the resulting products were sedimented through sucrose gradients, a 30S complex was detected that contained precursor RNA. The combination of fractions Ib and II resulted in the production of a 55S complex that contained the 5' exon as a prominent RNA species. The combination of fractions I (containing Ia and Ib) and II resulted in the formation of the 55S complex and material sedimenting between 40 S and 20 S, in which the predominant RNA species was spliced RNA.

Isolation and characterization of two fractions from HeLa cells required for mRNA splicing in vitro.

A nuclear extract from HeLa cells has been separated by DEAE-cellulose chromatography into two fractions, both of which are required for mRNA splicing in vitro. Both fractions are heat labile and sensitive to N-ethylmaleimide. The activity of one of the fractions was abolished by preincubation with micrococcal nuclease, while the other fraction was unaffected by this treatment. This abolition indicates an essential nucleic acid component. Fractions I and II are required for the in vitro splicing of human beta-globin and adenovirus transcripts.

Detection of the anti-Hu antibody in the serum of patients with small cell lung cancer--a quantitative western blot analysis.

We looked for the presence of the anti-Hu antibody in the sera from 50 normal subjects; 44 patients with small cell lung cancer, not associated with paraneoplastic disease; and 25 patients with small cell lung cancer associated with paraneoplastic sensory neuropathy, encephalomyelitis, or both. Using the avidin-biotin immunoperoxidase method and a highly sensitive quantitative Western blot analysis, the anti-Hu antibody was not detected in the 50 normal human sera. Seven of the 44 patients with small cell lung cancer but no paraneoplastic syndrome had detectable levels (average titer, 76 U/ml) of anti-Hu antibody on Western blot. These levels are significantly lower than the average titer of the 25 patients who had small cell lung cancer and paraneoplastic sensory neuropathy or encephalomyelitis (average titer, 4,592 U/ml). In the group with nonparaneoplastic small cell lung cancer (low anti-Hu titer) there was a predominance of women (5 women: 2 men), and all patients had "limited" disease when diagnosed. In the antibody-negative group the sex ratio was 16 women to 21 men and 51% of the patients had "extensive" disease. None of the 7 patients with a low-titer anti-Hu antibody developed a paraneoplastic syndrome by the time of writing. The anti-Hu antibody appears, when present, to be a good marker for small cell lung cancer and, when present at high titer, for small cell lung cancer associated with a paraneoplastic syndrome.

The expression of the Hu (paraneoplastic encephalomyelitis/sensory neuronopathy) antigen in human normal and tumor tissues.

Using immunohistochemistry or Western blot analysis, the authors have studied the expression of the Hu antigen (a neuronal protein identified by the serum of patients with small cell lung cancer and paraneoplastic encephalomyelitis/sensory neuronopathy) in normal human tissues and 115 tumors of different histologic types. In normal tissue, the Hu antigen is highly restricted to the nervous system. In lung tumors, the Hu antigen is restricted in its expression to all small cell carcinomas. A few other neuroendocrine-related tumors, especially neuroblastomas (50%), also express the antigen.

Poly(adenosine diphosphate ribose) synthetase activity in nuclei of dividing and of non-dividing but differentiating intestinal epithelial cells.

Poly(ADP-ribose) synthetase activity is found in nuclei of regenerating epithelial cells in the lower half of the crypts of guinea-pig small intestine. Nuclei from non-dividing but differentiating and maturing cells in the upper crypts and on the villi contain no more than about 10% of the synthetase activity of lower-crypt cell nuclei. The product in the active nuclei is shown to be 80% poly(ADP-ribosylated) protein and 20% mono(ADP-ribosylated) protein; 60% of thetotal labelled product was attached to acid-soluble proteins (including histones), and 40% to acid-insoluble (non-histone) proteins. The average number of ADP-ribosyl units in the oligomeric chains of the poly(ADP-ribosylated) proteins was 15 but the range of sizes of (ADP-ribose) oligomers attached to nuclear proteins was smaller in villus than in crypt cell nuclei.

Mapping of the gene coding for a paraneoplastic encephalomyelitis antigen (HuD) to human chromosome site 1p34.

A cDNA clone coding for a human brain protein (HuD), which is highly homologous to the Drosophila neuronal determination protein Elav and elicits antibody formation in a high proportion of patients with paraneoplastic encephalomyelitis, was used to isolate a lambda phage recombinant clone, including a large fragment of the relevant human genomic region. The fragment proved to be an efficient probe for the precise subregional mapping of the gene by molecular in situ hybridization onto extended human prometaphase chromosomes. Analysis of banded metaphases with clear hybridization signals pointed unequivocally to the localization of the HuD gene to human chromosome band 1p34.

Cloning of a leucine-zipper protein recognized by the sera of patients with antibody-associated paraneoplastic cerebellar degeneration.

Antibody-associated paraneoplastic cerebellar degeneration (the Yo syndrome) is an uncommon disorder in which an immune response is specifically directed against tumor tissue and the cerebellum. Screening of a lambda expression library has resulted in the isolation of cDNA clones that encode the major antigen recognized by serum from these patients. The fusion protein produced by the cDNA clones provides the basis of a simple diagnostic assay for this neurological syndrome. The occurrence of leucine-zipper and zinc-finger motifs in the predicted open reading frame suggests that this protein plays a role in the regulation of gene expression.

Hu neuronal proteins are expressed in proliferating neurogenic cells.

We have utilized immunochemical techniques to investigate the developmental expression of the Hu proteins, a neuron-specific family of RNA binding proteins in vertebrates. Previous work suggests that these proteins may play an important role in neuronal development and maintenance. For the present study, we developed a monoclonal antibody (MAb 16A11) that binds specifically to an epitope present in gene products of all known Hu genes, including HuD, HuC, and Hel-N1. Using brief pulses (1-2 h) of the DNA precursor analog bromodeoxyuridine (BrdU) in conjunction with MAb 16A11, we observed Hu+/BrdU+ cells in nascent sensory and sympathetic ganglia in vivo, and in populations of cultured neural crest cells. In addition, a few Hu+ cells were ambiguously BrdU+ in the neural tube. We conclude that Hu+ cells first appear in avian neurogenic populations immediately before neuronal birthdays in the peripheral nervous system, and at the time of withdrawal from the mitotic cycle in the central nervous system. Consistent with these conclusions, we have also observed neural crest-derived cells that are both Hu+ and in metaphase of the cell cycle. We suggest that Hu proteins function early in neurogenic differentiation.

Selective expression of Purkinje-cell antigens in tumor tissue from patients with paraneoplastic cerebellar degeneration.

Paraneoplastic cerebellar degeneration is a rare syndrome that occurs in patients with gynecologic cancer and is characterized by widespread loss of Purkinje cells. To determine whether Purkinje-cell antigens are selectively expressed in the tumors of patients with the syndrome, we examined tumor tissue from 10 patients whose serum contained anti-Purkinje-cell (anti-Yo) antibodies. The origins of the cancers were the breast (five patients), ovary (three), endometrium (one), and fallopian tube (one). We used as controls tumor tissue from 11 patients with ovarian cancer and 10 patients with breast cancer who were neurologically normal. Using immunohistochemical and Western blot analysis, we found that Purkinje-cell antigens were expressed in all the tumors from the 10 patients with paraneoplastic cerebellar degeneration but in none of the tumors from the 21 neurologically normal patients. When IgG from patients with paraneoplastic cerebellar degeneration was affinity-purified to cerebellar Purkinje-cell antigen, immunohistochemical analysis showed that it reacted specifically with the tumor tissue from those patients. We conclude that in patients with paraneoplastic cerebellar degeneration, the anti-Yo antibody results from an immune response to neural antigens expressed by the gynecologic tumors in the patients.

HuD, a paraneoplastic encephalomyelitis antigen, contains RNA-binding domains and is homologous to Elav and Sex-lethal.

A neuronal antigen (HuD) recognized by the sera of patients with antibody-associated paraneoplastic encephalomyelitis has been isolated by screening a lambda cerebellar expression library. The recombinant antigen provides an unambiguous assay for this rare condition associated with small cell lung cancer. The recombinant antigen has been used to identify specific infiltrating lymphocytes in tumors and affected brain tissues of patients with antibody-associated paraneoplastic encephalomyelitis and sensory neuronopathy. HuD mRNA is uniquely expressed in brain tissue. The HuD protein shows a remarkable homology to the Drosophila proteins Elav and Sex-lethal and is likely to play a role in neuron-specific RNA processing.