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PubMed1983

Mast-cell precursors in various haematopoietic colonies of mice produced in vivo and in vitro.

We have examined mast-cell precursors in various in vivo and in vitro haematopoietic colonies of mice. Cells from haematopoietic colonies of (WB x C57BL/6)F1- + +/+ mouse origin were injected into the skin of the congeneic W/Wv mice which are genetically depleted of tissue mast cells. The appearance of mast-cell clusters at the injection site indicated the presence of mast-cell precursors in the injected cell suspension. More than 40% of 12 d exogenous spleen colonies and 14 d in vitro mixed colonies contained mast-cell precursors, but only a small proportion of 7 d exogenous spleen colonies, 9 d in vitro erythroid bursts or 14 d large in vitro neutrophil-macrophage colonies contained mast-cell precursors. Neither transient endogenous erythroid spleen colonies examined at the fifth day nor 7 d in vitro neutrophil-macrophage colonies contained mast-cell precursors. Among 12 d spleen colonies mast-cell precursors were more frequent in the predominantly neutrophil than the predominantly erythroid colonies but 14 d in vitro mixed colonies, in which erythroid cells were predominant, contained mast-cell precursors more frequently than 14 d in vitro neutrophil-macrophage colonies. Therefore, differentiation of mast cells seems to be independent of either the neutrophil-macrophage or erythroid cell lineages.

Animals↗
PubMed2007

Progression of astrocytomas and meningiomas: an evaluation in vitro.

OBJECTIVES/BACKGROUND: In biological terms, progression means that malignancy increases as genetic mutations accumulate leading to increased proliferation and invasion capacity. By verifying the proliferation capacity, human telomerase reverse transcriptase (hTERT) expression and in vitro invasion, in a group of highly malignant glioblastomas, benign meningiomas and astrocytomas, at the initial stage of progression, we have analysed putative progression in vitro for proliferation and telomerase expression. MATERIALS AND METHODS: The relative proliferation status (visualized with Ki-67 antibodies) and presence of hTERT protein was analysed in 27 intracranial tumours (6 astrocytomas, 8 glioblastomas and 13 meningiomas) by immunohistochemistry on paraffin-embedded biopsy tissue, as well as on primary tumour-derived cell cultures. A confrontation model was used to analyse invasiveness in vitro. RESULTS: The mean proliferation indices were 22.3 (SD = 18.1) for glioblastomas and 2.1 (SD = 1.9) for low-grade (LG) astrocytomas. The group of benign meningiomas had a labelling index of 2.2 (SD = 2.7). Mean percentages of staining for hTERT varied between 36.5 (SD = 28.4) for glioblastomas and 10.2 (SD = 8.6) for LG astrocytomas. The group of benign meningiomas had a labelling index of 12.4 (SD = 19.2) for hTERT. A significant difference was seen for Ki-67 (P < 0.05) and hTERT (P < 0.001) in vivo versus in vitro. No difference was seen between the group of invasive and non-invasive tumour-derived cell cultures for the histopathological markers Ki-67 and hTERT (P > 0.05) in vitro. CONCLUSIONS: The elevated expression of hTERT and Ki-67 in vitro provides a potential prognostic tool for early detection of the progression of brain tumours. As tumour cells require telomerase for continued proliferation, the expression of hTERT may mark immortality, leading to indefinite life span. On the other hand, hTERT expression and cell proliferation are not linked directly to invasion in vitro.

Adolescent↗
PubMed2006

Adjuvant intravesical therapy based on an in vitro cytotoxicity assay in the management of superficial transitional cell cancer of the urinary bladder.

OBJECTIVE: To investigate the utility of an individualized chemo/immunotherapy regimen of intravesical therapy based on the results of an assessment of in vitro cytotoxicity. PATIENTS AND METHODS: Intravesical adjuvant chemo/immunotherapy was given to 47 patients based on the results of in vitro cytotoxicity assay of the responses of cultured autologous tumour cells to various cytotoxic drugs (mitomycin-C, doxorubicin and cisplatin) and immunomodulating agents (bacillus Calmette-Guérin, BCG and interferon-alpha2b). Intravesical therapy was given as single- or double-drug regimens according to the assay results: 16 (34%) patients showed cytotoxicity to a single drug and 31 (66%) showed maximum cytotoxicity to a combination of immunomodulators and cytotoxic agents. The efficacy of treatment in terms of tumour-free survival and recurrence rate was compared with 40 patients receiving intravesical BCG according to International Protocol (control group). RESULTS: In the in vitro assay group, seven patients (15%) had tumour recurrence, compared to 15 (38%) in the control group (P = 0.02). In the in vitro group, one of 16 patients on a single drug and six on the double-drug regimen had a recurrence. The patients given BCG with cytotoxic drugs had no recurrences, but 29% of patients given interferon-alpha2b combinations had recurrences. Kaplan-Meier analysis showed a longer recurrence-free survival in the in vitro group (75%) than in the control group (49%) at 48 months of follow-up. CONCLUSION: Intravesical therapy based on an in vitro cytotoxicity assay is an attempt to give individualized therapy, and to increase tumour-free survival in these patients, with no side-effects. Recurrences in seven patients in the in vitro group might be due to a defective host immune response, or to expansion of a subclone of tumour cells resistant to all treatment.

Adjuvants, Immunologic↗
PubMed1989

Comparative effects of the alpha-adrenoceptor agonists noradrenaline, phenylephrine and clonidine in the human saphenous vein in vivo and in vitro.

The alpha-adrenoceptor-mediated contractile effects of noradrenaline (alpha 1 + alpha 2), phenylephrine (alpha 1) and clonidine (alpha 2) on human saphenous veins were investigated in vivo and in vitro. By infusion (0.3 ml min-1) of the drugs (increasing concentrations in the infused solution) into distended (40 mmHg) saphenous veins in six healthy subjects, local vasoconstriction was induced, measured by a photo-electric device. The drugs induced dose-dependent contractions in all subjects, and dose-response curves were constructed. These were compared with concentration-response curves based on in-vitro results. Macroscopically normal saphenous veins, taken at saphenousectomies, were used, and the preparations were contracted isometrically in organ baths by the agonists. From the curves obtained in vivo and in vitro, the relative potencies of phenylephrine and clonidine (in relation to noradrenaline) were calculated and compared. The relative potencies of phenylephrine in vivo (76%) and in vitro (82%) did not differ significantly. However, the relative potency of clonidine was significantly (P less than 0.05) lower in vivo (90%) than in vitro (99%). Thus, it is concluded that there are differences between the results obtained in vivo and in vitro, stressing the importance of comparative in vivo-in vitro studies.

Adrenergic alpha-Agonists↗
PubMed2004

Comparison of fresh and cryopreserved rat hepatocyte suspensions for the prediction of in vitro intrinsic clearance.

Freshly isolated hepatocytes are currently regarded as the most superior in vitro model for use in prediction studies, in particular to provide estimates of in vivo intrinsic clearance (CL(int)). However, due to their loss of viability within 4 h and a decrease in cytochrome P450-dependent metabolism upon culture, newer cellular models are being developed. Cryopreserved hepatocytes have several potential advantages, but to date evaluation of the utility of this model for estimating in vitro CL(int) has been limited to the substrate depletion approach. We have incubated eight compounds with suspensions of freshly isolated and cryopreserved rat hepatocytes and obtained in vitro CL(int) via metabolite formation kinetics (for 14 pathways). A substantial range of in vitro CL(int) values (0.1-98 microl/min/10(6)cells) was obtained in both models, and the freshly isolated suspension data were in good agreement with the literature. Cryopreserved suspensions were able to give a comparable estimation (within 2-fold) of in vitro CL(int) to fresh cells for six pathways, namely tolbutamide, three diazepam metabolites, propranolol, and 7-hydroxylation of warfarin. A higher estimation of in vitro CL(int) was obtained for the three other metabolites of warfarin due to a decrease in the K(M) values. Lower estimations of in vitro CL(int) were observed for four compounds (six pathways), and this was particularly pronounced (4-16%) for pathways showing atypical Michaelis-Menten kinetic profiles (dextromethorphan, nordiazepam) but less so (25-45%) for pathways showing biphasic Michaelis-Menten kinetics (7-ethoxycoumarin and phenytoin).

Animals↗
PubMed2005

Evaluation of drug penetration into the brain: a double study by in vivo imaging with positron emission tomography and using an in vitro model of the human blood-brain barrier.

The blood-brain barrier (BBB) permeabilities of 11 compounds were measured both in vitro with a newly developed coculture-based model of human BBB and in vivo with positron emission tomography (PET). The 11 compounds were fluoropyridinyl derivatives labeled with the positron-emitter fluorine-18, [(18)F]F-A-85380 [2-[(18)F]fluoro-3-[2(S)-2 azetidinylmethoxy]pyridine], and 10 selected N-substituted-azetidinyl and pyrrolidinyl closely related [(18)F]fluoropyridinyl derivatives (including [N'-aromatic/aliphatic]-thioureas, -ureas, and -amides). The in vitro BBB model, a new coculture system of primary human brain endothelial cells and astrocytes, was used to measure the permeability coefficient for each compound. Dynamic PET studies were performed in rats with the same compounds, and a two-compartment model analysis was used to calculate their in vivo permeability coefficients. The 11 derivatives differed in their degree of BBB passage and transport mechanism. The analysis of PET data showed a significant cerebral uptake for six derivatives, for which the in vitro evaluation indicated active influx or free diffusion. Five derivatives displayed low in vivo cerebral uptake, in agreement with the observation of an in vitro active efflux. Overall, there was a remarkable correlation between the in vitro and in vivo permeability coefficients (r = 0.99). This double study proves a close correlationship between the assessment of the BBB passage in vitro and in vivo. The in vitro model of human BBB offers the possibility of subtle discrimination of various BBB permeability degrees and transport mechanisms. Conversely, small animal PET imaging appears suitable to screen directly in vivo brain targeting of drugs or radiopharmaceutical candidates.

Algorithms↗
PubMed2000

In vitro pharmacodynamic parameters of sordarin derivatives in comparison with those of marketed compounds against Pneumocystis carinii isolated from rats.

Pneumocystis carinii pneumonia remains one of the most serious complications of immunosuppressed patients. In this study, the in vitro pharmacodynamic parameters of four sordarin derivatives (GM 191519, GM 237354, GM 193663, and GM 219771) have been evaluated by a new quantitative approach and compared with the commercially available drugs pentamidine, atovaquone, and trimethoprim-sulfamethoxazole (TMP-SMX). In vitro activities and in vivo therapeutic efficacies of sordarin derivatives against P. carinii were also evaluated. In vitro activity was determined by the broth microdilution technique, comparing the total number of microorganisms in treated and drug-free cultures by using Giemsa staining. The in vitro maximum effect (E(max)), the drug concentrations to reach 50% of E(max) (EC(50)), and the slope of the dose-response curve were then estimated by the Hill equation (E(max) sigmoid model). Sordarin derivatives were the most potent agents against P. carinii, with EC(50)s of 0.00025, 0.0007, 0.0043, and 0. 025 microg/ml for GM 191519, GM 237354, GM 193663, and GM 219771, respectively. The EC(50)s of pentamidine, atovaquone, and TMP-SMX were 0.025, 0.16, and 26.7/133.5 microg/ml, respectively. The results obtained with this approach showed GM 237354 and GM 191519 to be approximately 35- and 100-fold more active in vitro than pentamidine, the most active marketed compound. All sordarin derivatives tested were at least 5,000-fold more active in vitro than TMP-SMX. The three sordarin derivatives tested in vivo-GM 191519, GM 237354, and GM 219771-showed a marked therapeutic efficacy, defined as reduction of cyst forms per gram of lung. GM 191519 was the most potent (daily dose reducing 50% of the P. carinii burden in the lungs [ED(50)], 0.05 mg/kg/day) followed by GM 237354 and GM 219771 (ED(50)s, 0.30 and 0.49 mg/kg/day, respectively). Good agreement between in vitro parameters and in vivo outcome was obtained when P. carinii pneumonia in rats was treated with sordarin derivatives.

Animals↗
PubMed1975

Bacteriocinogenic Clo DF13 minicells of Escherichia coli synthesize a protein that accounts for immunity to bacteriocin Clo DF16: action of the immunity protein in vivo and in vitro.

The Clo DF13 plasmid-specific immunity protein is able to prevent the inhibitory effect of cloacin DF13 on in vitro protein synthesis. We have shown, by gel filtration, that direct binding of the Clo DF13 immunity protein to cloacin occurs in vitro. This cloacin DF13-immunity protein complex is rather stable, and the cloacin present in the complex is no longer able to cause inhibition of in vitro protein synthesis. The binding of immunity protein to cloacin DF13 is rather specific because the Clo DF13 immunity protein does not bind to in vitro inactive cloacin and binds very poorly to the closely related bacteriocin colicin E3. Furthermore, we present data which strongly suggest that in vitro at least a fourfold excess of immunity protein is required to ensure that every cloacin molecule is inactivated by cloacin-immunity protein complex formation. Only a fraction (an about equimolar amount) of the immunity protein molecules, however, actually binds to cloacin DF13. The existence of an immunity protein-cloacin complex in vivo was concluded from the observation that cloacin, purified by chromatography on diethyl-(2-hydroxypropyl)-aminoethyl Sephadex in the absence of urea, contains an about equimolar amount of a second protein which comigrates with immunity protein on sodium dodecyl sulfate-polyacrylamide and urea-polyacrylamide gels. In an in vitro protein-synthesizing system, this component appeared to behave identical to the Clo DF13 immunity protein. The purified immunity protein-containing cloacin was at least 80 times less active in inhibiting in vitro protein synthesis, compared to cloacin, free of immunity protein. These data imply that few, if any, cloacin DF13 molecules are present in cloacinogenic cells as active, free cloacin molecules.

Bacterial Proteins↗
PubMed1980

Species specificity of in vitro Escherichia coli adherence to host intestinal cell membranes and its correlation with in vivo colonization and infectivity.

We have previously described an in vitro assay for examining the mucosal adherence of a rabbit diarrheagenic Escherichia coli, RDEC-1. That assay defined the in vitro characteristics of RDEC-1 adherence to brush borders isolated from rabbit ileal epithelial cells. The present study was conducted to examine the species specificity of both in vitro RDEC-1 adherence and in vivo infectivity of RDEC-1 and to compare these specificities. Species specificity in vitro adherence was examined by using brush borders prepared from intestinal epithelial cells of rats, guinea pigs, and rabbits, as well as from a surgically resected specimen of human ileum. Strain RDEC-1 adherence to rabbit brush borders in vitro was significantly greater (P < 0.001) than its adherence to brush borders from any of the other species. Regional specificity of in vitro adherence of RDEC-1 to ileal segments of rabbit intestinal mucosa was also demonstrated. There was significantly greater adherence of RDEC-1 to rabbit ileal brush borders as compared to rabbit jejunal brush borders (P < 0.05). In vivo infectivity was assessed by inoculating RDEC-1 into rats, guinea pigs, and rabbits. RDEC-1 elicited diarrhea in all inoculated rabbits with the mean onset of illness occurring 5 days after inoculation. In contrast, none of the RDEC-1-inoculated rats or guinea pigs developed diarrhea. Furthermore, colonization studies in these animals revealed that RDEC-1 heavily colonized the ileum and cecum (10(9) RDEC-1 colony-forming units/g of tissue) of rabbits; however, only minimal colonization was observed in guinea pigs and rats. In conclusion, the correlation between in vitro adherence and in vivo infectivity that we have observed suggests that the presence of receptors, specific for bacteria, on the surface of the host intestinal mucosa determines species susceptibility to enteric colonization and infectivity by certain strains of enteropathogenic E. coli.

Animals↗
PubMed1974

In vitro polyoma DNA synthesis: characterization of a system from infected 3T3 cells.

A lysate from hypotonically swollen polyoma-infected BALB/3T3 cells incorporated labeled deoxynucleotide triphosphates into both viral and cellular DNAs. The incorporation was stimulated by the presence of ATP, deoxynucleotide triphosphates, thiols, and magnesium ions. Strong inhibition of incorporation was observed with thiol reagents and arabinosyl nucleotide triphosphates. The rate of in vitro synthesis increased with the temperature of incubation as expected. Incorporation into cellular DNA for up to 2 h was observed in lysates from virus-infected and serum-stimulated cells but not from resting cells. Synthesis in the system, therefore, appeared to reflect the physiological state of the cells before preparation of the lysate. Incorporation into viral DNA stopped far sooner than that into cellular DNA. During the initial phase of the in vitro incubation, incorporation occurred into viral replicative intermediates (RI). These RIs had identical properties to those isolated after in vivo pulse labeling and a substantial proportion of them was matured to form I DNA at later times in the incubation through all the stages known to occur in vivo. Density labeling of the in vitro product showed that practically all of the RIs pre-existing in the infected cell took part in the in vitro reaction. Analysis of DNA labeled in vitro in the presence of 5-bromodeoxyuridine triphosphate showed that synthesis occurred on RIs at all stages of replication and that the progeny strands were elongated by up to 80% of unit viral DNA length. Pre-existing RIs, pulse labeled in vivo, showed evidence of a pool at a late stage of replication which required elongation of their progeny strands by approximately 25% during conversion to form I molecules. From density-labeling experiments, we were also able to show that viral DNA synthesis in vitro was semiconservative. The major reason for cessation of viral DNA synthesis in vitro was the very limited ability of the lysate to initiate new rounds of viral DNA synthesis.

Adenosine Triphosphate↗
PubMed1977

Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA.

The influenza virion transcriptase is capable of synthesizing in vitro complementary RNA (cRNA) that is similar in several characteristics to the cRNA synthesized in the infected cell, which is the viral mRNA. Most of the in vitro cRNA is large (approximately 2.5 X 10(5) to 10(6) daltons), similar in size to in vivo cRNA. The in vitro transcripts initiate in adenosine (A) or guanosine (G) at the 5' end, as also appears to be the case with in vivo cRNA (R.M. Krug et al., 1976). The in vitro transcripts contain covalently linked polyadenylate [poly(A)] sequences, which are longer and more heterogeneous than the poly(A) sequences found on in vivo cRNA. The synthesis in vitro of cRNA with these characteristics requires both the proper divalent cation, Mg2+, and a specific dinulceside monophosphage (DNMP), ApG or GpG. These DNMPs stimulate cRNA synthesis about 100-fold in the presence of Mg2+ and act as primers to initiate RNA chains, as demonstrated by the fact that the 5'-phosphorylated derivatives of these DNMP's, 32pApG or 32pGpG, are incroporated at the 5' end of the product RNA. The RNA synthesized in vitro differs from in vivo cRNA in that neither capping nor methylation of the in vitro transcripts has been detected. The virion does contain a methylase activity, as shown by its ability to methylate exogenous methyl-deficient Escherichia coli tRNA.

Cell Line↗
PubMed1983

Synthesis and processing of glycoprotein D of herpes simplex virus types 1 and 2 in an in vitro system.

We carried out studies of in vitro translation and processing of glycoprotein D (gD) of herpes simplex virus types 1 and 2 by using mRNA from cells infected for 6 h and a reticulocyte lysate translation system. Polypeptides of 49,000 daltons were immunoprecipitated with anti-gD-1 sera. Each in vitro-synthesized molecule had the same methionine tryptic peptide profile as the respective in vivo precursors, pgD-1 and pgD-2. In addition, the polypeptides synthesized in vitro were larger than the corresponding molecules synthesized in the presence of tunicamycin. This suggested that each of the gD polypeptides synthesized in vitro contained a transient N-terminal signal sequence. When the translation mixture was supplemented with pancreatic microsomes, each of the gD polypeptides was converted cotranslationally to a larger-molecular-weight form. Processing involved addition of three N-asparagine-linked oligosaccharides and removal of the signal peptide. When trypsin was added after in vitro processing, a polypeptide which was 3,000 daltons smaller than the in vitro-modified form of gD was immunoprecipitated. Experiments with endo-beta-N-acetylglucosaminidase H showed that this polypeptide still contained the three N-asparagine-linked oligosaccharides. Two monoclonal antibodies, 57S (group V) and 17O (group VII), were used to further orient gD in microsomes. The group V determinant was located in the trypsin-sensitive 3,000-dalton fragment, and the group VII determinant was located in the portion of gD which was protected from trypsin. We concluded that gD is oriented with the three glycosylation sites inside the vesicles and that 3,000 daltons containing the group V determinant are located outside. Immunofluorescence studies indicated that the group V determinant of gD is inside the plasma membrane of herpes simplex virus-infected cells and that the group VII determinant is outside. This cellular orientation is consistent with predictions based on the in vitro experiments.

Acetylglucosaminidase↗
PubMed1985

Phosphorylation in vitro of Escherichia coli-produced 235R and 266R tumor antigens encoded by human adenovirus type 12 early transformation region 1A.

The tumor (T) antigens encoded by the human adenovirus early transforming region 1A (E1A) are gene regulatory proteins whose functions can immortalize cells. We have recently described the synthesis in Escherichia coli and the purification of the complete T antigens encoded by the adenovirus type 12 (Ad12) E1A 12S mRNA (235-residue [235R] T antigen) and 13S mRNA (266R T antigen). In this study, we show that the Ad12 E1A T antigens are extensively phosphorylated in Ad12-infected mammalian cells but are not phosphorylated in E. coli. Inasmuch as posttranslational phosphorylation at specific amino acid sites may be important for biological activity, we have studied the phosphorylation of the E. coli-produced T antigens in vitro by using a kinase activity isolated from cultured human KB cells. The kinase was purified about 300-fold and appears to be a cyclic AMP-independent, Ca2+-independent protein kinase requiring only ATP and Mg2+ for activity. To determine which amino acids are phosphorylated and whether phosphorylation in vitro occurs at the same amino acid sites that are phosphorylated in vivo, the Ad12 E1A T-antigen species synthesized by infected cells were metabolically labeled with 32Pi and compared with the E. coli-produced E1A T antigens labeled in vitro with [gamma-32P]ATP by using the partially purified kinase. Partial V8 proteolysis analysis gave similar patterns for in vivo- and in vitro-phosphorylated T antigen. Two-dimensional maps of tryptic phosphopeptides and of chymotryptic phosphopeptides suggested that mainly the same amino acid sites are phosphorylated in vitro and in vivo and that phosphorylation occurred at multiple sites distributed throughout the T-antigen molecule. Serine was the only amino acid that was phosphorylated both in vivo and in vitro, and, surprisingly, most serines appeared to be phosphorylated. The feasibility of faithfully phosphorylating T antigens in vitro suggests that the E. coli-produced Ad12 E1A 235R and 266R T antigens may prove useful for molecular studies on T-antigen function.

Adenoviruses, Human↗
PubMed1988

Expression of herpes simplex virus type 1 DNA polymerase gene by in vitro translation and effects of gene deletions on activity.

A cloned herpes simplex virus type 1 DNA polymerase gene which is biologically functional was inserted into SP6 and T7 promoter-containing vectors for in vitro transcription-translation. pol-specific RNA synthesized in vitro will direct the synthesis of a 140-kilodalton polypeptide in rabbit reticulocyte lysates. RNAs prepared from pol templates linearized at internal restriction sites specified deleted polypeptides with sizes consistent with colinearity of the pol gene and the 140-kilodalton primary translation product. The in vitro translated pol gene product was enzymatically active, with salt resistance and sensitivity to acyclovir triphosphate, similar to the enzyme activity in crude extracts of herpes simplex virus type 1-infected Vero cells. An in-frame deletion of 78 residues (amino acids residues 881 to 959) was introduced into the expression vectors to investigate the function of a region of the polypeptide (amino acids residues 881 to 895) which is conserved in nine other DNA polymerases. In a complementation assay, this mutation abolished biological activity as well as the enzymatic activity of the in vitro translated product. A BAL31 mutation deleting the upstream open reading frame of pol had no effect on biological activity in a complementation assay but was found to increase the efficiency of in vitro translation of pol RNA. Two amino-terminal deletions of 27 and 67 residues were found to greatly enhance the enzymatic activity of the in vitro translated product, while all carboxy-terminal deletions examined (the smallest being 164 residues) abolished in vitro enzymatic activity. Expression of the 67-residue amino-terminal deleted pol gene in Escherichia coli, using a bacteriophage T7-based system, resulted in accumulation of large amounts of an insoluble fusion protein. An antiserum prepared against this fusion protein precipitated the 140-kilodalton DNA polymerase from herpes simplex virus type 1-infected cell lysates.

Animals↗
PubMed1996

Functional implications of mutations within polyomavirus large T antigen Rb-binding domain: effects on pRb and p107 binding in vitro and immortalization activity in vivo.

In this study, we have extensively modified the Rb-binding domain of polyomavirus large T antigen. Mutant polyomavirus large T antigens were tested for their ability to bind pRb and p107 in vitro and assayed for their capacity to immortalize primary rat embryo fibroblasts in vivo. Polyomavirus large T antigen bound pRb and p107 through a common region located between amino acids 141 to 158, containing the consensus Rb-binding sequence D/N-L-X-C-X-E. Substitution of any amino acid within the core Rb-binding sequence abolished pRb and p107 binding in vitro and immortalization activity in vivo. Substitution of amino acids outside the core Rb-binding sequence reduced pRb and p107 binding in vitro and decreased or abolished immortalization of rat embryo fibroblasts in vivo. Although duplication of the Rb-binding domain within the polyomavirus large T antigen results in a molecule that can bind at least twice as much pRb and p107 in vitro, this mutant displayed an essentially wild-type level of immortalization activity. More importantly, we found that the addition of acidic residues within the casein kinase II consensus phosphorylation region flanking the Rb-binding domain, or the deletion of amino acids 256 to 272, increased the immortalizing activity of the mutant polyomavirus large T antigen. These two mutants displayed a greater than wild-type level of pRb binding in vitro, while in contrast, a decreased affinity for p107 binding in vitro was observed. Together, these results indicate that while pRb binding appears to be an essential event for immortalization, there is no tight correlation between the frequency of immortalization and the absolute level of pRb binding in vitro, indicating that other large T antigen functions are important for cellular immortalization.

Amino Acid Sequence↗
PubMed1990

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription.

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Base Sequence↗
PubMed1998

Analysis of effects of stimulation in vitro of ovalbumin primed lymph node cells on adoptive transfer of experimental immune mediated blepharoconjunctivitis in Lewis rats.

AIM: To analyse the role of stimulation in vitro of lymphocytes on the augmentation of experimental immune mediated blepharoconjunctivitis (EC, formerly EAC) in Lewis rats induced by adoptive transfer. METHODS: Two weeks after immunisation with ovalbumin (OVA), rat draining lymph nodes were collected and 50 x 10(6) cells were injected into naive syngeneic recipients either directly or after culture in vitro with OVA, concanavalin A (Con A), or purified protein derivative (PPD) for 3 days. Four days after injection the rats were topically challenged with OVA. 24 hours later, they were sacrificed and eyes and spleens were harvested for histology and proliferation assay. In some experiments, naive recipient rats were irradiated with 7 Gy gamma ray before transfer. The expression of adhesion molecules and cytokine profile of OVA primed lymph node cells were also investigated. RESULTS: Both infiltrated cell number and splenocyte proliferation in the recipients of stimulated cells were higher than those of unstimulated cells. In vitro stimulation with OVA or Con A induced a severe cellular infiltration, while stimulation with PPD did not. Irradiation markedly diminished cellular infiltration. Stimulation in vitro upregulated the CD4/CD8 ratio by four times and augmented expression of CD25, I-A, ICAM-1 molecules on OVA primed lymph node cells by about five times. IFN-gamma was detected in OVA primed cells by stimulation in vitro, while IL-4 mRNA was extinguished by stimulation in vitro. CONCLUSIONS: Augmentation of EC by stimulation in vitro of transferred lymphocytes might depend on the upregulation of expression of cell surface molecules and cytokine shift as well as augmented antigen specificity.

Adoptive Transfer↗
PubMed1994

Comparison of the effects of salbutamol and adrenaline on airway smooth muscle contractility in vitro and on bronchial reactivity in vivo.

BACKGROUND: The effect of adrenergic agonists in asthma depends on their net effect on microvascular leakage, mucosal oedema, vascular clearance of spasmogens, inhibition of cholinergic neurotransmission, and airway smooth muscle contractility. It has been postulated that adrenaline, by virtue of its alpha effects on the vasculature and cholinergic neurotransmission, may have additional useful properties in asthma compared with selective beta agonists such as salbutamol. METHODS: The airway effects of adrenaline (a non-selective adrenoreceptor agonist) were compared with the selective beta 2 agonist salbutamol. Their airway smooth muscle relaxant potencies and effect on histamine contraction in human bronchi in vitro were compared with their effects on airway calibre and histamine reactivity in asthmatic subjects in vivo. For the in vitro studies changes in tension were measured in response to these agents in thoracotomy specimens of human airways. In vivo the effects of adrenaline and salbutamol on airway calibre and histamine reactivity were measured in eight subjects with mild to moderate asthma in a randomised crossover study. RESULTS: Salbutamol and adrenaline had approximately equivalent airway smooth muscle relaxant potencies in vitro and bronchodilator potency in vivo. However, their effects on histamine induced contraction in vitro were significantly different from their effects on histamine reactivity in vivo. Salbutamol was less potent in vitro producing a mean (SE) 2.4 (0.15) doubling dose increase in the histamine EC20 and adrenaline a 5.2 (0.18) doubling dose increase (mean difference between salbutamol and adrenaline 2.8 doubling doses; 95% CI 1.1 to 4.5). Salbutamol had no effect on the maximal response to histamine whereas adrenaline reduced it by 54%. In contrast, salbutamol was more potent in vivo producing a mean (SE) increase in PD20 histamine of 1.84 (0.5) doubling doses whereas adrenaline was without effect increasing PD20 by only 0.06 (0.47) doubling doses (mean difference between adrenaline and salbutamol 1.78, 95% CI 0.26 to 3.29 doubling doses). CONCLUSIONS: These findings suggest that the alpha adrenergic airway effects of non-selective adrenoreceptor agonists such as adrenaline offer no additional protection against histamine-induced broncho-constriction in vivo than beta 2 selective drugs such as salbutamol, despite adrenaline providing greater protection against histamine-induced contraction in vitro. The differences between the effects of these agents in vitro and in vivo may be related to their opposing vascular effects in vivo.

Adult↗