Cellular Distribution of G Protein Goa in Pituitary Lactotrophs: Effects of Dopamine

Membrane-bound GTP-binding (G) proteins mediate signal transduction in a variety of cell systems. The exact mechanisms of G proteins action are still under investigation but they appear to involve effectors located in the plasma membrane as well as in other parts of the cell. With this study, we investigated the cellular and ultrastructural localization of G protein subunits, and particularly of Goa, in normal rat anterior pituitaries and in estrone-induced rat adenomatous lactotrophs. We also evaluated the effects of Goα cellular redistribution in rat adenomatous lactotrophs following short-term exposure to dopamine (DA). Using the Protein A-gold (PAG) methodology, Goα was found to be present in the cysternae of the endoplasmic reticulum of normal pituitary cells and of adenomatous lactotrophs. In the latter, Goα could be co-localized with prolactin (PRL). By immunoblots, using specific antisera, significant amounts of Goα and Gs42α, together with smaller amounts of Giα, Gs47α and Gβ were found to be present in the uncontaminated supernatant fraction of adenomatous lactotrophs. Unexpectedly, exposure of the cells to DA induced a rapid and short-lived decrease in the cytosolic fraction of Goα and Gβ associated with a decrease of PRL release. Since cytosolic Goα can be ADP-ribosylated by pertussis toxin (PT) and is therefore in a heterotrimeric form, our data suggest that the soluble Go protein may play a role during lactotrophs' exposure to an inhibitor of PRL release, perhaps through its relocalization after being internalized with the D₂ receptor or by being used for interaction with intracellular and/or membrane-bound effectors.     

Metabolism of methyl-branched iodo palmitic acids in cultured hepatocytes

The metabolic fate of methyl-branched iodo fatty acids was studied in primary culture of rat hepatocytes. We compared 16-iodo-2-R,S-methyl palmitic acid (2-Me), which can be oxidized, with 16-iodo-3-R,S-methyl palmitic acid (3-Me) which can be oxidized only after an initial oxydation and with 16-iodo-2,2-dimethyl palmitic acid (2,2-Me₂) and 16-iodo-3,3-dimethyl palmitic acid (3,3-Me₂) which cannot be oxidized at all. The normal fate of natural fatty acids was given by comparative experiments with [1-¹⁴C] palmitic acid. Monomethyl-branched iodo fatty acids were taken up in the same range as palmitic acid but more than dimethyl-branched iodo fatty acids. After a 15-h incubation, acido-soluble products (ASP) accounted for 75% of the radioactivity taken up as 16-iodo-2-methyl palmitic acid, 50% as other methyl-branched iodo fatty acids and only 30% as palmitic acid, which indicated that all the methyl-branched iodo fatty acids underwent a strong deiodination process. Fatty acids were esterified in the following order: palmitic acid >16-iodo-3-R,S-methyl palmitic acid>16-iodo-2-R,S-methyl palmitic acid>16-iodo-2,2-dimethyl palmitic acid>16-iodo-3,3-dimethyl palmitic acid. Cultured hepatocytes, labelled for 3 h with the various fatty acids and reincubated for 12 h without fatty acid, secreted large amounts of free dimethylbranched iodo fatty acids as compared to the monomethyl ones and palmitic acid. Only hepatocytes prelabelled with 16-[¹²⁵I]iodo-2,2-dimethyl palmitic acid exhibited an appreciable secretion of labeled triglycerides, but at a lower rate than with [1-¹⁴C] palmitic acid. Conversely, the 16-iodo-monomethyl palmitic acids remained chiefly in hepatocyte triglycerides. Minute amounts of 16-iodo-methyl-branched-palmitic acids were found in hepatocyte or secred phospholipids as compared with palmitic acid. This metabolic fate of methyl-branched iodo palmitic acids argues against their utilization as imaging probes to monitor in vivo the synthesis and the secretion of triglycerides by the liver.     

Noninfectious Virus-Like Particle Antigen for Detection of Swine Vesicular Disease Virus Antibodies in Pigs by Enzyme-Linked Immunosorbent Assay

An inactivated SVDV antigen is used in current enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies to swine vesicular disease virus (SVDV). To develop a noninfectious recombinant alternative, we produced SVDV-like particles (VLPs) morphologically and antigenically resembling authentic SVDV particles by using a dual baculovirus recombinant, which expresses simultaneously the P1 and 3CD protein genes of SVDV under different promoters. Antigenic differences between recombinant VLPs and SVDV particles were not statistically significant in results obtained with a 5B7-ELISA kit, indicating that the VLPs could be used in the place of SVDV antigen in ELISA kits. We developed a blocking ELISA using the VLPs and SVDV-specific neutralizing monoclonal antibody 3H10 (VLP-ELISA) for detection of SVDV serum antibodies in pigs. The VLP-ELISA showed a high specificity of 99.9% when tested with pig sera that are negative for SVDV neutralization (n = 1,041). When tested using sera (n = 186) collected periodically from pigs (n = 19) with experimental infection with each of three different strains of SVDV, the VLP-ELISA detected SVDV serum antibodies as early as 3 days postinfection and continued to detect the antibodies from all infected pigs until termination of the experiments (up to 121 days postinfection). This test performance was similar to that of the gold standard virus neutralization test and indicates that the VLP-ELISA is a highly specific and sensitive method for the detection of SVDV serum antibodies in pigs. This is the first report of the production and diagnostic application of recombinant VLPs of SVDV. Further potential uses of the VLPs are discussed.     

Selection by phage display of llama conventional V(H) fragments with heavy chain antibody V(H)H properties

A llama single domain antibody (dAb) library designed and constructed to contain only heavy chain antibody variable domains (V(H)Hs) also contained a substantial number of typical conventional antibody heavy chain variable sequences (V(H)s). Panning the library against two carbohydrate-specific antibodies yielded anti-idiotypic dAbs and enriched solely for sequences from the V(H) subpopulation of the library. The conventional antibody origin of these V(H)s was confirmed by using oligonucleotide probes, specific for the enriched V(H)s, to identify the parental sequences in the message employed in library construction. Surprisingly, these V(H) dAbs, which are produced in high yield in Escherichia coli, are highly soluble, have excellent temperature stability profiles and do not display any aggregation tendencies. The very close similarity of these molecules to human V(H)s makes them potentially very useful as therapeutic dAbs.     

Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins

We examined by immunofluorescence the distribution of vimentin, desmin, alpha-smooth muscle actin and alpha-sarcomeric actin in normal human soft tissues and in pathologic tissues containing myofibroblasts, including normally healing granulation tissue, hypertrophic scar, and fibromatosis. The pattern of actin isoforms was also documented biochemically by two-dimensional gel electrophoresis. Fibroblastic and/or myofibroblastic cells in each setting always expressed vimentin and never alpha-sarcomeric actin. Moreover, these cells showed an heterogeneous cytoskeletal composition which defined four phenotypes: (a) cells expressing only vimentin; (b) cells expressing vimentin, alpha-smooth muscle actin and desmin; (c) cells expressing vimentin and alpha-smooth muscle actin; and (d) cells expressing vimentin and desmin. Given this, two groups of lesions are distinguished: the first contains only vimentin cells and consists of normally healing granulation tissue, eschars and normally healed scars; the second contains vimentin cells admixed with variable proportions of vimentin, alpha-smooth muscle actin and desmin, vimentin and alpha-smooth muscle actin, and vimentin and desmin cells and consists of hypertrophic scars and fibromatoses. Immunogold electron microscopy showed that alpha-smooth muscle actin was present in a proportion of cells with ultrastructural features of myofibroblasts. Our findings suggest that contrary to myofibroblasts of normally healing granulation tissue and normally healed scars, myofibroblasts of pathologic conditions characterized by chronic retraction express always immunochemical features indicative of smooth muscle differentiation.     

Does malignant fibrous histiocytoma exist?

Malignant fibrous histiocytoma (MFH) has come to be regarded as the most common malignant neoplasm of the mesenchymal soft tissues. It designates a spectrum of tumors which share morphologic features that allow their inclusion in a distinct clinicopathologic setting, although being not uniform in their histogenesis and pathogenesis. Clinicopathologic variants include the following: the storiform-pleomorphic form of MFH, the myxoid type of MFH, the giant cell type of MFH and the inflammatory type. The latter group, the angiomatoid variant, has been reclassified within the fibrohistiocytic tumors of low malignant potential. Tissue culture, ultrastructural and immunohistochemical studies have both endorsed or refuted the validity of the concept. As a whole, these morphologic studies which attempted to characterize MFH were not able to delineate specific markers or to describe the phenotype of this sarcoma of supposed fibrohistiocytic lineage. There is growing evidence that MFH is a second component in another sarcoma and represents a morphologic modulation resulting from tumor progression. Recent cytogenetic and molecular genetic investigations are consistent with that hypothesis: a comparative analysis between the most frequent genomic imbalances observed in series of MFH and leiomyosarcomas (LMS) demonstrated that both tumors had similar recurrent imbalances. Immunohistochemical and molecular biologic investigations have shown similar targets of chromosome deletions in both tumors. A new classification of soft tissue sarcoma based on molecular parameters is nevertheless premature. The morphologic characterization of MFH and its sub-types provides the clinician with unique information in the management of these tumors, by identifying a spectrum of tumors with well-recognized clinical profiles.     

Fine-needle sampling of primary neuroendocrine carcinomas of salivary glands: cytohistological correlations and clinical analysis

Fine-needle samplings of nine examples of primary neuroendocrine carcinoma of salivary glands were evaluated for their cytologic characteristics and were correlated with the corresponding histological sections. Consistent cytological findings were dispersed or loose clusters of poorly differentiated small- to intermediate-sized cells and occasional smudged nuclei. Mild to moderate nuclear pleomorphism, scant or absent cytoplasm, and nuclear molding were also observed. Rosette-like patterns and multinucleated cells were occasionally seen. Immunostaining of one recent case showed positivity for chromogranin and keratin. The differential diagnosis of primary and metastatic tumors with neuroendocrine features of the salivary glands is discussed.     

Morphological and quantitative study of spirochetes in the feces of normal and infected SPF pigs during the incubation period of swine dysentery.

Spirochetes found in feces of normal and infected pigs during the eight days preceding the onset of the clinical signs of swine dysentery have been studied using electron and phase contrast microscopy. According to their dimensions, diameter, length and pitch, three morphological groups have been described: small, intermediate and large spirochetes. On the basis of their axial filaments arrangement, eight types have been observed. Compared to the control pigs, there was no increase in the total number of spirochetes in infected pigs, except at day 6 before the onset of clinical signs. However, infected pigs have shown a wide spectrum of different morphological types of spirochetes in comparison to control, uninfected pigs, which showed only the type "1-2-1" spirochete of axial filaments arrangement. This last type was predominant in infected swine during the entire incubation period as compared to other spirochete types described.     

Compliance with consensus recommendations for systemic therapy is associated with improved survival of women with node-negative breast cancer.

PURPOSE: The impact of consensus recommendations for systemic therapy on outcome of disease is unclear. We evaluated if compliance with guidelines for systemic adjuvant treatment is associated with improved survival of women with node-negative breast cancer. PATIENTS AND METHODS: The study population included women diagnosed with invasive node-negative breast cancer in Québec, Canada, in 1988 to 1989, 1991 to 1992, and 1993 to 1994. Information was collected by chart review, linkage with administrative databases, and queries to attending physicians. Guidelines from the 1992 St Gallen conference were used as standard of care. Survival was estimated by Kaplan-Meier and Cox proportional hazards analyses. RESULTS: Among 1,541 women, 358 died before December 1999. Median follow-up was 6.8 years. Seven-year event-free and overall survivals were 66% and 81%, respectively. Survival was 88%, 84%, and 74% in women at minimal, moderate, or high risk of recurrence. Virtually all women at minimal risk were treated according to the consensus (98.4% of 370). In comparison, adjusted hazard ratios of death were 1.0 (95% CI, 0.6 to 1.7) and 2.3 (95% CI, 1.3 to 4.0) among women at moderate risk treated according to the consensus or not, respectively. Among women at high risk, adjusted hazard ratios of death were 2.0 (95% CI, 1.4 to 2.8) and 2.7 (95% CI, 1.9 to 3.9), respectively. Both risk category (P <.0005) and compliance with guidelines (P <.0005) were independent significant predictors of survival. CONCLUSION: Treatment according to consensus recommendations is associated with improved survival of women with breast cancer in the community. Promoting the adoption of guidelines for treatment is an effective strategy for disease control.