Biomedical subjects

A B Rossi

Publications and source records attributed to A B Rossi.

8 recordsLinked to original sources

PubMed2000

Optimization of regulated LTR-mediated expression.

Retroviral vectors are ideally suited to the study of gene function, allowing efficient, stable expression. Many biological systems (e.g., cell cycle, apoptosis) require the use of regulated expression systems. We therefore developed a regulated retroviral vector system, TRA99, based on a tetracycline transactivator-dependent LTR, where the MMLV enhancer was replaced with a tetracycline-response element. Using fluorescence-activated flow cytometric analysis of a destabilized green fluorescent protein to monitor expression levels, we optimized the minimal promoter configuration with respect to both activated and repressed transcription. The TRA99 vectors demonstrate regulated expression with activated levels comparable to those of standard retroviral vectors and repressed levels indistinguishable from background. This was achieved without using an internal promoter cassette, thus retaining the cis-packaging elements requisite for helper-mediated transfer.

Animals↗

PubMed2000

Rab37 is a novel mast cell specific GTPase localized to secretory granules.

GTPases regulate a myriad of cellular functions including signal transduction, cytoskeletal organization and membrane trafficking. Rab GTPases act to coordinate the membrane dynamics of cells by organizing and regulating the activity of effector proteins important in vesicle trafficking. Rab37 is a novel Rab GTPase specifically expressed in the MC-9 mast cell line and bone marrow mast cells. Rab37 is 74% identical to Rab26 and 47% identical to Rab8, a GTPase important in Golgi to plasma membrane vesicle trafficking in mammalian cells. When green fluorescent protein tagged Rab37 is expressed in bone marrow mast cells, the secretory granules are labeled. These data suggest that Rab37 may play an important role in mast cell degranulation making this protein a potentially important target for therapeutic intervention in the treatment of allergy.

Amino Acid Sequence↗

PubMed2000

Retroviral delivery of peptide modulators of cellular functions.

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.

Amino Acid Motifs↗

PubMed2000

Cellulite: a review.

Gynoid lipodystrophy (cellulite) is an extremely controversial topic. A lack of knowledge regarding specific aetiopathogenic factors, as well as the opportunism of some professionals and the media, has fuelled debate regarding the scientific basis of this condition. This article reviews the clinical, epidemiological, histopathological and therapeutic aspects of cellulite.

Adipose Tissue↗

PubMed1999

Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay.

BACKGROUND: Mast cells are primary mediators of allergic inflammation. Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) receptors results in degranulation and the release of proinflammatory mediators including histamine, tumor necrosis factor-alpha, and leukotrienes. METHODS: Mast cells were stimulated to degranulate by using either IgE crosslinking or ionophore treatment. Exogenously added annexin-V was used to stain exocytosing granules, and the extent of binding was measured flow cytometrically. Release of the enzyme beta-hexosaminidase was used for population-based measurements of degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were used as controls to validate the annexin-V binding assay. RESULTS: Annexin-V specifically bound to mast cell granules exposed after stimulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing liposomes, consistent with specific binding to this membrane lipid. Visualization of annexin-V staining showed granular cell surface patches that colocalized with the exocytic granule marker VAMP-green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding and beta-hexosaminidase release in RBL-2H3 cells, as did the expression of a dominant negative rab3d mutant protein. CONCLUSIONS: The annexin-V binding assay represents a powerful new flow cytometric method to monitor mast cell degranulation for functional analysis.

Androstadienes↗

PubMed1996

Human bleomycin hydrolase: molecular cloning, sequencing, functional expression, and enzymatic characterization.

We have cloned the cDNA of human bleomycin hydrolase (hBH), a protease which is thought to be involved in the metabolic inactivation of the antineoplastic drug bleomycin. The open reading frame consists of 1365 base pairs and is predicted to encode a 52 kDa protein. The protein shares 40% identity with yeast bleomycin hydrolase and contains the conserved active site residues (Cys, His, Asn) characteristic for cysteine proteases of the papain superfamily. Human bleomycin hydrolase has been functionally expressed in Spodoptera frugiperda Sf9 cells using the Autographa californica nuclear polyhedrosis virus. The 52 kDa recombinant protein forms a hexamer of 310 kDa and acts strictly as an aminopeptidase with a broad substrate specificity. The lack of a leader sequence and its pH optimum at 7.2 suggest a cytosolic/nuclear localization. Human bleomycin hydrolase was detected at low to moderate expression levels in most of the human organs tested. Significantly higher RNA levels have been observed in a variety of tumor cell lines. The human enzyme effectively degrades both forms of bleomycin (A2 and B2) in vitro and could indeed be responsible for the resistance of various tumors to this widely used anticancer drug.

Amino Acid Sequence↗

PubMed1990

Expression of functional mouse muscle acetylcholine receptors in Chinese hamster ovary cells.

CHO cells were transfected with cDNAs for all 4 subunits of the mouse muscle acetylcholine receptor (AChR) to obtain a stable cell line (CHO-AR) expressing the AChR on its surface. Immunoprecipitation experiments established that the AChR contained alpha- and beta-subunits assembled with gamma- and/or delta-subunits. In addition, one of the toxin-binding sites of the AChR was blocked by a myasthenic serum that specifically recognizes AChR containing the gamma-subunit. AChR from the CHO-AR cells had the same sedimentation rate, association rate constant for the binding of alpha-bungarotoxin (alpha BTX), and the same metabolic half-life as the AChR in myotubes of the mouse muscle cell line C2C12. Electrophysiological assay of CHO-AR cells by single-channel recording showed the presence of ACh-responsive ion channels with the characteristics of the embryonic AChR (gamma = 40 pS, tau = 5.6 msec). In some patches a smaller conductance channel was also seen that may represent partially assembled receptor. Fluorescence microscopy of fixed, permeabilized cells stained with rhodamine-alpha-BTX demonstrated both perinuclear and diffuse surface staining. The expression of fully assembled, functional mammalian muscle AChR in nonmuscle cells will allow detailed investigation of its properties and interactions with other cellular components.

Animals↗

PubMedDate not supplied

Use of autologous fibrin glue in dermatologic surgery: application of skin graft and second intention healing.

OBJECTIVE: To evaluate the efficiency of biological sealant, an autologous fibrin glue, in dermatological surgery. DESIGN: Randomized clinical trial. SETTING: The Dermatology Service of Hospital das Clinicas, Universidade de Campinas (UNICAMP), referral center. PATIENTS: 14 patients with malign epithelial cutaneous tumors participated in the evaluation, each having two tumors, generally facial and symmetrical, in order to perform a comparative evaluation on the same individual. PROCEDURES: The glue was prepared beforehand with a sample of autologous blood. Surgical extirpation of the tumor was followed by grafts or second intention healing. OUTCOMES: The efficiency of the sealant was then evaluated in relation to hemostasis, adhesion, surgical time and evolution of the granulation tissue, clinically and histologically. RESULTS: Immediate hemostasis and graft adhesion, with a significant reduction of surgical time, and in the open wounds there was immediate hemostasis and a clinical increase in granulation tissue, but with no histological differences among the groups on the 7th day. CONCLUSION: It is an adjuvant resource in skin cancer surgery.

Adult↗