An altered intracellular distribution of the autoantigen La/SS-B when translated from a La mRNA isoform

This study reports the successful growth suppression of a rat glioblastoma model (RT-2) both in vitro and in vivo by the insertion of p21(WAF1/CIP1), a negative cell cycle regulatory gene, into the tumor cells. Greater than 95% of the tumor cells expressed p21 protein after being infected with pCL based p21 retrovirus at 4x M.O.I. (multiplicity of infection). The p21-infected cells showed a 91% reduction in colony forming efficiency and a 66% reduction in growth rate. More prominent p21 staining was found in cells exhibiting histologic evidence of senescence. Intracranial implantation of the infected cells showed complete disappearance of the p21-infected cells at day 10 and long-term survival of the animals compared to controls. Injection of pCLp21 virus into tumor established in situ showed tumor necrosis and gene expression. In a clonogenic radiation survival assay, a 93% reduction of surviving colonies of p21-infected cells was seen in comparison to vector-infected control cells and to p53-infected cells after exposure to 8 Gy (800 rads).     

Activation of a murine autoreactive B cell by immunization with human recombinant autoantigen La/SS-B: characterization of the autoepitope

Immunization of Balb/c mice with a homogeneously purified recombinant human La/SS-B protein resulted in activation of an autoreactive B cell secreting a novel monoclonal anti-La antibody termed La4B6. La4B6 reacted with La protein from a variety of sources including human, bovine, rat and mouse. ATP blocked the binding of La4B6 to recombinant La protein. The human epitope was identified as consisting of the amino acid sequence SKGRRFKGKGKGN, which includes the proposed ATP-binding site of the La protein. In the human and bovine La protein, the epitope exists as a continuous amino acid sequence. In rat and mouse the epitope was found to consist of the amino acid sequence SKG interrupted by a species-specific insert of 16 amino acids, and followed by the second half of the epitope, the amino acid sequence RRFKGKGKGN. Our data suggest that in the case of the rat and mouse La proteins the two separated parts of the epitope are able to form a conformational epitope which looks similar to the continuous human epitope.     

Analysis of neurotrophin-3 expression using the lacZ reporter gene suggests its local mode of neurotrophic activity

We replaced the mouse neurotrophin-3 gene with the Escherichia coli-derived lacZ gene by means of homologous recombination. The mice with this mutation were useful models for studying the distribution of neurotrophin-3 expression in vivo, because visualization by 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-Gal) staining was simple and rapid compared with in situ hybridization or immunohistochemistry. Whole-mount staining of mutant embryos at embryonic day 10 revealed that lacZ, a reporter for the neurotrophin-3 gene, was expressed in the mesencephalon, mandibular arch and somites. In the embryos at days 13-17, lacZ was markedly expressed in the peripheral target tissues of sensory and sympathetic neurons. We also found that spinal motor neurons and sensory neurons in trigeminal and dorsal root ganglia express lacZ. Some of these X-Gal staining regions overlapped with the sites expressing trkC, a high-affinity receptor for neurotrophin-3. The distribution of X-Gal staining in heterozygotes and homozygotes was similar to that of neurotrophin-3 messenger RNA detected by in situ hybridization. However, there was less lacZ expression in the dorsal root ganglia of homozygotes than neurotrophin-3 expression in wild-type mice. These results suggest that the neurotrophin-3 produced in the dorsal root ganglia also plays a role in the survival of some of the neurotrophin-3-positive neurons and that the local mode of neurotrophic activity is widely distributed.     

Dual-function reporter protein for analysis of gene expression in living cells

The firefly luciferase (Luc) protein and the jellyfish green fluorescent protein (GFP) are two commonly used molecular reporters that can be detected noninvasively in living cells. The properties that make GFP or Luc useful for a particular experimental application are quite distinct. A recombinant protein with both fluorescent and bioluminescent characteristics might take advantage of the strengths of both reporters. An expression vector encoding a chimeric protein in which GFP was tethered to Luc through a 19-amino acid linker was prepared and characterized. Western blotting with antibodies specific for either GFP or Luc showed that a protein of appropriate size was expressed in transfected cells. Fluorescence microscopy revealed bright green fluorescence from transfected cells, indicating proper formation of the GFP chromophore. Luc enzymatic activity in protein extracts from transfected cells showed that Luc was fully functional. The treatment of living cell cultures stably expressing the GFP-Luc fusion protein with the protein translation-inhibitor cycloheximide (Chx) was used to show that the half-life for Luc protein activity was approximately 2 h at 37 degrees C. The utility of this dual-function reporter protein was shown by the identification of single living cells expressing the chimeric protein within a population by fluorescence microscopy, followed by quantification of Luc activity from the same living cells.     

Second gene expression in bicistronic constructs using short synthetic intercistrons and viral IRES sequences

The authors addressed 5 issues bearing on the validity of the construct of depressive personality disorder (DPD): its relationship with the Diagnostic and Statistical Manual of Mental Disorders (3rd ed., rev.; American Psychiatric Association, 1987) mood and personality disorders and normal personality dimensions of negative and positive affectivity, its stability over 30-months, and its impact on the course of Axis I depressive disorders. Two samples were used: 156 outpatients with mood disorders, personality disorders, or both, and 267 of their 1st-degree relatives. The association between DPD and dysthymia was fairly modest, whereas the associations with major depression and the personality disorders were quite low. DPD was moderately correlated with both negative and positive affectivity; however, it contributed unique information beyond that available from the 2 emotional superfactors. Finally, DPD was moderately stable over a 30-month period and was associated with a poorer course of depression.     

Dicistronic LacZ and alkaline phosphatase reporter constructs permit simultaneous histological analysis of expression from multiple transgenes

We report here the development of convenient dicistronic transgenic markers for the rapid and efficient simultaneous analysis of transgene activity in transgenic mice. Two sensitive histological markers, the beta-galactosidase (beta-gal)-encoding lacZ gene and the human placental alkaline phosphatase (hpAP) gene, have been fused to the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus, which directs efficient mRNA cap-independent entry of the translation apparatus in mammalian cells. The IRES permits efficient translation of either lacZ or hpAP when placed anywhere within transgene exonic sequences, including both 5' and 3' untranslated regions. In addition, the production of constructs for transgenic analysis of DNA regulatory elements is greatly facilitated with IRES-lacZ or IRES-hpAP, since the IRES relieves the need for complicated in frame transgene protein fusions to produce a functional beta-gal or hpAP protein.     

Construct for assessment of transfected secretory proteins using an independently secreted reporter gene

PURPOSE: To report a noninvasive method for evaluating eyes with cystoid macular edema. METHODS: We obtained infrared images of cystoid macular edema in eight eyes of eight patients using a scanning laser ophthalmoscope with the dark-field mode of a 780-nm diode laser. Differences between infrared images and fluorescein angiograms in the imaging of cystoid changes were examined. RESULTS: With the scanning laser ophthalmoscope, we observed cystoid macular changes as images that resembled three-dimensional pictures in the dark-field mode with infrared light. Cystoid changes observed by this method generally agreed with changes observed by fluorescein angiography. CONCLUSIONS: Scanning laser ophthalmoscopy with infrared light in a dark-field mode is noninvasive, and the results in eyes with cystoid macular edema generally agreed with results obtained by fluorescein angiography. This method is useful for examining eyes with cystoid macular edema.     

Clinical relevance of antibodies to Ro/SS-A and La/SS-B in subacute cutaneous lupus erythematosus and related conditions

Ro/SS-A autoantibodies are frequently associated with subacute cutaneous lupus erythematosus, neonatal lupus erythematosus and Sj?gren's syndrome. The Ro/SS-A autoantigen is a ribonucleoprotein complex consisting of at least four protein components and four small cytoplasmic RNA components designated hY RNA 1, 3, 4 and 5. Three of the Ro/SS-A peptides have been isolated and cloned. The function of this ribonucleoprotein complex is as yet unknown.     

Effects of the human immunodeficiency virus type 1 Rev protein on reporter gene and host T-cell gene expression

The mechanism of DNA mismatch repair has been modeled upon biochemical studies of the E. coli DNA adenine methylation-instructed pathway where the initial recognition of mismatched nucleotides is performed by the MutS protein. MutS homologs (MSH) have been identified based on a highly conserved region containing a Walker-A adenine nucleotide binding motif. Here we show that adenine nucleotide binding and hydrolysis by the human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch. The hMSH2-hMSH6 complex is ON (binds mismatched nucleotides) in the ADP-bound form and OFF in the ATP-bound form. These results suggest a new model for the function of MutS proteins during mismatch repair in which the switch determines the timing of downstream events.     

Different La/SS-B mRNA isoforms are expressed in salivary gland tissue of patients with primary Sj?gren's syndrome

Recently we isolated a La/SS-B mRNA isoform from a cDNA library made from peripheral blood lymphocytes of a patient with primary Sj?gren's Syndrome. In the La/SS-B mRNA isoform the exon 1 was replaced. The alternative exon was termed exon 1'. Genomic analysis showed that the exon 1' La mRNA was the result of a promoter-switch in combination with alternative splicing. Due to the unusual structure of the exon 1' La/SS-B mRNA, the function and the behaviour under physiological and pathophysiological conditions in tissue of patients with primary Sj?gren's syndrome or Systemic Lupus Erythematosus remained obscure. Therefore assays were established allowing a qualitative and quantitative estimation of expression of the exon 1 and 1' La mRNA form, including in situ and dot blot hybridization as well as reversed PCR. Both mRNA forms were found to represent finally processed cytoplasmic mRNAs belonging to the abundant class of mRNAs. They were expressed and regulated in parallel. A ratio exon 1 to 1' between 1:1 and 5:1 was determined. Both mRNA forms were downregulated in quiescent cells and upregulated in activated and proliferating cells including non-keratized stratified squamous epithelial, endothelial, salivary gland as well as infiltrating cells.     

Structure and expression of the tobacco nuclear gene encoding RNA-binding protein RZ-1: the existence of an intron in the 3''-untranslated region

We investigated prolactin (PRL) and growth hormone (GH) secretion during acute and late abstinence following methylphenidate (MP) administration. Ten male patients who were undergoing acute cocaine abstinence and nine control subjects were randomly assigned into one of two possible sequences of MP and placebo, with each experimental condition occurring on two successive days. This procedure was repeated after 7 days for the patients. Baseline measures were analyzed by analysis of variance (ANOVA) with post hoc tests. Measures of MP challenge were analyzed by analysis of covariance (ANCOVA) with baseline as the covariate. Acute abstinence was compared with control values and then to late abstinence. Plasma levels of PRL, GH, and MP were measured along with a measure of clinical symptoms. Patients had higher basal PRL concentrations during acute abstinence compared with controls, and patients showed no difference when compared to themselves after 7 days (late abstinence). Provocation with MP yielded exaggerated PRL and GH responses in patients during acute abstinence compared with control values, and ANCOVA also revealed a significant increase in PRL response during late abstinence compared with acute abstinence. GH was a less sensitive indicator than PRL. Craving was exacerbated by MP during both acute and late abstinence and was possibly increased at late abstinence. This indicates that the perturbation in dopamine regulation persists and may be increased as clinical recovery occurs for most subjective symptoms. Blood pressure changes were variable and interpretation was uncertain.     

A microplate fluorimetric assay for transfection of the beta-galactosidase reporter gene

Despite having been first marketed in the 1950s, new designs of progressive addition spectacle lens continue to appear. Some of the recent patent literature is reviewed on the design of such lenses. As well as a number of improvements to general purpose designs, specifically to include aspheric surfaces for the distance portion of progressive lenses, the literature includes a recent patent on an improved version of the Alvarez lateral translation lens system. The optimisation of single vision lens forms in order to extend the depth of field for early presbyopes is also discussed.     

Replication-competent retroviral vectors encoding alkaline phosphatase reveal spatial restriction of viral gene expression/transduction in the chick embryo

Replication-competent avian retroviruses, capable of transducing and expressing up to 2 kb of nonviral sequences, are now available to effect widespread gene transfer in chicken (chick) embryos (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We have constructed novel avian retroviral vectors that encode human placental alkaline phosphatase as a marker whose expression can be histochemically monitored. These vectors have been tested for expression by introducing them into the embryonic chick nervous system. They have revealed that the expression of retrovirally transduced genes can be spatially and temporally limited without the need for tissue-specific promoters. By varying the site and time of infection, targeted gene transfer can be confined to selected populations of neural cells over the course of several days, a time window that is sufficient for many key developmental processes. The capability of differentially infecting specific target populations may avoid confounding variables such as detrimental effects of a transduced gene on processes unrelated to the cells or tissue of interest. These vectors and methods thus should be useful in studies of the effect of transduced genes on the development of various organs and tissues during avian embryogenesis. In addition, the vectors will facilitate studies aimed at an understanding of viral infection and expression patterns.