Membrane-anchored form of v-sis/PDGF-B induces mitogenesis without detectable PDGF receptor autophosphorylation.

The v-sis protein is structurally and functionally related to PDGF. Forms of the v-sis protein which are anchored to the cell membrane via the transmembrane domain of the vesicular stomatitis virus G protein have been previously described (Hannink, M., and D.J. Donoghue. 1986. J. Cell Biol. 103:2311-2322). Several of these fusion proteins were shown to interact productively with the PDGF receptor (PDGFR) based on their ability to transform NIH 3T3 cells. In this report, we further characterized one of these membrane-anchored v-sis proteins, designated v-sis239-G. The gene encoding v-sis239-G was placed under control of the Drosophila melanogaster hsp70 promotor and synthesis of this protein was shown to induce a mitogenic response in NIH 3T3 cells. Unexpectedly, v-sis239-G did not induce detectable autophosphorylation of the PDGFR, in contrast to a similarly expressed secreted form of the v-sis protein. Thus, it appears that a PDGFR-mediated mitogenic response may be dissociated from detectable receptor autophosphorylation. Furthermore, induced synthesis of v-sis239-G was shown to lead to c-fos expression even in the absence of detectable receptor autophosphorylation. Interestingly, a nonmitogenic membrane-anchored form of the v-sis protein, designated v-sis239-G338, also induced c-fos without receptor autophosphorylation. These results raise interesting questions regarding the roles of autophosphorylation and c-fos induction in PDGFR-mediated signal transduction and suggest the possibility of an autophosphorylation-independent signal transduction pathway.

Intracellular retention of membrane-anchored v-sis protein abrogates autocrine signal transduction.

An important question regarding autocrine transformation by v-sis is whether intracellularly activated PDGF receptors are sufficient to transform cells or whether activated receptor-ligand complexes are required at the cell surface. We have addressed this question by inhibiting cell surface transport of a membrane-anchored v-sis protein utilizing the ER retention signal of the adenoviral transmembrane protein E3/19K. A v-sis fusion protein containing this signal was retained within the cell and not transported to the cell surface as confirmed by immunofluorescent localization experiments. Also, proteolytic maturation of this protein was suppressed, indicating inefficient transport to post-Golgi compartments of the secretory pathway. When compared with v-sis proteins lacking a functional retention signal, the ER-retained protein showed a diminished ability to transform NIH 3T3 cells, as measured by the number and size of foci formed. In newly established cell lines, the ER-retained protein did not down-regulate PDGF receptors. However, continued passage of these cells selected for a fully transformed phenotype exhibiting downregulated PDGF receptors and proteolytically processed v-sis protein. These results indicate that productive autocrine interactions occur in a post-ER compartment of the secretory pathway. Transport of v-sis protein beyond the Golgi correlated with acquisition of the transformed phenotype. Furthermore, suramin treatment reversed transformation and upregulated the expression of cell surface PDGF receptors, suggesting an important role for receptor-ligand complexes localized to the cell surface.

The v-sis protein retains biological activity as a type II membrane protein when anchored by various signal-anchor domains, including the hydrophobic domain of the bovine papilloma virus E5 oncoprotein.

Membrane-anchored forms of the v-sis oncoprotein have been previously described which are oriented as type I transmembrane proteins and which efficiently induce autocrine transformation. Several examples of naturally occurring membrane-anchored growth factors have been identified, but all exhibit a type I orientation. In this work, we wished to construct and characterize membrane-anchored growth factors with a type II orientation. These experiments were designed to determine whether type II membrane-anchored growth factors would in fact exhibit biological activity. Additionally, we wished to determine whether the hydrophobic domain of the E5 oncoprotein of bovine papilloma virus (BPV) can function as a signal-anchor domain to direct type II membrane insertion. Type II derivatives of the v-sis oncoprotein were constructed, with the NH2 terminus intracellular and the COOH terminus extracellular, by substituting the NH2 terminal signal sequence with the signal-anchor domain of a known type II membrane protein. The signal-anchor domains of neuraminidase (NA), asialoglycoprotein receptor (ASGPR) and transferrin receptor (TR) all yielded biologically active type II derivatives of the v-sis oncoprotein. Although transforming all of the type II signal/anchor-sis proteins exhibited a very short half-life. The short half-life exhibited by the signal/anchor-sis constructs suggests that, in some cases, cellular transformation may result from the synthesis of growth factors so labile that they activate undetectable autocrine loops. The E5 oncoprotein encoded by BPV exhibits amino acid sequence similarity with PDGF, activates the PDGF beta-receptor, and thus resembles a miniature membrane-anchored growth factor with a putative type II orientation. The hydrophobic domain of the E5 oncoprotein, when substituted in place of the signal sequence of v-sis, was indistinguishable compared with the signal-anchor domains of NA, TR, and ASGPR, demonstrating its ability to function as a signal-anchor domain. NIH 3T3 cells transformed by the signal/anchor-sis constructs exhibited morphological reversion upon treatment with suramin, indicating a requirement for ligand/receptor interactions in a suramin-sensitive compartment, most likely the cell surface. In contrast, NIH 3T3 cells transformed by the E5 oncoprotein did not exhibit morphological reversion in response to suramin.

The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex.

The location of autocrine interactions between the v-sis protein and PDGF receptors remains uncertain and controversial. To examine whether receptor-ligand interactions can occur intracellularly, we have constructed fusion proteins that anchor v-sis to specific intracellular membranes. Fusion of a cis-Golgi retention signal from a coronavirus E1 glycoprotein to v-sis protein completely abolished its transforming ability when transfected into NIH3T3 cells. Fusion proteins incorporating mutations in this retention signal were not retained within the Golgi complex but instead were transported to the cell surface, resulting in efficient transformation. All chimeric proteins were shown to dimerize properly. Derivatives of some of these constructs were also constructed bearing the cytoplasmic tail from the glycoprotein of vesicular stomatitis virus (VSV-G). These constructs allowed examination of subcellular localization by double-label immunofluorescence, using antibodies that distinguish between the extracellular PDGF-related domain and the VSV-G cytoplasmic tail. Colocalization of sis-E1-G with Golgi markers confirmed its targeting to the early Golgi complex. The sis-E1 constructs, targeted to the early Golgi complex, exhibited no proteolytic processing whereas the mutant forms of sis-E1 exhibited normal proteolytic processing. Treatment with suramin, a polyanionic compound that disrupts ligand/receptor interactions at the cell surface, was able to revert the transformed phenotype induced by the mutant sis-E1 constructs described here. Our results demonstrate that autocrine interactions between the v-sis oncoprotein and PDGF receptors within the early Golgi complex do not result in functional signal transduction. Another v-sis fusion protein was constructed by attaching the transmembrane domain and COOH-terminus of TGN38, a protein that localizes to the trans-Golgi network (TGN). This construct was primarily retained intracellularly, although some of the fusion protein reached the surface. Deletion of the COOH-terminal region of the TGN38 retention signal abrogated the TGN-localization, as evidenced by very prominent cell surface localization, and resulted in increased transforming activity. The behavior of the sis-TGN38 derivatives is discussed within the context of the properties of TGN38 itself, which is known to recycle from the cell surface to the TGN.

Coupling termination of transcription to messenger RNA maturation in yeast.

The direct association between messenger RNA (mRNA) 3'-end processing and the termination of transcription was established for the CYC1 gene of Saccharomyces cerevisiae. The mutation of factors involved in the initial cleavage of the primary transcript at the poly(A) site (RNA14, RNA15, and PCF11) disrupted transcription termination at the 3' end of the CYC1 gene. In contrast, the mutation of factors involved in the subsequent polyadenylation step (PAP1, FIP1, and YTH1) had little effect. Thus, cleavage factors link transcription termination of RNA polymerase II with pre-mRNA 3'-end processing.

The alternatively spliced exon of the platelet-derived growth factor A chain encodes a nuclear targeting signal.

We have previously shown that the SIS/platelet-derived growth factor B chain contains a nuclear targeting signal near its C terminus. Here we show that the platelet-derived growth factor A chain also contains a nuclear targeting signal encoded by an exon which is subject to alternative splicing. This sequence is capable of targeting a nonsecreted form of the A chain to the nucleus and can also target the cytoplasmic proteins dihydrofolate reductase, chloramphenicol acetyltransferase, and pyruvate kinase to the nucleus.

Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules.

The v-vis gene encodes p28sis, the transforming protein of simian sarcoma virus. This gene resulted from a fusion of the env gene of simian sarcoma-associated virus and the woolly monkey gene for the B chain of platelet-derived growth factor (PDGF). Previous work has shown that the v-sis gene product undergoes signal sequence cleavage, glycosylation, dimerization, and proteolytic processing to yield a secreted form of the protein. It transport across the endoplasmic reticulum is blocked by the introduction of a charged amino acid residue within the signal sequence, the protein does not dimerize, is not secreted, and is no longer transforming as assayed by focus-forming ability in NIH 3T3 cells. Instead, this mutant protein localizes to the nucleus as demonstrated by both indirect immunofluorescence and cell fractionation. Using a series of deletion mutations, we delimited an amino acid sequence within this protein which is responsible for nuclear localization. This region is completely conserved in the predicted human c-sis protein, although it lies outside of regions required for transformation by the v-sis gene product. This nuclear transport signal is contained within amino acid residues 237 to 255, RVTIRTVRVRRPPKGKHRK. An amino acid sequence containing these residues is capable of directing cytoplasmic v-sis mutant proteins to the nucleus. This sequence is also capable of directing less efficient nuclear transport of a normally cytoplasmic protein, pyruvate kinase. Pulse-chase experiments indicate that the half-lives of nuclear and cytoplasmic v-sis mutant proteins are approximately 35 min. Using the heat-inducible hsp70 promoter from Drosophila melanogaster, we showed that the nuclear v-sis protein accumulates in the nucleus within 30 min of induction. The identification of a nuclear transport signal in the v-sis gene product raises interesting questions regarding the possibility of some function for PDGF or PDGF-related molecules in the nucleus.

A clinically variant fibrosis syndrome in a Turkish family maps to the CFEOM1 locus on chromosome 12.

OBJECTIVES: To describe the phenotype of a Turkish family with variably expressed congenital fibrosis of the extraocular muscles (CFEOM), and to determine the genetic location of their disorder. METHODS: Participants were examined and had blood extracted for genetic analysis. The clinical features of the family's disorder were studied, and the disorder was tested for linkage to the 3 known CFEOM loci (CFEOM1, CFEOM2, and CFEOM3). RESULTS: Twenty-nine affected and 31 unaffected family members participated in the study. Eighteen affected individuals had congenital bilateral ptosis and restrictive infraductive (downward) ophthalmoplegia, consistent with the published descriptions of classic CFEOM families linked to the CFEOM1 locus. Eleven affected individuals, however, had eye(s) in a neutral primary position, residual upgaze, and/or absence of ptosis, thus deviating from previous descriptions of CFEOM1-linked families. Analysis of the autosomal dominant variably expressed disorder in this family revealed linkage to the CFEOM1 locus on chromosome 12 with a maximum lod score of 10.8 at D12S85. CONCLUSIONS: This Turkish family segregates a variably expressed form of CFEOM that most closely resembles CFEOM3-linked CFEOM, but maps to the CFEOM1 locus. CLINICAL RELEVANCE: These data establish that there is much greater phenotypic heterogeneity at the CFEOM1 locus than previously reported, and this may blur our ability to distinguish the different CFEOM loci based solely on clinical presentation. Arch Ophthalmol. 2000;118:1090-1097

Evidence of genetic heterogeneity in autosomal recessive congenital fibrosis of the extraocular muscles.

PURPOSE: Autosomal recessive congenital fibrosis of the extraocular muscles (CFEOM2) has been described in families from Saudi Arabia. Affected individuals have ptosis and exotropic ophthalmoplegia, and their disease has been mapped to chromosome 11q13. Here, we describe the phenotypic findings in a similarly affected Yemenite family and analyze the family for linkage to the CFEOM2 locus, as well as to the autosomal dominant CFEOM1 and CFEOM3 loci on chromosomes 12cen and 16q24, respectively. METHODS: The family was ascertained through two affected daughters. There are four unaffected siblings, and the parents are consanguineous. Each family member was examined, and linkage analysis was performed using markers from the CFEOM1, CFEOM2, and CFEOM3 loci. RESULTS: Both affected daughters have congenital bilateral ophthalmoplegia. The 15-month-old proband has restrictive exotropia. She fixates with either eye in abduction and with a compensatory head turn to the opposite side. Her 4-year-old sister has a small exotropia and severely limited eye movements. All other family members have normal ophthalmologic examinations. Genetic analysis excluded linkage of the family's disease to the CFEOM2 and CFEOM3 loci. A lod score of 2.0 (the maximum possible, given the family size and structure), was obtained at the CFEOM1 locus, and the alleles reduced to homozygosity in both affected daughters and none of the other children. CONCLUSIONS: These data establish that there is genetic heterogeneity in autosomal recessive CFEOM and suggest that this second recessive locus may be allelic to the autosomal dominant CFEOM1 locus at 12cen.

Identification of duck T lymphocytes using an anti-human T cell (CD3) antiserum.

Duck lymphocytes have not been classified into cells resembling B or T cells of mammals. Reagents used in the past to identify lymphocyte populations in other species have not been useful for this purpose and antibodies raised to duck immunoglobulin bind in high proportions to blood and organ lymphocytes of ducks as well as to their red blood cells. Here we report that a polyclonal rabbit antiserum reacting to the CD3 marker on human T cells has been used to identify duck T lymphocytes. These antibodies react with the intracytoplasmic portion of the human CD3 epsilon chain (amino acids 156-168), an epitope highly conserved between mammals. Immunohistochemical staining with this antiserum of sections of duck lymphoid organs and FACScan analysis of duck lymphoid cell suspensions identified a population of duck lymphocytes with a staining pattern similar to that seen for mammalian T cells. This anti-human CD3 immunoprecipitated a 23 kDa protein from a duck lymphoblast lysate: a size similar to the human CD3 epsilon chain. This is the first direct identification of duck T lymphocytes.

The use of time-expanded speech in the identification of part-word repetitions of stutterers.

The purpose of this investigation was to explore the possibility of using time-expanded speech to aid clinicians in the identification of one-, two-, and three-unit repetitions of stutterers. Tape-recorded samples of nine stutterers (five adults and four children) were time-expanded to 0%, 150%, and 200% of original time. Thirty graduate student clinicians listened individually to the speech samples under the three listening conditions. The three conditions were presented in a counterbalanced order. Clinicians were asked to indicate each occurrence of one-, two-, or three-unit part-word repetitions on a response sheet. Results indicated that time expansion of the stutterers' speech by 150% or 200% of the original time resulted in significantly (P less than 0.05) more accurate identification one-unit part-word repetitions. Time expansion did not, however, provide a perceptual advantage for the identification of two- and three-unit part-word repetitions. Implications for clinical application and further investigation are discussed.

Residential mobility on skid row: disaffiliation, powerlessness, and decision making.

Individual-level models of residential mobility emphasize (a) the stabilizing effects of various social, demographic, and housing characteristics and (b) the important mediating role played by decision-making variables. Data from a sample of skid row residents are analyzed to determine if these models retain their accuracy under conditions of disaffiliation and powerlessness. The findings indicate that, while older age, employment, and other characteristics may encourage residential stability on skid row, such factors influences mobility behavior in a direct fashion rather than through the intervening decision variables of residential evaluation and mobility expectation. In general, persons with weakened social attachments and little control over their lives and resources find it difficult to engage in the calculated, long-term type of decision-making process implied by mobility theory.

Stability and change in an urban homeless population.

Few studies have produced the over-time observational data needed to draw valid conclusions about changes in urban homeless populations during the 1980s. One place for which such data exist is Nashville, Tennessee. An ongoing series of enumerations lends little support to Nashvillians' perception that the number of homeless in their city is growing rapidly. Enumeration results also (1) contradict expectations regarding the rise of "new homeless" groups and (2) show two types of spatial redistribution--from indoor to outdoor and core to peripheral locations--to be under way. The applicability of the enumeration methodology to other communities is discussed, as are the discrepancies between purported and measured demographic changes in homelessness.

Is neighborhood racial succession place-specific?

This paper evaluates the geographic generality of the succession model of neighborhood racial change for the period 1970 to 1980. Using census data on racially mixed tracts, we determine whether white-to-black compositional shifts were equally common across the four regions and 58 central cities in our sample. Substantial variation occurred by region in the incidence and magnitude of racial succession; tracts in western cities departed most markedly from expectations. Even in other regions, some cities experienced more numerous instances of stability and displacement than of succession. These region and city effects persist when neighborhood characteristics believed to influence racial transition are controlled.

A basic system for improving quality of care in nursing homes.

This article describes a computer-based system for storing, retrieving, and analyzing several related parts of the medical record in long-term care facilities--physicians' orders, diagnoses and sensitivities. The system is in response to a number of problems in the areas of governmental regulation, documentation, on-going review, prospective and retrospective medical care evaluation. (The proposed changes in regulations may dictate further refinements, which are relatively simple to program.) It has been tested and installed in two Maryland facilities of 82 and 143 beds respectively, and is well accepted by medical, nursing, pharmaceutical and administrative staff members in these institutions. The system is believed to have satisfied all of its initial objectives, including making a significant contribution to the quality of patient care in these nursing homes.

Heterolocalism: an alternative model of the sociospatial behaviour of immigrant ethnic communities.

"This paper evaluates critically the applicability of the well-known assimilation and pluralist models to the contemporary ethnic landscape of the U.S.... We then consider an alternative model, labelled heterolocalism, which suggests that members of certain newly arrived groups may be able to sustain their identity as an ethnic community despite immediate or rapid spatial dispersion. The applicability of the heterolocal perspective to non-metropolitan and transnational phenomena is evaluated in subsequent sections of the paper."

Multiple SP1 binding sites confer enhancer-independent, replication-activated transcription of HIV-1 and globin gene promoters.

We demonstrate that multiple SP1 protein:DNA binding sites confer enhancer-independent activation on the HIV-1 and globin gene promoters. This activation process can be achieved either by DNA replication of the promoter-containing plasmid or by high concentrations of input plasmid DNA used in the transfections. In the case of HIV-1, the three SP1 sites adjacent to the promoters TATA box are essential for this activation process. Furthermore, the human beta globin gene, which is normally dependent on a linked enhancer for transcriptional activity, can be made enhancer independent by insertion of SP1 binding sites adjacent to its TATA box. We speculate that (SP1)n-TATA type RNA polymerase II promoters may be generally permissive when present on actively replicating DNA templates and that this property of the HIV-1 promoter may be of importance to the activation of the DNA provirus in latently infected T cells.

Neighborhood revitalization and racial change: the case of Washington, D.C.

Using 1970 and 1980 census block data for Washington, D.C., we test several hypotheses about the racial residential consequences of neighborhood revitalization. Areas located in the revitalizing core of the city have a) become whiter in both absolute and proportional terms, consistent with the displacement hypothesis, and b) experienced substantial though difficult to interpret shifts in segregation. The types of racial changes occurring in the core are not apparent elsewhere in Washington, and tract data for 1940-1980 show the core changes to be temporally as well as spatially specific. Because different racial residential trends have accompanied revitalization in other cities, we treat Washington as an exceptional case that helps specify the conditions under which conventional wisdom about neighborhood change is least applicable.