Monkey Kidney Cell Line Research Articles

Decolourization and Detoxification of Methyl Red by Aerobic Bacteria from A Wastewater Treatment Plant

Bacterial cultures from a wastewater treatment plant degraded a toxic azo dye (methyl red) by decolourization. Complete decolourization using a mixed-culture was achieved at pH 6, 30 °C within 6 h at 5 mg/l methyl red concentration, and 16 h at 20—30 mg/l. Four bacterial species were isolated that were capable of growth on methyl red as the sole carbon source, and two were identified, namely Vibrio logei and Pseudomonas nitroreducens. The Vibrio species showed the highest methyl red degradation activity at the optimum conditions of pH 6--7, and 30—35 °C. Analysis by NMR showed that previously reported degradation products 2-aminobenzoic acid and N,N-dimethyl-1,4-phenylenediamine were not observed. The decolourized dye was not toxic to a monkey kidney cell line (COS-7) at a concentration of 250 μM.

SARS coronavirus: a new challenge for prevention and therapy.

A new and deadly clinical syndrome now called severe acute respiratory syndrome (SARS) was brought to the attention of the WHO by Dr. Carlo Urbani and his colleagues in a Vietnamese hospital in February 2003 (1). The WHO, the medical staffs in hospitals where the disease had appeared, and local and regional governments, together with a dozen cooperating laboratories across the globe, immediately responded. They provided a provisional case definition to identify the extent and geographic distribution of the outbreak (2), laboratory investigations to identify the infectious agent, and travel advisories and quarantines to limit the spread of the disease (3, 4). This extraordinary and effective collaboration limited the potentially explosive spread of the outbreak, while initial case reports with clinical and epidemiological information were quickly posted on the Internet to help physicians identify additional cases of the new syndrome (2, 4–9). The press and scientific journals played valuable roles in rapidly distributing accurate information about SARS to the frightened public and making key scientific publications about SARS available via the Internet before they could appear in print. A stroke of good fortune in this crisis was the discovery that a novel virus could be readily isolated from patients’ lungs and sputum and cultivated in a monkey kidney cell line (8, 10, 11). Laboratory investigations using electron microscopy, virus-discovery microarrays containing conserved nucleotide sequences characteristic of many virus families, randomly primed RT-PCR, and serological tests quickly identified the virus as a new coronavirus (8, 10, 11). Inoculation of monkeys with the SARS-associated coronavirus (SARS-CoV) caused interstitial pneumonia resembling SARS, and the virus was isolated from the nose and throat (12). No viral or bacterial copathogen was needed to induce the disease. These experiments fulfilled Koch’s postulates and proved that SARS-CoV is the cause of SARS.

Emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication

The subgenomic RNA 3 of IBV has been shown to be a tricistronic mRNA, encoding three products in IBV-infected cells. To explore if the least expressed ORF, ORF 3b, which encodes a nonstructural protein, is evolutionarily conserved and functionally indispensable for viral propagation in cultured cells, the Beaudette strain of IBV was propagated in chicken embryonated eggs for three passages and then adapted to a monkey kidney cell line, Vero. The 3b gene of passage 3 in embryonated eggs and passages 7, 15, 20, 25, 30, 35 50, and 65 in Vero cells were amplified by reverse transcription–polymerase chain reaction and sequenced. The results showed that viral RNA extracted from passages 35, 50, and 65 contained a single A insertion in a 6A stretch of the 3b gene between nucleotides 24075 and 24080, whereas the early passages carried the normal 3b gene. This insertion resulted in a frameshift event and therefore, if expressed, a C-terminally truncated protein. We showed that the frameshifting product, cloned in a plasmid, was expressed in vitro and in cells transfected with the mutant construct. The normal product of the 3b gene is 64 amino acids long, whereas the frameshifting product is 34 amino acids long with only 17 homogeneous amino acid residues at the N-terminal half. Immunofluorescent studies revealed that the normal 3b protein was localized to the nucleus and the truncated product showed a “free” distribution pattern, indicating that the C-terminal portion of 3b was responsible for its nuclear localization. Comparison of the complete genome sequences (27.6 kb) of isolates p20c22 and p36c12 (from passages 20 and 36, respectively) revealed that p36c12 contains three amino acid substitutions, two in the 195-kDa protein (encoded by gene 1) and one in the S protein, in addition to the frameshifting 3b product. Further characterization of the two isolates demonstrated that p36c12 showed growth advantage over p20c22 in both Vero cells and chicken embryos and was more virulent in chicken embryos than p20c22. These results suggest that the 3b gene product is not essential for the replication of IBV.

Intracellular localization of the p35 subunit of murine IL-12.

Production of interleukin-12 (IL-12), a heterodimer of p35 and p40 subunits, is limited by p35 expression. A long and a short murine p35 mRNA potentially encoding proteins differing in pre-sequence size are produced. Increased pre-sequence size could convert a cleaved signal peptide to an uncleaved signal peptide, raising the possibility that a membrane-bound form of p35 is produced. The intracellular localization of the p35 encoded by each mRNA isoform was determined by constructing cDNAs containing the long or short p35 cDNA isoform fused in-frame to a cDNA encoding green fluorescent protein (GFP). After transfection of a CV-1 African green monkey kidney cell line with the constructs, confocal microscopy and immunoblotting of extracted microsomal membranes demonstrated that the p35-GFP fusion protein encoded by the long or short mRNA accumulates in the Golgi apparatus as an endoglycosidase H-sensitive glycosylated integral membrane protein. In contrast, a p40-GFP fusion protein accumulates in the Golgi apparatus as a soluble protein. Since assembly of the p35 and p40 subunits to form bioactive IL-12 occurs in the ER, release of membrane-tethered IL-12 by proteolytic cleavage in a late Golgi or post-Golgi compartment may represent an as yet unidentified level at which bioactive IL-12 secretion is regulated.

Embryotrophic factor-3 from human oviductal cells affects the messenger RNA expression of mouse blastocyst.

Our previous results showed that embryotrophic factor-3 (ETF-3) from human oviductal cells increased the size and hatching rate of mouse blastocysts in vitro. The present study investigated the production of ETF-3 by an immortalized human oviductal cell line (OE-E6/E7) and the effects of ETF-3 on the mRNA expression of mouse embryos. The ETF-3 was purified from primary oviductal cell conditioned media using sequential liquid chromatographic systems, and antiserum against ETF-3 was raised. The ETF-3-supplemented Chatot-Ziomek-Bavister medium was used to culture Day 1 MF1 x BALB/c mouse embryos for 4 days. The ETF-3 treatment significantly enhanced the mouse embryo blastulation and hatching rate. The antiserum, at concentrations of 0.03-3%, abolished the embryotrophic effect of ETF-3. Positive ETF-3 immunoreactivity was detected in the primary oviductal cells, OE-E6/E7, and blastocysts derived from ETF-3 treatment. Vero cells (African Green Monkey kidney cell line), fibroblasts, and embryos cultured in control medium did not possess ETF-3 immunoreactivity. The mRNA expression patterns of the treated embryos were studied at the blastocyst stage by mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR). The DDRT-PCR showed that some of the mRNAs were differentially expressed after ETF-3 treatment. Twelve of the differentially expressed mRNAs that had high homology with cDNA sequences in the GenBank were selected for further characterization. The differential expression of seven of these mRNAs (ezrin, heat shock 70-kDa protein, cytochrome c oxidase subunit VIIa-L precursor, proteinase-activated receptor 2, eukaryotic translation initiation factor 2beta, cullin 1, and proliferating cell nuclear antigen) was confirmed by semiquantitative RT-PCR. In conclusion, immortalized oviductal cells produce ETF-3, which influences mRNA expression of mouse blastocyst.

Role of human type 3 3α-hydroxysteroid dehydrogenase (AKR1C2) in androgen metabolism of prostate cancer cells

Four human aldo–keto reductases (AKRs) that belong to the AKR1C subfamily function in vitro as 3-keto-, 17-keto- and 20-ketosteroid reductases or as 3α-, 17β- and 20α- hydroxysteroid oxidases to varying degrees. By acting as ketosteroid reductases or hydroxysteroid oxidases these AKRs can either convert potent sex hormones (androgens, estrogens and progestins) into their inactive metabolites or they can form potent hormones by catalyzing the reverse reaction. In this manner they may regulate occupancy and trans-activation of steroid hormone receptors. Tissue distribution studies previously indicated that AKR1C2 (type 3 3α-hydroxysteroid dehydrogenase (3α-HSD)) and AKR1C3 (type 2 3α-HSD) are highly expressed in human prostate. An assessment of the directionality of these AKR1C isozymes in a cellular environment would help identify which isozymes are responsible for 5α-dihydrotestosterone (5α-DHT) formation or its elimination in the prostate. An imbalance in 5α-DHT levels has been implicated in development of prostate carcinoma and benign prostatic hyperplasia. We focused our attention on AKR1C2 since this is the isoform that will oxidize 3α-androstanediol (3α-diol) to 5α-DHT in vitro, suggesting it could elevate 5α-DHT levels. To determine whether AKR1C2 preferentially functions as a reductase or an oxidase in a cellular context, we transiently transfected AKR1C2 (pcDNA3-AKR1C2) into COS-1 cells and stably transfected pcDNA3-AKR1C2 and pLNCX-AKR1C2 constructs into PC-3 and LNCaP cells, respectively. COS-1 is a monkey kidney cell line, while PC-3 and LNCaP cells are androgen receptor (−) and (+) prostate adenocarcinoma cell lines, respectively. In transient COS-1-AKR1C2 and in stable PC3-AKR1C2 transfectants, AKR1C2 functioned as a 3-ketosteroid reductase inactivating 5α-DHT. In androgen dependent human prostate cancer cells LNCaP, it was not possible to ascertain the preferred direction of AKR1C2 by stable transfection due to the high rate of 5α-DHT and 3α-diol glucuronidation. Based on these findings AKR1C2 may diminish 5α-DHT and prevent this ligand from activating the androgen receptor in situ.

Cytotoxic activity of some lichen extracts on murine and human cancer cell lines

Eight lichens were extracted successively with n-hexane, diethyl ether and methanol using a Soxhlet process. The cytotoxic activity of the 24 lichen extracts was evaluated in vitro using two murine (the L1210: lymphocytic leukaemia, and the 3LL: Lewis lung carcinoma) and four human (the K-562: chronic myelogenous leukaemia, the U251: glioblastoma, the DU145: prostate carcinoma, and the MCF7: breast adenocarcinoma) cancer cell lines and non-cancerous cells, the Vero cell line (African green monkey kidney cell line). The MTT assay revealed significant cytotoxicity (IC50 < or = 20 microg/ml) on one of the tested cancer cell lines for at least one extract of each lichen species. Some extracts of Cladonia convoluta, Cladonia rangiformis, Parmelia caperata, Platismatia glauca and Ramalina cuspidata demonstrated interesting activities particularly on human cancer cell lines as good selectivity indices were recorded (SI > 3).

Induction of marked apoptosis in mammalian cancer cell lines by antisense DNA treatment to abolish expression of DENN (differentially expressed in normal and neoplastic cells).

We previously reported the isolation of the novel human DENN gene, which is differentially expressed in normal and neoplastic cells. DENN is identical to MADD (mitogen-activated protein kinase-activating death domain), which interacts with tumor necrosis factor receptor 1 through their death domains. DENN is also homologous to Rab3 GEP, a rat Rab3 GDP/GTP exchange protein. Real-time reverse transcription-polymerase chain reaction analysis showed that DENN expression in cancer cell lines was 26-50 times that in normal cells. The Jurkat human leukemia, PLC/PRF/5 human hepatoma, and NS-1 mouse myeloma cell lines as well as the MRC-5 human fetal lung and Vero monkey kidney cell lines were treated successfully with four separate DENN-targeted antisense oligodeoxynucleotides (ODNs) to abrogate DENN expression. Quantitative assessment of cell viability and apoptosis by flow cytometry via fluorescein diacetate and propidium iodide membrane-integrity tests, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end-labeling, and annexin V assays showed that antisense silencing of DENN resulted in markedly more pronounced cell death in cancer cells compared with nonmalignant cells. Antisense-treated cell lines exhibited extensive loss of DNA content, forming distinct sub-G(1) peaks, while cell proliferation diminished significantly. Ultrastructural features of programmed cell death in cells subjected to antisense ODNs were authenticated by electron microscopy. In contrast, transfection of cell lines with a plasmid construct to achieve DENN overexpression augmented cellular proliferation and could reverse the apoptotic effect of antisense and staurosporine treatment. Our findings suggest that DENN is intimately involved in anti-apoptotic and cell-survival processes.

Characterization of a monoclonal antibody specific for a novel primate cell surface marker with distinct biochemical properties on human erythroleukemia and myeloid cell lines.

A hybridoma, CSC-1, which secretes monoclonal antibody (MAb) specific for a cell surface molecule on African green monkey kidney cell line, BGMK, was isolated and characterized. The cell surface molecule recognized by CSC-1 is widely expressed on a variety of human cell lines. Among the hematopoietic cell lines examined, the CSC-1 marker seems to be preferentially expressed by lymphoid cell lines (e.g., Raji, CEM-SS, Jurkat, and MOLT-3). Although CSC-1 also recognizes some uncommitted myeloid and erythroleukemia cell lines (e.g., U-937 and K562), the CSC-1 marker expression on these cell lines is subjected to PMA-induced down-regulation. In addition, the PMA-induced cell surface down-regulation of the CSC-1 marker required more than 24 h. Proteolytic analysis shows that the CSC-1 marker can be categorized into proteinase K-resistant and-sensitive phenotypes. Furthermore, the CSC-1 marker exhibits a slow cell surface turnover rate after proteinase K treatment. Our data suggest that the CSC-1 cell surface molecule might be useful as a megakaryocytic or monocytic differentiation marker.

Measles Virus Receptor SLAM (CD150)

Measles virus (MV), a member of the Morbillivirus genus in the Paramyxoviridae family, is an enveloped virus with a nonsegmented negative-strand RNA genome and infects humans and nonhuman primates (Griffin, 2001). Despite the availability of vaccines, MV remains a major cause of childhood mortality, claiming roughly one million lives a year worldwide. The transient immunosuppression that accompanies and follows measles renders the patients susceptible to secondary infections accounting for most of measles-related complications and deaths. MV also causes postinfectious encephalitis, and in rare instances, subacute sclerosing panencephalitis, a persistent infection in the central nervous system. This review is concerned with the identification of a new MV receptor and its implication for understanding the pathology and pathogenesis of MV infection. MV was first isolated in primary human kidney cells inoculated with the blood and throat washings of a child with measles (Enders and Peebles, 1954). This first isolate, the Edmonston strain, was subsequently adapted to chicken embryo fibroblasts and became the progenitor for currently used attenuated vaccines. The Edmonston strain also grows well in continuous cell lines and has become the most extensively studied MV strain in laboratories. Vero cells, an African green monkey kidney cell line, had been commonly used for MV isolation until a decade ago, but several blind passages were usually required before virus propagation and development of cytopathic effect (CPE). Kobune et al. (1990) reported that an Epstein–Barr virus (EBV)-transformed marmoset B cell line B95-8 and its adherent subline B95a were highly sensitive to MV. Furthermore, they showed that MV strains isolated in B95a cells, but not Vero cell-isolated

Cell type-specific replication of simian virus 40 conferred by hormone response elements in the late promoter.

The late genes of SV40 are not expressed at significant levels until after the onset of viral DNA replication. We previously identified two hormone response elements (HREs) in the late promoter that contribute to this delay. Mutants defective in these HREs overexpress late RNA at early, but not late, times after transfection of CV-1PD cells. Overexpression of nuclear receptors (NRs) that recognize these HREs leads to repression of the late promoter in a sequence-specific and titratable manner, resulting in a delay in late gene expression. These observations led to a model in which the late promoter is repressed at early times after infection by NRs, with this repression being relieved by titration of these repressors through simian virus 40 (SV40) genome replication to high copy number. Here, we tested this model in the context of the viral life cycle. SV40 genomes containing mutations in either or both HREs that significantly reduce NR binding without altering the coding of any proteins were constructed. Competition for replication between mutant and wild-type viruses in low-multiplicity coinfections indicated that the +1 HRE offered a significant selective advantage to the virus within a few cycles of infection in African green monkey kidney cell lines CV-1, CV-1P, TC-7, MA-134, and Vero but not in CV-1PD' cells. Interestingly, the +55 HRE offered a selective disadvantage in MA-134 cells but had no effect in CV-1, CV-1P, TC-7, Vero, and CV-1PD' cells. Thus, we conclude that these HREs are biologically important to the virus, but in a cell type-specific manner.

Porcine reproductive and respiratory syndrome virus-induced cell death exhibits features consistent with a nontypical form of apoptosis

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, belongs to a group of RNA viruses that are cytopathic for macrophages and establish persistent infections. Apoptosis is the presumed mechanism of cell death in monkey kidney cell lines and porcine alveolar macrophages after infection with European PRRSV isolates. However, evidence from in vivo and in vitro studies using North American strains have failed to identify apoptosis in cells supporting virus replication and suggest that apoptosis is present in only uninfected bystander cells. The purpose of this study was to evaluate the mechanism of cell death following the infection of MARC-145 cells with wild-type (P6) and a cell culture-adapted (P136) strains derived from the North American isolate SDSU-23983. At 2 days after infection with P136, cytoplasmic blebbing and nuclear condensation were absent in monolayers containing almost 90% infected cells. By day 3, these infected cells detached and showed evidence of apoptosis, including nuclear condensation and inter-nucleosomal DNA fragmentation. Apoptosis in single infected floating cells was confirmed by the co-localization of FITC-anti-digoxigenin antibody, used to detect uridine-digoxigenin-labled nuclear DNA in a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling assay, and Texas red-labeled PRRSV antibody. A majority of infected floating cells were also positive for the uptake of trypan blue, an indicator of necrotic cell death. These results demonstrate that apoptosis does occur in PRRSV infected cells, but is a late event during PRRSV replication and rapidly culminates in a necrotic-like death.

Presence of Cytokines in Biological Preparations

In vaccines produced in eukaryote cells as well as in commercial medical preparations of leukocyte interferon a number of cytokines such as IL-1β, IL-6 and TNF-α have been detected. Among the vaccines examined in this study the highest level of IL-1β was demonstrated in inactivated hepatitis A vaccine prepared in the green monkey kidney cell line 4647, that of IL-6 in inactivated rabies vaccine produced in Syrian hamster kidney (SHK) cell culture, and that of TNF-α in live poliomyelitis vaccine manufactured in VERO cells. A spontaneous and poliovirus-induced capacity of cell cultures to produce cytokines was detected. The level of cytokines produced depend on the kind of cell culture and the type of virus, a more pronounced effect being generated by types 1 and 2 poliovirus as compared with type 3. The presence of highly active cytokines in virus vaccines and interferon preparations points to the necessity of investigating the influence of the presence of cytokines on the biological activity of these preparations and to the advisability of standardizing and controlling the cytokine content.

Molecular cloning and expression of the porcine trigeminal ganglion cDNA encoding a 5-ht 1F receptor

Using a combination of reverse transcription polymerase chain reaction (RT-PCR) and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-hydroxytryptamine 1F (5-ht 1F) receptor complementary DNA (cDNA) derived from porcine trigeminal ganglion. Sequence analysis revealed 1101 base pairs (bp) encoding an open reading frame of 366 amino acids showing a high similarity (>90%) with the 5-ht 1F receptor sequences from other species, including human. The recombinant porcine 5-ht 1F receptor was expressed in African green monkey kidney cell lines (COS-7 cells) and its ligand binding profile was determined using [ 3H]5-HT. The affinities of several agonists (LY334370 (5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate)>CP122638 ( N-methyl-3 [pyrrolidin 2( R)-yl methyl]-1H-indol-5-ylmethyl sulphonamide)=naratriptan=5-HT>eletriptan>sumatriptan>frovatriptan=avitriptan>dihydroergotamine>zolmitriptan>5-carboxamidotryptamine>rizatriptan>alniditan=donitriptan>L694247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine) and putative antagonists (methiothepin>GR127935 ( N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2′-methyl 4′-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide hydrochloride)>ritanserin>SB224289 (2,3,6,7-tetrahydro-1′-methyl-5-[2′-methyl-4′(5-methyl-1,2,4-oxadiazol-3-yl) biphenyl-4-carbonyl] furo [2,3-f] indole-3-spiro-4′-piperidine hydrochloride)>BRL155572 ([1-(3-chlorophenyl)-4-[3,3-diphenyl (2-( S, R) hydroxypropanyl)piperazine] hydrochloride)>ketanserin=pindolol) correlated highly with those described for the recombinant human 5-ht 1F receptor (Spearman correlation coefficient; r s=0.942). Nevertheless, as compared to the human homologue, some triptans (i.e. sumatriptan, zolmitriptan and rizatriptan) displayed a 10- to 15-fold lower affinity for the porcine 5-ht 1F receptor. Using RT-PCR technique, the expression of porcine 5-ht 1F receptor mRNA was observed in cerebral cortex, trigeminal ganglion and several blood vessels, but not in skeletal muscles. In conclusion, we have cloned and established the amino acid sequence and ligand binding profile of the porcine 5-ht 1F receptor as well as the distribution of its mRNA. This information may be helpful in exploring the role of 5-ht 1F receptor in physiological processes and diseases, such as migraine.

The Histone Acetyltransferase Domains of CREB-binding Protein (CBP) and p300/CBP-associated Factor Are Not Necessary for Cooperativity with the Class II Transactivator

The class II transactivator (CIITA) is a transcriptional co-activator regulating the constitutive and interferon-gamma-inducible expression of class II major histocompatibility complex (MHC) and related genes. Promoter remodeling occurs following CIITA induction, suggesting the involvement of chromatin remodeling factors. Transcription of numerous genes requires the histone acetyltransferase (HAT) activities of CREB-binding protein (CBP), p300, and/or p300/CBP-associated factor (pCAF). These co-activators cooperate with CIITA and are hypothesized to promote class II major histocompatibility complex transcription through their HAT activity. To directly test this, we used HAT-defective CBP and pCAF. We demonstrate that cooperation between CIITA and CBP is independent of CBP HAT activity. Further, although pCAF enhances CIITA-mediated transcription, pCAF HAT domain dependence appears contingent upon the concentration of available CIITA. When HAT-defective CBP and pCAF are both present, cooperativity with CIITA is maintained. Consistent with a recent report, we show that nuclear localization of CIITA is enhanced by lysine 144, an in vitro target of pCAF-mediated HAT. Yet we find that neither mutation of lysine 144 nor deletion of residues 132-209 affects transcriptional cooperation with CBP or pCAF. Thus, acetylation of this residue may not be the primary mechanism for pCAF/CBP cooperation with CIITA. In conclusion, the HAT activities of the co-activators are not necessary for cooperation with CIITA.

βIII Spectrin Binds to the Arp1 Subunit of Dynactin

Cytoplasmic dynein is an intracellular motor responsible for endoplasmic reticulum-to-Golgi vesicle trafficking and retrograde axonal transport. The accessory protein dynactin has been proposed to mediate the association of dynein with vesicular cargo. Dynactin contains a 37-nm filament made up of the actin-related protein, Arp1, which may interact with a vesicle-associated spectrin network. Here, we demonstrate that Arp1 binds directly to the Golgi-associated betaIII spectrin isoform. We identify two Arp1-binding sites in betaIII spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to betaIII spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and betaIII spectrin co-purify on vesicles isolated from rat brain, and betaIII spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, betaIII spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, betaIII spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for betaIII spectrin. We hypothesize that the interaction between betaIII spectrin and Arp1 recruits dynein and dynactin to intracellular membranes and provides a direct link between the microtubule motor complex and its membrane-bounded cargo.

A Critical Role for Phosphoinositide 3-Kinase Upstream of Gab1 and SHP2 in the Activation of Ras and Mitogen-activated Protein Kinases by Epidermal Growth Factor

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.

Effects of Cyclosporin A on Cell Fusion in a Monkey Kidney Cell Line Persistently Infected with Measles Virus

The authors studied the effects of immunosuppressive peptide cyclosporin A (CsA) on cell fusion efficiency in cells persistently infected with measles virus (448-PI-Vero cells). Treatment of 448-PI-Vero cells with 5 µM CsA enhanced the infusion. In addition, the expression of measles virus antigen on cell surface was increased by treatment with CsA. The addition of phenothiazine, an anti-calmodulin drug, enhanced the fusion of 448-PI-Vero cells in the presence of CsA, although treatment with phenothiazine alone did not affect polykaryocyte formation. The enhancement of fusion efficiency in 448-PI-Vero cells by CsA was suppressed by oligopeptide Z-D-Phe-Phe-Gly, a synthetic oligopeptide that inhibits fusion induced by measles virus. Since the cell content of major virus-specific polypeptides, such as hemagglutinin, nucleoprotein or matrix protein is the same as in untreated controls, this fusion enhancement may be related to transport and accumulation of measles virus glycoproteins.

Synthesis, Structure, and Antitumor Activity of a Novel Tetranuclear Titanium Complex

The coordination complex cyclo-tetrakis[bis(1-phenyl-3-methyl-4-benzoylpyrazolon-5-ato++ +)mu-o xotitanium(IV)] has been synthesized and characterized with IR and NMR spectroscopies and X-ray diffraction. The core of this species consists of an eight-membered Ti-mu-oxo ring with alternate short-long Ti-O bond lengths. Besides these two O ligands, each metal is bound octahedrally to four O atoms from two chelating 1-phenyl-3-methyl-4-benzoylpyrazolon-5-ato anions. Several sets of Ti-O bond lengths are present: the shortest are the two Ti-O(oxo) (which are cis to each other), the longest are the two Ti-O(acyl) (cis to each other), and the two Ti-O(pyrazolonato) (trans to each other) are intermediate. The beta-diketonate ligand asymmetry, a feature considered essential in other antitumor Ti compounds, induces the short-long Ti-O(oxo) sequence of bond lengths. The antitumor activity of this compound, encapsulated in a dipalmitoylphosphatidylcholine liposome, has been studied in vitro using TA-3 (mouse mammary adenocarcinoma), HEP-2 (human epithelial larynx carcinoma), and VERO (African green monkey kidney) cell lines and in vivo in CF-1 and AJ female mice ip inoculated with TA-3. In vitro cytotoxicity is greater for TA-3 than for HEP-2 and null for VERO cell lines. In vivo results show a marked increase in survival time (T/C = 293% for AJ and 208% for CF-1), whereas tumor weight decrease was observed for CF-1-treated mice. These results suggest the Ti complex-liposome system may be promising as an antitumor drug.

Preliminary characterization of protein binding factor for porcine reproductive and respiratory syndrome virus on the surface of permissive and non-permissive cells.

Summary. In its natural host, porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to have a restricted tropism for cells of the monocyte/macrophage lineage. To date, cloned monkey kidney cell lines, such as MARC-145 and CL2621 cells which have been established from MA-104 cells, are the only non-porcine cells known to support PRRSV replication. In the present study, a binding assay was set up to follow by flow cytometry the attachment of PRRSV on the surface of porcine and non-porcine cells. PRRSV was found to be able to bind permissive cells like porcine alveolar macrophages and MARC-145. Further binding assays with porcine peripheral blood leukocytes showed that only monocytes can attach the virus. By their lack of binding factor, lymphocytes appeared to be refractory to PRRSV infection. Pre-incubation of MARC-145 cells with chymotrypsin and pronase E, but not neuraminidase, blocked their binding activity for PRRSV. The binding activity of the protease-treated cells was regenerated 8 hours after treatment, but cells remained unable to bind PRRSV if maintained in the presence of cycloheximide, thus confirming the proteinic nature of the specific binding factor(s). Experiments conduced with cells that have been previously characterized as non-permissive to PRRSV infection showed that many of them were able to bind the virus. Data obtained suggest that interaction of PRRSV with a specific binding factor on the surface of some cells is not sufficient to lead to a productive infection, and that a second putative receptor or other phenomena are probably required to pursue later events.