Mouse nerve growth factor gene: structure and expression.

The organization and biologically significant sequences of the entire mouse nerve growth factor (NGF) gene have been determined. The gene spans 45 kilobases and contains several small 5' exons. Transcription of the gene results in four different mRNA species, which can be accounted for by alternative splicing and independent initiation from two promoters. These transcripts encode proteins which have divergent N termini and the NGF moiety at their C termini. The levels of the various NGF transcripts have been determined in different tissues and throughout postnatal development. We have also examined the expression of these transcripts in the brain in response to specific early sensory deprivation. The results suggest that the expression of NGF mRNA during postnatal development is regulated independently of the formation of complex neural networks.

Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector.

To study posttranslational mechanisms for the control of nerve growth factor (NGF), we used a recombinant vaccinia virus vector to independently express the two major NGF transcripts in a variety of mammalian cell lines. The two major transcripts contain NGF (12.5 kilodaltons [kDa]) at the C-terminus and differ by alternative splicing of an N-terminal exon, so that the large precursor (34 kDa) had 67 amino acids upstream of an internal signal peptide and the smaller precursor (27 kDa) had this signal peptide at its N-terminus. In L929 cells, expression of either NGF transcript with the vaccinia virus vector gave rise to an apparently identical intracellular 35-kDa glycosylated precursor formed by cleavage of the primary gene product after the signal peptide. These cells also secreted biologically active NGF. To determine whether NGF processing is restricted by cell type, we infected a variety of mammalian cell lines with both recombinant viruses; all accumulated the same 35-kDa precursor and secreted NGF. Thus, many types of cells have the machinery to process and secrete NGF. However, NGF accumulated intracellularly (presumably in secretory granules) in cells with a regulated pathway of secretion (e.g., AtT-20 and HIT cells). In these cells, a membrane-permeable cyclic AMP analog, 8-bromo-cyclic AMP, stimulated NGF secretion. This suggests a mechanism for the regulation of NGF levels in which specific secretagogues, e.g., neurotransmitters, control NGF secretion.

Synergism between Tat and VP16 in trans-activation of HIV-1 LTR.

When tethered to heterologous DNA both Tat and VP16 can activate transcription from the HIV-1 LTR. To determine if they act by similar mechanisms, we constructed several hybrid effectors between Tat or VP16 and DNA-binding domains of GAL4 or LexA proteins. We tested these effectors on substituted reporter targets, which contained one to six GAL4 or LexA DNA-binding sites placed upstream of the HIV-1 promoter. Whereas Tat acted very inefficiently via DNA even with five DNA-binding sites, effects of VP16 were observed with a single DNA-binding site and increased with increasing number of sites. More importantly, effects of VP16 via DNA were synergistic with those of Tat via TAR RNA when both proteins were expressed simultaneously. We next created a tripartite fusion protein, which contained the GAL4 DNA-binding domain and activation domains of both Tat and VP16, which could be targeted to the HIV-1 LTR either via DNA or RNA. By introducing individual deleterious mutations into either Tat or VP16, we confirmed that effects of VP16 predominated via DNA whereas Tat but not VP16 acted via TAR RNA. Thus, Tat and VP16 act at different steps of the transcription process and increase expression from the HIV-1 LTR by different mechanisms.

Neuropsychological functioning in drug abusers.

The present study examined differences in neuropsychological performance among chronic cocaine, alcohol, and polysubstance abusers. A comprehensive neuropsychological battery was completed by 355 incarcerated adult male felons who were classified by DSM-IV criteria into four subgroups: (1) alcohol dependence or abuse (ETOH) (n = 101), (2) cocaine dependence or abuse (COC) (n = 60), (3) polysubstance dependence or abuse (POLY (n = 56), and (4) a group of age and education matched adult male felons with no history of drug abuse (n = 138). Results showed no significant differences in neuropsychological performance between COC and control subjects. However, both the POLY and ETOH groups were found to perform significantly worse on nearly all measures compared to the COC and control groups. Further, analysis of neuropsychological domains showed the POLY group to perform significantly worse compared to the other groups in the areas of short-term memory, long-term memory and visual motor ability. Correlations between neuropsychological performance and length of abstinence from drug use showed the ETOH group to have made the greatest amount of improvement on individual measures and domains. The COC group showed the least amount of improvement, but their performance was not significantly different from controls. Results provide further support for the differential effects of drug use on neuropsychological functioning.

Assessment of memory in multiple sclerosis patients using the Memory Assessment Scale.

Previous assessment of memory function in multiple sclerosis patients has yielded mixed findings regarding the type and severity of memory deficits, which may be due to (1) differential selection of scales for memory assessment; (2) limited, inconsistent or weak reliability and validity data for the memory scales employed; (3) poor standardization techniques; (4) lack of theoretical foundation for the measure; and (5) limited control of confounding variables, e.g., education, age and the use of nonverbal memory tests. The purpose of the present study was to assess memory function in multiple sclerosis subjects using the verbal subtests of the Memory Assessment Scale, a relatively new measure designed to overcome many of the aforementioned problems. Participants included 57 patients diagnosed as relapsing-remitting, 47 diagnosed as chronic progressive (two generally recognized types of multiple sclerosis), and 132 control participants. A multivariate analysis controlling for age and verbal IQ was significant (Wilks = 5.64, p < .001). One way follow-up tests showed both groups with multiple sclerosis had significantly diminished performance across all memory variables when compared with controls, with the exception of List Clustering Acquisition. This indicated that the patients used clustering (mentally grouping similar words together) as often as controls did. These findings provide support for the presence of significant and consistent verbal memory impairment in multiple sclerosis patients and the particular importance of using psychometrically sound measures in the assessment of this population.

Interferon beta 1-b in verbal memory functioning of patients with relapsing-remitting multiple sclerosis.

The effects of interferon Beta 1-b (Betaseron) on verbal memory functioning was examined in 167 patients with relapsing-remitting multiple sclerosis and 112 matched normal controls. Subjects were administered 10 verbal memory tests from the Memory Assessment Scales and the Verbal subtests from the Wechsler Adult Intelligence Scale. Analysis showed subjects treated with Betaseron (n = 73) did not perform significantly better on measures of verbal memory or verbal ability than subjects not receiving the drug (n = 94), although the mean performance of treated subjects was higher across all verbal memory tests. Both groups of patients performed significantly worse on verbal memory subtests measuring list acquisition, delayed list recall, delayed cued recall, and the immediate and delayed recall of names and faces than control subjects. Although patients had lower performance scores across all memory tests than the control subjects, their scores were not within the impaired range. These results do not permit a clear conclusion about the effects of Betaseron on verbal memory for any effect is probably obscured by the relatively preserved cognitive functioning of this outpatient sample.

Hostility in depression.

Subjects (39 men and 30 women) from two university counseling centers and one university medical center were administered the Hamilton Rating Scale for Depression, the Buss-Durkee Hostility Inventory, the State-Trait Anger Scale, and the Hostility and Direction of Hostility Questionnaire. Results showed significant positive correlations between self-reported severity of depression and all subtypes of hostility including behavior, attitude, affect, intropunitiveness, and extrapunitiveness. Hierarchical regression analysis using demographic and hostility variables as predictors of depression scores showed increasing age, lower education, and female gender to account for 50% of the explained variance. The Intropunitive subscale from the Hostility and Direction of Hostility Questionnaire accounted for an additional 19% of the explained variance and was the single most powerful predictor of depression. Correlational analysis showed women tending to have higher scores on most hostility measures. Implications of these results with respect to theory and clinical practice are discussed.

Assessment of violence potential using measures of anger, hostility, and social desirability.

The assessment of violence potential was studied using the following scales: The Novaco Anger Inventory, the Buss-Durkee Hostility Inventory, the MMPI Hostility Control Scale (Hc), the MMPI Overt Hostility Scale (Ho), and the Marlowe-Crowne Social Desirability Scale. The five measures were evaluated for their ability to discriminate between violent and nonviolent criminals, and between criminal and normal population samples. Normative data was collected for 204 adult male felons. A correlation matrix presents interrelationships among the five scales. A comparison of mean scale scores between violent and nonviolent groups resulted in a significant discrimination in the expected direction for all but the Hc scale. A discriminant analysis procedure applied to individual items from the Novaco Anger Inventory resulted in the selection of 25 variables which identified violence prone individuals with 90% accuracy. Clinical implications are noted and recommendations for future research are made.

Personal and familial substance misuse patterns among eating disordered and depressed subjects.

A personal and familial history of substance misuse problems was obtained from 25 anorectic, 43 bulimic, and 33 obese women admitted to an inpatient treatment unit for eating disorders. Similar data was also collected on 40 noneating disordered, depressed women admitted to a general psychiatric ward at the same facility. Results showed significant differences between groups, with bulimic subjects reporting greater frequency of both personal and familial substance misuse problems across all groups. In addition, bulimic subjects with personal substance misuse problems reported a significantly greater frequency of familial substance misuse. However, bulimic subjects with and without substance misuse problems reported similar frequencies of familial substance misuse problems. Results point out the complex interaction of dispositional and situational variables in the formation of substance misuse problems in eating disorders, and the need to examine more completely the substance misuse and developmental histories of eating disordered patients.

Evaluation of a rapid method for the quantitative estimation of coliforms in meat by impedimetric procedures.

A 24-h instrumental procedure is described for the quantitative estimation of coliforms in ground meat. The method is simple and rapid, and it requires but a single sample dilution and four replicates. The data are recorded automatically and can be used to estimate coliforms in the range of 100 to 10,000 organisms per g. The procedure is an impedance detection time (IDT) method using a new medium, tested against 131 stock cultures, that markedly enhances the impedance response of gram-negative organisms, and it is selective for coliforms. Seventy samples of ground beef were analyzed for coliforms by the IDT method and the conventional three-dilution, two-step most-probable-number test tube procedure. Seventy-nine percent of the impedimetric estimates fell within the 95% confidence limits of the most-probable-number values. This corresponds to the criteria used to evaluate other coliform tests, with the added advantage of a single dilution and more rapid results.

Trans-activation by HIV-1 Tat via a heterologous RNA binding protein.

The HIV-1 trans-activator Tat increases levels of viral gene expression and replication. The target for Tat is an RNA stem-loop called TAR, located at the 5' end of all viral transcripts. To study the mechanism of action and map functional domains of Tat, we fused Tat to the coat protein of bacteriophage MS2, an RNA binding protein. TAR in the HIV-1 LTR was replaced by the operator, the RNA target of the coat protein. The hybrid Tat-coat protein trans-activated HIV-1 LTRs containing either TAR or operator sequences. Mutations in the operator that weaken binding of the coat protein in vitro led to decreased levels of trans-activation in vivo. Deletions in Tat within the hybrid Tat-coat protein identified activation and RNA binding domains of Tat. These experiments suggest that trans-activation by Tat can occur independently of TAR RNA and DNA binding proteins and that Tat exerts its effects on HIV-1 transcription by directly interacting with the TAR RNA stem-loop.

Measurement of hostility, anger, and depression in depressed and nondepressed subjects.

This study examined the relationship between hostility and depression in depressed and nondepressed subjects as well as the reliability and validity of several measures of anger, hostility and depression. Sixty-nine subjects were evaluated for depression using the Hamilton Rating Scale for Depression (HRSD; Hamilton, 1960). These subjects were then administered the Beck Depression Inventory (BDI; Beck, Ward, Mendelson, Mock, & Ergaugh, 1961), Buss-Durkee Hostility Inventory (BDHI; Buss & Durkee, 1957), Hostility and Direction of Hostility Questionnaire (HDHQ; Foulds, Caine, & Creasy, 1960) and the State-Trait Anger Scale (STAS; Spielberger, Jacobs, Russell, & Crane, 1983). Results showed the BDI, STAS-TRAIT, HDHQ, and BDHI to have good temporal stability. Support was found for the convergent validity of all measures of depression, hostility, and anger. Limited discriminant validity was found between measures of anger and hostility and measures of depression. This latter finding was interpreted as lending support for the relationship between hostility and depression rather than as an indication of limited construct validity for the measures. Intercorrelations among hostility, anger, and depression scales offer some support for the hypothesis that depression is linked most strongly with attitudinal versus motoric forms of hostility. However, normative data suggests that both forms of hostility increase with severity of depression. Clinical implications and directions for further research are discussed.

Human immunodeficiency virus type 1 Tat does not transactivate mature trans-acting responsive region RNA species in the nucleus or cytoplasm of primate cells.

Human immunodeficiency virus (HIV)-encoded transactivator Tat is essential for viral gene expression and replication. By interacting with a nascent RNA stem-loop called the trans-acting responsive region (TAR). Tat increases rates of initiation and/or elongation of HIV transcription. Several reports have also suggested that Tat has additional effects on mature HIV RNA species including modification of primary transcripts in the nucleus and their increased translation in the cytoplasm. These posttranscriptional effects are most pronounced in the Xenopus oocyte. To investigate directly whether Tat has similar effects on viral transcripts in cells that are permissive for HIV replication, we cotransfected and microinjected human and monkey cells with Tat and TAR in the form of DNA or RNA. Whereas Tat transactivated TAR DNA targets, it did not transactivate TAR RNA targets in the nucleus of microinjected cells or in the cytoplasm of transfected cells. We conclude that in cells permissive for viral replication, Tat exerts its effect primarily at the level of HIV transcription.

Complex processing and protein:protein interactions in the E2:NS2 region of HCV.

Hepatitis C virus (HCV), the principal cause of parenteral non-A, non-B hepatitis, is an RNA virus and a member of the Flaviviridae family. Its genome is translated into a single polyprotein that is processed co- and post-translationally into both structural and nonstructural (NS) proteins. There are three putative structural proteins, consisting of the nucleocapsid protein and two envelope glycoproteins, E1 and E2. Analysis of transient transfections of serially extended templates covering the E2/NS2 region provided evidence for three E2 species with distinct C-termini. One form is E2 terminating at amino acid 729, while the larger two species represent fusions with the downstream NS2A and NS2A/NS2B proteins terminating at amino acids 809 and 1026, respectively. Using the same E2 templates, we defined a region of E2 important for co-immunoprecipitation of E1 and observed that this region also prevents E2 secretion. The N-terminus of NS2B was determined by radiosequencing and a novel association of NS2B and probable NS4B with E2 was observed; the regions of NS2B and E2 important for this association have been mapped. These data indicate that complex processing and protein:protein interactions occur during HCV morphogenesis.

Differential RNA splicing predicts two distinct nerve growth factor precursors.

Nerve growth factor (NGF) has a crucial role in the development of sensory and sympathetic neurones. However, although it can affect other neural cell types under certain experimental conditions, no biological role has been convincingly demonstrated elsewhere in the nervous system. The 5' end of the mouse NGF gene contains several relatively short exons. The NGF messenger RNA contains two in-frame initiator methionine codons; the second precedes the signal peptide sequence. Studies of the translation of other eukaryotic mRNAs indicate that the first AUG is preferred, suggesting that the signal for secretion might be ambiguous. We have analysed the NGF mRNA species from various cell types, some of which (clonal myoblast and fibroblast cell lines) are known to secrete NGF, to search for different NGF transcripts. One pathway of RNA splicing generates the transcript already described from a submaxillary gland complementary DNA clone. We demonstrate here that there is another splicing pathway, leading to a shorter transcript that lacks the second exon. This short transcript is the major form in most other mouse tissues and in the tissues of several other species, but both transcripts are usually present. In the short transcript, the initiator methionine is immediately upstream from a signal peptide-like sequence whereas in the long transcript the first methionine is 62 amino acids upstream from the signal peptide-like sequences. This may result in a different cellular localization of the NGF or alter the biological activity of the NGF precursor.

Interaction between hepatitis C virus core protein and E1 envelope protein.

Hepatitis C virus has three structural genes named C, E1, and E2. The C gene encodes the core (capsid) protein and the E1 and E2 genes encode the envelope proteins. In an immunoprecipitation experiment, the E1 protein was found to be precipitated by an anti-core antibody in the presence but not in the absence of the core protein, indicating that the E1 protein can interact with the core protein. This interaction is independent of whether the E1 and the C genes are linked in cis or separated in different DNA constructs for expression. The interaction between the core and the E1 proteins is confirmed by the observation that a hybrid protein derived from the core protein and the tissue plasminogen activator is localized in the nucleus in the absence of the E1 protein and in the perinuclear region in the presence of the E1 protein. Deletion-mapping studies indicate that the carboxy-terminal sequences of both the core and the E1 proteins are important for their interaction. Since little E1 sequence is exposed on the cytosolic side of the membrane of the endoplasmic reticulum, the interaction between the core and the E1 proteins most likely takes place in the endoplasmic reticulum membrane. The E2 protein could not be coprecipitated with the core protein by the anti-core antibody in a similar assay and likely does not interact with the core protein. The implications of these findings on the morphogenesis of the hepatitis C virus virion are discussed.

Virus-specific cytotoxic T-lymphocyte activity elicited by coimmunization with human immunodeficiency virus type 1 genes regulated by the bacteriophage T7 promoter and T7 RNA polymerase protein.

Cytotoxic T lymphocyte (CTL) activity was assessed in mice immunized with DNA plasmids containing the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (pTMIgp120) or p55gag (pTMIgag) gene regulated by the bacteriophage T7 promoter. Immunization with either plasmid resulted in CTL activity against class I major histocompatibility complex-restricted viral epitopes when coadministered with a recombinant vaccinia virus expressing the T7 RNA polymerase protein (T7 RNAP) but not a control vaccinia virus. Recombinant vaccinia-T7 RNAP virus (VTF7-3) could be replaced with a noninfectious source of T7 RNAP. A three-component vaccine consisting of pTMIgag, a recombinant subunit T7 RNAP protein, and a plasmid (pT7T7) encoding T7 RNAP under the control of its own promoter induced gag-specific CTL activity. Intramuscular immunization with the pTMIgag plasmid delivered with either the T7 RNAP protein or pT7T7 plasmid alone also induced HIV-1-specific CTL. Thus, there is adventitious expression of the pT7T7 plasmid in vivo, and enough T7 RNAP is produced to result in production of p24gag protein from the pTMIgag plasmid. The results demonstrate that regulated expression of genes in vivo is possible with this T7-based expression system, and may be useful in vaccine settings where short-term cytoplasmic expression of protein in antigen presenting cells is desired.

Cobra nerve growth factor: structure and evolutionary comparison.

We have cloned and sequenced an NGF cDNA from the cobra venom gland. The structure of the predicted cobra precursor resembles that of the mouse prohormone, with a highly conserved mature NGF protein at the C-terminus. In the non-NGF moiety, a putative signal peptide and two sets of basic residues presumably reflecting cleavage sites that are more conserved, while large interstitial regions are poorly conserved. This moiety may be involved in defining the folding of the precursor for subsequent proteolytic maturation and/or encode a bioactive polypeptide(s). Primer extension analysis indicates a single NGF transcript in the cobra venom gland.

Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

The human immunodeficiency virus (HIV-1)-encoded trans-activator (tat) increases HIV gene expression and replication. Previously, we demonstrated that tat facilitates elongation of transcription through the HIV-1 long terminal repeat (LTR) and that short transcripts corresponding to prematurely terminated RNA are released and accumulate in the absence of tat. Here, using a transient expression assay, we tested clustered and compensatory mutations, as well as 3' deletions, in the trans-acting responsive region (tar) and observed that the primary sequence in the loop and secondary structure in the stem of the stem-loop in tar are required for trans-activation by tat. Insertions in the 5' region of tar revealed that tar must be near the site of HIV-1 initiation of transcription for trans-activation by tat. Deletions (3') and an insertion in tar demonstrated that an intact stem-loop is required for the recovery of prematurely terminated transcripts. Short and full-length transcripts were observed also with HIV type 2 (HIV-2) in the absence and presence of tat, respectively. We conclude that an intact stem-loop in tar is essential for trans-activation by tat and that initiation of transcription by HIV-1 promoter factors and elongation of transcription by tat are coupled.