Mevalonate dependency of the early cell cycle mitogenic response to epidermal growth factor and prostaglandin F2α in Swiss mouse 3T3 cells

Lovastatin (LOV), a hydroxy-methylglutaryl-coenzyme A (HMGCoA) reductase competitive inhibitor, blocks epidermal growth factor (EGF)— or prostaglandin F_2α (PGF_2α)—induced mitogenesis in confluent resting Swiss 3T3 cells. This inhibition occurs even in the presence of insulin, which potentiates the action of these mitogens in such cells. LOV exerts its effect in a 2–80 μM concentration range, with both mitogens attaining 50% inhibition at 7.5 μM. LOV exerted its effect within 0–8 h following mitogenic induction. Mevanolactone (10–80 μM) in the presence of LOV could reverse LOV inhibition within a similar time period. LOV-induced blockage of PGF_2α response is reflected in a decrease in the rate of cell entry into S phase. Neither cholesterol, ubiquinone, nor dolichols of various lengths could revert LOV blockage. In EGF- or PGF_2α-stimulated cells, LOV did not inhibit [³H]leucine or [³H]mannose incorporation into proteins, while tunicamycin, an inhibitor of N′ glycosylation, prevented this last phenomenon. Thus, it appears that LOV exerts its action neither by inhibiting unspecific protein synthesis nor by impairing the N′ glycosylation process. These findings strongly suggest that either EGF or PGF_2α stimulations generate early cell cycle signals which induce mevalonate formation, N′ glycoprotein synthesis, and proliferation. The causal relationship of these events to various mechanisms controlling the onset of DNA synthesis is also discussed. © 1995 Wiley-Liss, Inc.     

Functional properties of FC-2.15, a monoclonal antibody that mediates human complement cytotoxicity against breast cancer cells

FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15⁺) cells were investigated. A⁵¹Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15⁺ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1–50 g/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8±2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15^–. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with¹²⁵I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9×10⁷ M^–1 and 2.8×10⁶ antigenic sites/cell.¹²⁵I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15⁺ cells.     

Partial characterization of the cohemolytic factor produced by Streptococcus uberis and comparison with the CAMP-factor

Abstract Exosubstances (cohemolysins) produced by Streptococcus agalactiae (CAMP-factor) and Streptococcus uberis (Uberis-factor) showing hemolytic synergism with β-lysin produced by Staphylococcus aureus were compared. Cohemolytic activity was evaluated in the supernatants of bacterial cultures, before and after ammonium sulfate precipitation. Sheep erythrocytes sensitized with β-lysin were used as substrate. The assays were performed in microtiter plates and results were expressed as cohemolytic units/ml. Maximum cohemolytic activity was detected, respectively, after 8 h and 14 h of growth in Columbia broth in S. uberis and S. agalactiae cultures. Cohemolytic activities of both microorganisms showed similarities when submitted to various physical and chemical treatments. They were significantly decreased by heating at 60°C and 100°C, or in presence of trypsin, and were abolished in the presence of Tween 20. Activities were found to be stable in crude supernatants and concentrated preparations maintained at −20°C for 3 months. Differences were related to levels of activity and kinetics of detection during the growth cycle. The results indicate the proteic nature, at least in part, of the Uberis factor. Analysis by PAGE in the presence or absence of SDS allowed us to correlate Uberis activity with a protein band with apparent molecular mass of 42 kDa, while CAMP activity was associated with a protein band of 27 kDa.     

Group-specific antigen shared by the members of the reticuloendotheliosis virus complex.

The polypeptides of reticuloendotheliosis virus (REV) were separated by gel filtration in the presence of guanidine hydrochloride. The eight peaks obtained by gel filtration were then analyzed by polyacrylamide gel electrophoresis and four appeared to contain single polypeptides. The material identified as p29 was used to prepare antiserum. This protein constitutes the major internal non-glycosylated polypeptide in the virion. Double immunodiffusion indicated that the antiserum was specific for p29. Using this antiserum, cross-reactivity was demonstrated between REV, chick syncytial virus, duck infectious anemia virus, and spleen necrosis virus. Antiserum to p29 failed to cross-react with Rous sarcoma virus. This indicates that p29 is a group-specific antigen shared by the viruses of the REV complex. A microcomplement fixation test was developed with this antiserum that will be useful in the quantitation of REV and the identification of other members of this newly defined group.     

Distinct functions of maternal and somatic Pat1 protein paralogs

We previously identified Xenopus Pat1a (P100) as a member of the maternal CPEB RNP complex, whose components resemble those of P-(rocessing) bodies, and which is implicated in translational control in Xenopus oocytes. Database searches have identified Pat1a proteins in other vertebrates, as well as paralogous Pat1b proteins. Here we characterize Pat1 proteins, which have no readily discernable sequence features, in Xenopus oocytes, eggs, and early embryos and in human tissue culture cells. xPat1a and 1b have essentially mutually exclusive expression patterns in oogenesis and embryogenesis. xPat1a is degraded during meiotic maturation, via PEST-like regions, while xPat1b mRNA is translationally activated at GVBD by cytoplasmic polyadenylation. Pat1 proteins bind RNA in vitro, via a central domain, with a preference for G-rich sequences, including the NRAS 5′ UTR G-quadruplex-forming sequence. When tethered to reporter mRNA, both Pat proteins repress translation in oocytes. Indeed, both epitope-tagged proteins interact with the same components of the CPEB RNP complex, including CPEB, Xp54, eIF4E1b, Rap55B, and ePAB. However, examining endogenous protein interactions, we find that in oocytes only xPat1a is a bona fide component of the CPEB RNP, and that xPat1b resides in a separate large complex. In tissue culture cells, hPat1b localizes to P-bodies, while mPat1a-GFP is either found weakly in P-bodies or disperses P-bodies in a dominant-negative fashion. Altogether we conclude that Pat1a and Pat1b proteins have distinct functions, mediated in separate complexes. Pat1a is a translational repressor in oocytes in a CPEB-containing complex, and Pat1b is a component of P-bodies in somatic cells.     

A Chemical Proteomics Approach for the Search of Pharmacological Targets of the Antimalarial Clinical Candidate Albitiazolium in Plasmodium falciparum Using Photocrosslinking and Click Chemistry

Plasmodium falciparum is responsible for severe malaria which is one of the most prevalent and deadly infectious diseases in the world. The antimalarial therapeutic arsenal is hampered by the onset of resistance to all known pharmacological classes of compounds, so new drugs with novel mechanisms of action are critically needed. Albitiazolium is a clinical antimalarial candidate from a series of choline analogs designed to inhibit plasmodial phospholipid metabolism. Here we developed an original chemical proteomic approach to identify parasite proteins targeted by albitiazolium during their native interaction in living parasites. We designed a bifunctional albitiazolium-derived compound (photoactivable and clickable) to covalently crosslink drug–interacting parasite proteins in situ followed by their isolation via click chemistry reactions. Mass spectrometry analysis of drug–interacting proteins and subsequent clustering on gene ontology terms revealed parasite proteins involved in lipid metabolic activities and, interestingly, also in lipid binding, transport, and vesicular transport functions. In accordance with this, the albitiazolium-derivative was localized in the endoplasmic reticulum and trans-Golgi network of P. falciparum. Importantly, during competitive assays with albitiazolium, the binding of choline/ethanolamine phosphotransferase (the enzyme involved in the last step of phosphatidylcholine synthesis) was substantially displaced, thus confirming the efficiency of this strategy for searching albitiazolium targets.     

Drought tolerance increases with seed size in a semiarid grassland from southern Mexico

Plant Ecology - Due to increased reserves available to face adverse conditions, drought tolerance should increase with seed size. This has been confirmed in the laboratory, but under field...     

Correction to: Application of a multiphase microreactor chemostat for the determination of reaction kinetics of Staphylococcus carnosus

Bioprocess and Biosystems Engineering - Unfortunately, in the “How to Cite as” section, the given and the family name of the author was incorrectly published, the correct name is...