J Cell Biol, 1997 Dec 15, 139(6), 1533 - 43 Thyroid hormone induces apoptosis in primary cell cultures of tadpole intestine: cell type specificity and effects of extracellular matrix; Su Y et al.; Thyroid hormone (T3 or 3,5,3'-triiodothyronine) plays a causative role during amphibian metamorphosis . To investigate how T3 induces some cells to die and others to proliferate and differentiate during this process, we have chosen the model system of intestinal remodeling, which involves apoptotic degeneration of larval epithelial cells and proliferation and differentiation of other cells, such as the fibroblasts and adult epithelial cells, to form the adult intestine . We have established in vitro culture conditions for intestinal epithelial cells and fibroblasts . With this system, we show that T3 can enhance the proliferation of both cell types . However, T3 also concurrently induces larval epithelial apoptosis, which can be inhibited by the extracellular matrix (ECM) . Our studies with known inhibitors of mammalian cell death reveal both similarities and differences between amphibian and mammalian cell death . These, together with gene expression analysis, reveal that T3 appears to simultaneously induce different pathways that lead to specific gene regulation, proliferation, and apoptotic degeneration of the epithelial cells . Thus, our data provide an important molecular and cellular basis for the differential responses of different cell types to the endogenous T3 during metamorphosis and support a role of ECM during frog metamorphosis. J Lipid Res, 1997 Dec, 38(12), 2473 - 82 Secretion from cell culture of HDL and VLDL bearing apoB-33 with a large internal deletion; Wu MJ et al.; Rat hepatoma McA-RH7777 cells synthesize and secrete two populations of apoB-containing lipoproteins: a larger, VLDL-sized population floating in the Sf 40-150 range and a smaller, LDL and HDL-sized population . Three permanently transfected cell lines of McA-RH7777 cells secreted (in addition to the endogenous lipoproteins) lipoproteins containing 1) a carboxyl-terminally truncated human apoB-53 (2377 amino acids in length); 2) a carboxyl-terminally truncated human apoB-31 (1420 amino acids in length); or 3) an internally deleted human apoB protein, apoB-18/95, containing a total of 1490 amino acid residues, equivalent in length to an apoB33 . The apoB-18/95 protein contained amino acid residues 1-782 joined to 708 residues near the C-terminus of apoB (residues 36364343) . All three of the apoB peptides, apoB53, apoB-31, and apoB-18/95, were present on smaller LDL-HDL-class lipoproteins, with buoyant densities in the HDL density range . The sizes of the HDL class lipoproteins agreed with prior observations that lipoprotein core circumference is directly proportional to apoB size . As HDL containing apoB-18/95 conformed to this rule, contiguous apoB amino acid sequence is not required for the rule to be obeyed . In addition, apoB-18/95, but not apoB-31, was also present on the VLDL-sized lipoproteins even in the absence of serum or oleate supplementation . As the latter two constructs encode equally sized apoB peptides, their particular amino acid sequences rather than just overall length must determine whether they can assemble into a VLDL particle. Fertil Steril, 1998 Jan, 69(1), 112 - 7 Follicular fluid and human granulosa cell cultures: influence on sperm kinetic parameters, hyperactivation, and acrosome reaction; Fabbri R et al.; OBJECTIVE: To evaluate the influence of human granulosa cell (GC) cultures and follicular fluid (FF) on sperm kinetic parameters, hyperactivation, and the acrosome reaction compared with the influence of human tubal fluid (HTF) and Ham's F-10 medium . DESIGN: Sperm kinetic parameters, hyperactivation, and the acrosome reaction were evaluated after 6 hours of incubation in HTF, Ham's F-10 medium, FF, and GC cultures . SETTING: Infertility and In Vitro Fertilization Centre, Reproductive Endocrinology Unit, Institute of Obstetrics and Gynaecology . PATIENT(S): Sixteen normal semen samples . INTERVENTION(S): Sperm kinetic parameters and hyperactivation were analyzed using an automated videomicrography system, the acrosome reaction was performed using a triple-stain technique, and progesterone and 17OH-progesterone levels were measured with the use of commercially available kits . MAIN OUTCOME MEASURE(S): Sperm kinetic parameters, hyperactivation, acrosome reaction . RESULT(S): The percentage of motile sperm, the mean curvilinear velocity, and the mean of the maximum amplitude of lateral head movement were increased significantly after 6 hours of incubation in FF or GC cultures compared with incubation in HTF or Ham's F-10 medium, whereas the mean linearity was decreased significantly . Follicular fluid and GC cultures significantly increased hyperactivation and the acrosome reaction compared with the values obtained using HTF and Ham's F-10 medium . Progesterone and 17OH-progesterone levels were increased significantly after incubation in FF and GC cultures compared with HTF and Ham's F-10 medium . CONCLUSION(S): Follicular fluid and GC cultures increase sperm motility parameters, hyperactivation, and the acrosome reaction . This effect may be related to GC detoxification of the microenvironement or GC secretion of peptides, glycoproteins, growth factors (insulin-like growth factors 1 and 2), or steroids (progesterone and 17OH-progesterone). Virology, 1998 Jan 20, 240(2), 193 - 201 Comparison of Sindbis virus-induced pathology in mosquito and vertebrate cell cultures; Karpf AR et al.; We have compared Sindbis virus-induced cytopathology in vertebrate and mosquito (Aedes albopictus) cell cultures . It has been shown that vertebrate cells undergo apoptosis when infected by Sindbis virus and this was confirmed here using hamster cells (BHK) . The occurrence of cell death in Sindbis virus-infected A . albopictus cells is a cell clone-specific phenomenon and, unlike in BHK cell cultures, mosquito cell death does not correlate with a large induction of apoptosis, as determined by assays testing for DNA fragmentation or reduced cellular DNA content . Cell cycle distribution changes were observed in Sindbis virus-infected BHK and C7-10 cell cultures, and the changes are distinct, both in the time of induction and the types of perturbations . In Sindbis virus-infected BHK cells, the major cell cycle profile change is the early accumulation of cells with sub-G1 DNA content and a corresponding reduction in the proportion of cells in G1 and G2/M . For Sindbis virus-infected C7-10 cells, the major perturbations are an increased proportion of cells showing G2/M or polyploid DNA content and a reduction in the proportion of G1 and S phase cells . These data suggest that the pathology induced in mosquito cell cultures by Sindbis virus infection may be distinct from the pathology which appears in vertebrate cell cultures. Infect Immun, 1998 Feb, 66(2), 835 - 8 Inducible nitric oxide synthase does not affect resolution of murine chlamydial genital tract infections or eradication of chlamydiae in primary murine cell culture; Ramsey KH et al.; Mice lacking inducible nitric oxide synthase (iNOS) or treated with iNOS inhibitors resolved chlamydial genital tract infections . Additionally, treatment of primary murine cell cultures with gamma interferon restricted chlamydial growth in the absence of nitric oxide . From these results, iNOS activity is unnecessary for the resolution of chlamydial genital tract infections in mice and inhibition of chlamydial growth in culture. Antisense Nucleic Acid Drug Dev, 1997 Dec, 7(6), 567 - 73 Stereodependent inhibition of plasminogen activator inhibitor type 1 by phosphorothioate oligonucleotides: proof of sequence specificity in cell culture and in vivo rat experiments; Stec WJ et al.; Hexadecadeoxyribonucleotides complementary to a fragment of human PAI-1 mRNA located upstream of the start codon and their phosphorothioate analogs were studied in cultured HUVECs as sequence-dependent inhibitors of PAI-1 expression . The activity of the random mixture of diastereomers of phosphorothioate hexadecanucleotide PS-16H has been compared with that of isosequential, stereoregular {All-Sp} and {All-Rp} isomers . The highest inhibitory effect on PAI-1 synthesis was observed with the {All-Sp} diastereomer . Stereorandom phosphorothioate oligonucleotide PS-16R complementary to the same region of rat PAI-1 mRNA, when injected into tail vein of rats, substantially decreased the level of PAI-1 in blood plasma. J Biol Chem, 1997 Dec 26, 272(52), 32836 - 46 A far upstream cis-element is required for Wilms' tumor-1 (WT1) gene expression in renal cell culture; Scholz H et al.; To identify novel cis-regulatory elements responsible for the tissue-restricted expression pattern of the Wilms' tumor-1 (WT1) gene, we mapped a total of 11 DNase I-hypersensitive sites in the 5'-flanking region and first intron of the human gene, six of which were specific for WT1 expressing cell lines . A 1.4-kilobase (kb) fragment from the mouse wt1 5'-flanking region contained cross-hybridizing sequence with significant homology to a region of DNase I hypersensitivity in the human WT1 gene which bound to nuclear matrix in human fetal kidney 293 cells . None of the DNase I-hypersensitive sites/matrix attachment regions, either alone or in combination, were sufficient for tissue-specific WT1 expression in transient and stably transfected cell lines . However, stable transfection of an approximately 620-kb yeast artificial chromosome (YAC) that carried the entire mouse wt1 locus into 293 cells resulted in wt1 (trans)gene expression at a level of approximately 30% of the endogenous human gene . Deletion of the 1.4-kb cross-hybridizing mouse fragment, located approximately 15 kb upstream of the transcription start site, caused complete loss of wt1 gene expression in the YAC-transfected 293 cells . In summary, we have identified a far upstream element that contains a region of DNase I hypersensitivity and that binds to nuclear matrix . This element includes phylogenetically conserved sequence and is required, although not sufficient, for mouse wt1 gene expression in human fetal kidney cells in culture. J Virol, 1998 Feb, 72(2), 1623 - 6 Essential roles of NF-kappaB and C/EBP in the regulation of intercellular adhesion molecule-1 after respiratory syncytial virus infection of human respiratory epithelial cell cultures; Chini BA et al.; To determine the molecular mechanism(s) of respiratory syncytial virus (RSV)-induced intercellular adhesion molecule-1 (ICAM-1) upregulation in respiratory epithelial cells (REC; A549 cell cultures), we investigated the roles of the transcription factors NF-kappaB and C/EBP . Increases in ICAM-1 message required de novo mRNA synthesis . ICAM-1 promoter constructs (luciferase reporter gene) transfected into A549 monolayers demonstrated promoter activation following RSV infection . Activation was abolished by site-specific mutation of the NF-kappaB (-228) or C/EBP (-239) sites . These data support the critical role of the activation of NF-kappaB and C/EBP in RSV-induced ICAM-1 expression by REC. Lasers Surg Med, 1998, 22(1), 30 - 6 Effects of Nd:YAG-laser irradiation on monolayer cell cultures; Gutknecht N et al.; BACKGROUND AND OBJECTIVE: The clinical applications of Nd:YAG lasers on oral soft tissues include a wide field of surgical and periodontal procedures . This in vitro study focuses on the histological effects of Nd:YAG-laser irradiation on a fibroblast monolayer cell culture especially with regard to thermal damage and cell necrosis . The results of this basic research study provide us with clear power settings for a safe soft tissue laser treatment . STUDY DESIGN/MATERIALS AND METHODS: Two hundred forty multiwell cell cultures and 24 micro-slide Leighton tubes were laser treated . Laser irradiation was performed with a commercial free-running pulse Nd:YAG laser and a quartz fiber with a diameter of 200 microns on L-929 fibroblast cell cultures . The variable parameters were pulse energy (30-120 mJ), pulse rate (20-100 Hz), power output (1.5-3.0 W), and time of irradiation (10-60 s) . The cultures were analyzed with help of vital staining, autoradiography, and cytomorphology examination . RESULTS: Depending on the different settings the laser irradiation caused inhibitions of the DNA metabolism rate and the cell division rate, a degeneratively changed cytomorphology up to cell pyknosis . An increasing pulse energy, pulse rate, or an increased time of irradiation created an extended diameter of the pyknotic cell zone . CONCLUSIONS: The laser beam creates an exactly bordered damage between cells . The cells had a very good inherent mobility, but the border between eliminated and unloaded cell zone was sharp, even after an incubation of 24 h . These stable results prove that the laser can be applied up to a micrometer distance . With the help of cell clusters it was proved that the laser beam is also able to eliminate exactly one monolayer . Cells which had been covered by another cell layer (in a cluster) were not eliminated. J Cell Biochem, 1997 Dec 15, 67(4), 528 - 40 Opposing effects by glucocorticoid and bone morphogenetic protein-2 in fetal rat bone cell cultures; Centrella M et al.; Glucocorticoid in excess produces bone loss in vivo . Consistent with this, it reduces the stimulatory effect of transforming growth factor beta (TGF-beta) on collagen synthesis in osteoblast-enriched cultures in vitro, where it also suppresses TGF-beta binding to its type I receptors . Analogous studies with bone morphogenetic protein-2 (BMP-2) show directly opposite results . These findings prompted us to assess the effect of glucocorticoid on BMP-2 activity in cultured bone cells, and whether either agent had a dominant influence on TGF-beta binding or function . BMP-2 activity was retained in part in osteoblast-enriched cultures pre-treated or co-treated with cortisol, and was fully evident when glucocorticoid exposure followed BMP-2 treatment . In addition, BMP-2 suppressed the effects of cortisol on TGF-beta activity, on TGF-beta binding, and on gene promoter activity directed by a glucocorticoid sensitive transfection construct . While BMP-2 also alters the function of less-differentiated bone cells, it only minimally prevented cortisol activity in these cultures . Our studies indicate that BMP-2 can oppose certain effects by cortisol on differentiated osteoblasts, and may reveal useful ways to diminish glucocorticoid-dependent bone wasting. J Neurosci, 1998 Jan 1, 18(1), 458 - 66 Involvement of presynaptic and postsynaptic mechanisms in a cellular analog of classical conditioning at Aplysia sensory-motor neuron synapses in isolated cell culture; Bao JX et al.; Temporal pairing of presynaptic activity and serotonin produces enhanced facilitation at Aplysia sensory-motor neuron synapses (pairing-specific facilitation), which may contribute to classical conditioning of the gill and siphon withdrawal reflex . This cellular analog of conditioning is thought to involve Ca2+ priming of the cAMP pathway in the sensory neurons . Consistent with that idea, we have found that pairing-specific facilitation by serotonin is greatly reduced by presynaptic injection of a slow Ca2+ chelator or a specific inhibitor of cAMP-dependent protein kinase and is accompanied by a transient increase in the frequency but by no change in the amplitude of spontaneous, miniature EPSPs . However, like post-tetanic potentiation (PTP) and long-term potentiation (LTP) at these synapses, pairing-specific facilitation is also greatly reduced by postsynaptic injection of a rapid Ca2+ chelator or by postsynaptic hyperpolarization during training, although postsynaptic hyperpolarization has no effect on the increase in frequency or on the amplitude of spontaneous EPSPs . These results suggest that pairing-specific facilitation by serotonin involves Hebbian postsynaptic as well as non-Hebbian presynaptic components that interact in some way, perhaps via retrograde signaling, to specifically enhance evoked, synchronized release of transmitter. Ciba Found Symp, 1997, 209, 142 - 54; discussion 154-7 Structure-activity relationships in cell culture; Wagner RW; The use of antisense oligonucleotides in cell culture relies on the development of potent modifications and cell delivery techniques . C-5 propyne pyrimidine phosphorothioate oligonucleotides bind selectively and with high affinity to RNA within cells, leading to potent antisense inhibition . The effect that increasing steric bulk of C-5-substituted deoxyuridine analogues has on the affinity for RNA and the ability to inhibit gene expression is discussed . The GS 2888 cytofectin agent delivers oligonucleotides to cells at high efficiency in the presence of serum in cell media . Modifications leading to the discovery of GS 2888 centered on the aliphatic chain length of the molecule and the pKa of the polar head group . Together, the C-5 propyne modifications and the GS 2888 cytofectin agent have been shown to be effective inhibitors of gene expression in cell culture, particularly in the area of cell cycle proteins involved in cancer progression. Ciba Found Symp, 1997, 209, 124 - 37; discussion 137-41 Differential oligonucleotide activity in cell culture versus mouse models; Wickstrom E et al.; The usual course of drug discovery begins with the demonstration of compound activity in cells and, usually, a lower level of activity in animals . Successive rounds of drug design may result in a compound with sufficient activity in animals to justify clinical trials . The basic endpoints of therapeutic oligonucleotide experiments include target antigen reduction, target messenger reduction and inhibition of transformed cell proliferation or viral replication . However, one should expect oligonucleotides to exhibit pleiotropic behaviour, as do all other drugs . In an animal oligonucleotides will necessarily bind to and dissociate from all macromolecules encountered in the blood, in tissues, on cell surfaces and within cellular compartments . Contrary to expectations, oligonucleotides designed to be complementary to certain transcripts have sometimes been found moderately effective in cell-free extracts, more effective in cell culture and most effective in animal models . If greater potency against standard endpoints is reported in mouse models than was observed in cell culture, critical examination must consider alternate modes of action in animals that may not apply in cell culture . This counterintuitive paradox will be examined, based on studies of Ha-ras expression in bladder cancer, Ki-ras expression in pancreatic cancer, erbB2 expression in ovarian cancer and c-myc expression in B cell lymphoma. Ciba Found Symp, 1997, 209, 47 - 54; discussion 54-9 Pharmacokinetics of oligonucleotides in cell culture; Lebleu B et al.; Synthetic oligonucleotides offer interesting perspectives for the regulation of gene expression in normal and pathological situations . Poor uptake in many cell types, inadequate intracellular compartmentalization, often fragmentary knowledge of intracellular behaviour and mechanism of action, and lack of specificity remain major challenges . These limitations strongly urge the design of new oligonucleotide analogues and more efficient antisense strategies . Present achievements and perspectives for further developments will be discussed with emphasis on cell delivery and intracellular fate. Pediatr Nephrol, 1997 Dec, 11(6), 778 - 83 Tubular toxicity of cyclosporine A and the influence of endothelin-1 in renal cell culture models (LLC-PK1 and MDCK); Zimmerhackl LB et al.; To evaluate the effect of cyclosporine A (CyA) at high concentrations (10(-4) and 10(-5) M) and the influence of endothelin-1 (ET-1) at physiological and pharmacological concentrations (10(-14) to 10(-6) M) on epithelial cell function, LLC-PK1 cells were studied as a model of the proximal tubule and MDCK cells as a model of the distal tubule/collecting duct . CyA caused time- and concentration-dependent acute toxicity . In LLC-PK1 cells, CyA caused a decrease in transepithelial resistance, indicating a loss of cell contacts, a release of lactate dehydrogenase (LDH) and villin into the supernatant, suggesting destruction of the apical membrane with loss of brush border, and finally release of uvomorulin, suggesting a disruption of the cell-cell adhesion, the zonula adherens . DNA synthesis, as evaluated by bromodeoxyuridine (BrdU) incorporation, was significantly affected at > or = 10(-5) M CyA . The toxicity of CyA was higher when given from the apical rather than the basolateral compartment . ET-1 alone was without effect, but in combination with CyA, ET-1 significantly enhanced toxicity . The ET-1 effect was partially inhibitable by an ET(B), but not an ET(A), antagonist . Immunofluorescence for alpha-catenin, another protein of the zonula adherens, demonstrated no change in polarity for this protein, and immunoprecipitation of the complex indicated relative stability of the zonula adherens despite loss of cadherin into the supernatant . In MDCK cells the effects were different . CyA was not associated with LDH release, but with an increase in transepithelial resistance, indicating increased paracellular resistance . Morphological alterations were significantly less, but BrdU incorporation was decreased . This pattern of toxicity is compatible with a direct toxic effect of CyA on cells of the proximal tubule, with predominant morphological destruction of the cells, with concomitant proximal tubular dysfunction, and a functional alteration in cells of the distal tubule associated with increased paracellular resistance, which may lead to solute and water loss. Mol Biotechnol, 1997 Dec, 8(3), 279 - 81 Separation of monoclonal antibodies from cell-culture supernatants and ascites fluid using thiophilic agarose; Bog-Hansen TC; A one-step purification procedure will yield monoclonal antibodies from cell-culture supernatants and ascites fluids . The chromatographic adsorbent is thiophilic argose, i.e., beaded agarose gel coupled with ligands of thiophilic nature, often with a sulfone group and a sulfur atom . The chromatographic procedure is simply adsorption, wash, elution . The procedure is simple, efficient, and inexpensive. Eur J Cell Biol, 1997 Dec, 74(4), 342 - 9 Functional downregulation of the E-cadherin/catenin complex leads to loss of contact inhibition of motility and of mitochondrial activity, but not of growth in confluent epithelial cell cultures; Bracke ME et al.; When epithelial cells reach confluency in vitro, a number of energy-requiring activities such as growth and motility are contact-inhibited . We investigated the possible role of the E-cadherin/catenin complex, which acts as an invasion suppressor, in contact inhibition . Three strategies for modulation of the complex were used . Firstly, the cell-cell adhesion and signal transduction functions of E-cadherin were neutralized immunologically in human MCF-7/6 mammary carcinoma cells possessing a complete complex . Secondly, the effect of E-cadherin transfection in E-cadherin negative cell lines was investigated . Thirdly, alpha-catenin deficient variants of the human HCT-8/S11 colon carcinoma cell line were compared with their parent cells . In confluent cultures functional downregulation of the E-cadherin/catenin complex did not alter cell growth nor saturation density . This was shown by cell number counts, protein staining assays, cell cycle analysis, proliferation markers (Ki67 and Proliferating Cell Nuclear Antigen) and apoptosis assays . However, confluent cells with a functionally deficient complex showed positional instability and enhanced succinate dehydrogenase-mediated mitochondrial 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl) tetrazolium bromide (MTT) conversion, as compared to cells with an active complex . Our data indicate that contact inhibition of motility and of mitochondrial enzyme activity, but not of growth is regulated by the E-cadherin/catenin complex in epithelial cells. Exp Gerontol, 1994 Nov-Dec, 29(6), 601 - 9 Decline in CD28+ T cells in centenarians and in long-term T cell cultures: a possible cause for both in vivo and in vitro immunosenescence; Effros RB et al.; The dramatic decline in immune function with age, especially in T cell proliferative activity, has been documented extensively in experimental animal models and in clinical studies of the elderly . A similar proliferative decline is also seen in long-term T lymphocyte cultures used to study in vitro cellular senescence . We have compared the peripheral blood T lymphocytes of centenarians and younger controls for the cell surface expression of CD28, a costimulatory molecule that is required for optimal activation and proliferation following engagement of the T cell receptor . Our analysis shows a significant decrease (p < 0.001) in the percentage of T cells expressing CD28 in the elderly cohort, with values ranging from 44% to 90%, as compared to the mean control value of 91% . The decline in the percentage of CD28+ T cells correlates with a reduction in the CD4/CD8 ratio (r2 = 0.695, p < 0.0001) . Concommitantly, experiments using an in vitro T cell culture system showed a progressive loss of CD28 expression with culture "age." The concordance of proliferative decline and loss of CD28 in the centenarians and in the in vitro cultures suggest that a Hayflick phenomenon may operate in vivo leading to immunosenescence. Adv Biochem Eng Biotechnol, 1998, 59, 225 - 49 Apoptosis and cell culture technology; al-Rubeai M; The importance of apoptosis in cell culture has now been widely recognised . Recent work has shown that this process is widely manifested during the in vitro cultivation of commercially important mammalian cell lines . In this review I summarise what is now known of the characteristics, significance and regulatory mechanisms of apoptosis . As the process of cell proliferation and cell death are now considered intimately related, particular attention is paid to highlight the progress and opportunities in the field of cell culture engineering . The strategies that have been undertaken to prevent the induction of apoptosis in cell culture and those which have been suggested as possibilities to improve culture productivity through the apoptosis route are discussed with given examples. Bone, 1997 Dec, 21(6), 491 - 500 Cell sorting enriches osteogenic populations in rat bone marrow stromal cell cultures; Herbertson A et al.; The presence of multiple cell types in bone marrow stromal populations complicates interpretation of cytokine and hormone effects on the osteoprogenitors present, indicating a need for a method for purification of the osteoprogenitor population . Flow cytometric sorting of 7 day primary rat bone marrow stromal cell cultures was performed on the basis of alkaline phosphatase (AP) expression with an antibody against AP (RBM 211.13) . The resultant AP(high), AP(low), or control cells were plated to determine osteoprogenitor, macrophage, and adipocyte distribution and frequency . Approximately 50% of osteoprogenitor/bone nodule-forming cells were lost during processing/sorting when compared with unsorted controls . Nevertheless, within the AP(high) fraction, the numbers of AP-positive colonies and osteoprogenitors (bone nodules) were significantly enriched compared with the unfractionated control; the increase in osteoprogenitor frequency ranged from approximately 2 to 100-fold . There were few assayable osteoprogenitors in either the AP(high) or AP(low) fractions in the absence of dexamethasone (dex), suggesting that RBM stroma contains largely dex-dependent osteoprogenitor populations, and that dex may regulate osteoprogenitors subsequent to the upregulation of AP . Osteoprogenitor/bone nodule numbers in either the AP(high) or AP(low) fraction did not follow a linear relationship with decreasing plating density . The AP(high) fraction of cells was depleted for adipocyte and macrophage colonies . In contrast, within the AP(low) fraction of cells, adipocyte and macrophage colonies were consistently enriched . We conclude that flow-cytometric sorting of RBM stromal populations according to high or low AP expression is an effective technique for enrichment of AP-positive colonies and osteoprogenitors/bone nodule-forming cells. Biol Cell, 1997 Jun, 89(3), 233 - 40 Primary human muscle satellite cell culture: variations of cell yield, proliferation and differentiation rates according to age and sex of donors, site of muscle biopsy, and delay before processing; Bonavaud S et al.; The present study was performed to determine the influence on human satellite cell yield, proliferation, and differentiation rates of: 1) sex and age of donors; 2) site of the muscle biopsy; and 3) delay before processing of the muscle biopsy sample . We used a standardized primary muscle cell culture procedure on 206 normal muscle samples obtained from different muscle groups of patients aged from 20 to 88 years, at time of orthopedic surgery . Sex of donors did not influence muscle culture parameters . In contrast, aging tended to affect muscle cell yield (age group 50-59 years vs 70-79 years, P < 0.08), but not myogenic cell abilities to proliferate and to fuse into myotubes . The anatomic origin of muscle samples used for culture appeared to influence culture parameters . In contrast with other tested muscles, the tensor fasciae muscle gave both a good cell yield (174 +/- 25 10(3) cells per gram) and homogeneous proliferation and differentiation rates . Storage of the muscle sample at 4 degrees C in transport medium was associated with a very high cell yield when processing was done in early hours after biopsy (277 +/- 50 10(3) cells/g), a high and stable cell yield when processing was done from day 1 to day 3 after biopsy (185 +/- 15 10(3) cells/g), and a poor cell yield when processing was done after day 4 (111 +/- 13 10(3) cells/g) . Storage of muscle biopsy samples at 4 degrees C for 1 to 4 days was associated with good proliferation and fusion rates . In conclusion, these data validate a convenient procedure of primary human muscle cell culture, using tensor fasciae muscle biopsy, which is easily done at time of orthopedic surgery, obtained from men and women of all ages (if possible less than 70 years to obtain good cell yield), and allowing of 1-3 days of storage before processing that may compensate uncertainty of the exact time of availability of muscle samples for the scientist. Neuroreport, 1997 Dec 1, 8(17), 3791 - 4 BDNF enhances neuronal growth and synaptic activity in hippocampal cell cultures; Bartrup JT et al.; Brain-derived neurotrophic factor (BDNF) (20 ng/ml) significantly enhanced the growth of the somata of GABA-immunoreactive neurones in primary cultures of hippocampal neurones from postnatal rats after only 24h . Whole-cell patch-clamp experiments showed an increase in spontaneous synaptic activity between neurones with time in culture . After 10 days in culture, 90% of neurones sampled in control cultures showed spontaneous synaptic activity, whereas in cultures treated with BDNF, 100% of neurones had synaptic inputs after only 6 days . This difference in spontaneous activity was not due to the lack of synaptic inputs as KCl-induced synaptic activity was equally effective in BDNF and control cultures . These experiments demonstrate the rapid rate at which BDNF can promote neuronal growth and also show that BDNF can promote long term synaptic activity. Plant Mol Biol, 1997 Dec, 35(6), 777 - 89 Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L; Yang Q et al.; Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.), and the product N-benzoylanthranilate is the precursor of several sets of dianthramides . The transferase activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract . The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa . Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of these sequences differed in a few amino acid residues only suggesting the presence of isoenzymes . A specific 0.8 kb cDNA probe was generated by RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)+ RNA from elicited carnation cells . Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1-3, were sequenced and shown to encode full-size N-benzoyltransferases . The translated peptide sequences revealed more than 95% identity among these three clones . The additional two clones harbored insert sequences mostly homologous with pchcbt 1 but differing in the 3'-flanking regions due to variable usage of poly(A) addition sites . The identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequences as well as by the activity of the benzoyltransferase expressed in Escherichia coli . Therefore, carnation encodes a small family of anthranilate N-benzoyltransferase genes . In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs . Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo . Thus the cDNAs described represent also the first hydroxycinnamoyl-transferases cloned from plants, which classifies the enzymes as hydroxycinnamoyl/benzoyltransferases. Mol Cell Endocrinol, 1997 Nov 15, 134(2), 119 - 27 Mitogenic effects of nerve growth factor on different cell types in reaggregate cell cultures of immature rat pituitary; Proesmans M et al.; Treatment of reaggregate pituitary cell cultures of 14-day-old female rats with nerve growth factor (NGF) augmented the number of {3H}thymidine ({3H}T)-incorporating lactotrophs in a dose-dependent manner (0.03-3 nM) . At least during short-term treatment NGF increased the total number of cells expressing prolactin (PRL) mRNA and enlarged the cytoplasmic area occupied by PRL mRNA but did not affect the number of cells and the cytoplasmic area containing PRL, suggesting that NGF recruits lactotrophs expressing PRL mRNA but not yet PRL . NGF also stimulated {3H}T incorporation in ACTH cells but not in somatotrophs, thyrotrophs and gonadotrophs . In addition, NGF augmented the total number of {3H}T-incorporating cells to a much higher extent than was expected from its effect on lactotrophs and ACTH cells, suggesting NGF also stimulates {3H}T-incorporation in non-hormone producing cells (progenitors or stem cells?) . Around 40% of these {3H}T-incorporating cells in both control and NGF treated cultures showed immunoreactivity for the transcription factor Pit-1 in the nuclei, which is twice the percentage expected (18%) if these {3H}T-incorporating cells were the only known Pit-1 expressing cells in the pituitary i.e . lactotrophs, somatotrophs and thyrotrophs . The present data suggest that NGF has a mitogenic effect on several cell lineages in the pituitary: lactotrophs, corticotrophs and non-hormone-containing cells . The high proportion of mitotic non-hormone containing cells that express Pit-1 is consistent with the proposed role of Pit-1 in cell proliferation in the developing lactosomatotroph lineage. J Pharm Sci, 1997 Dec, 86(12), 1448 - 57 Influence of the microporous substratum and hydrodynamics on resistances to drug transport in cell culture systems: calculation of intrinsic transport parameters; Yu H et al.; Although cell culture models are increasingly used to study drug transport and metabolism, the influence of the substratum on the transport properties of the cell monolayer has not been studied in great detail . Furthermore, the use of effective (or apparent) permeabilities (Peff) assumes that the contribution of the microporous filter substratum and the aqueous boundary layer (ABL) to transport are negligible or are at least constant for a series of drugs . In the present study, the permeabilities of the substratum, ABL, and monolayer were obtained for a series of compounds at variable flow rates in side-by-side diffusion chambers . Comparisons of transport properties were made between cell monolayers grown on substrata made of polycarbonate (PC) and polyester (PE) . All paracellular markers demonstrated a reduction in permeability and a corresponding increase in transepithelial electrical resistance (TEER) through PE-grown monolayers . The permeabilities of two carrier-mediated compounds, phenylalanine and proline, were 55% higher and 48% lower through PE-grown monolayers than through the PC-grown monolayers, respectively . The resistance to progesterone transport attributed to the PE and PC filters was large (71% and 27% of total resistance, respectively) at a flow rate of 20 mL/min, indicating that the monolayer was not the rate-limiting transport barrier . Therefore, for highly permeable compounds, reporting Peff has limited value since it is an indicator of the transport properties of the substratum rather than of the monolayer . These results demonstrate that substratum properties (e.g., membrane composition, pore size, etc.) significantly affect the barrier properties of the Caco-2 cell monolayer . The most probable mechanism is by the modulation of the functional expression of nutrient and ion transporters resulting in variable transcellular and paracellular transport properties . These results further demonstrate the importance of calculating intrinsic membrane transport parameters if the monolayer is not maintained as the rate-determining barrier in the transport experiment . Using higher flow rates and higher porosity substrata supports may help maintain the monolayer as the rate-limiting transport barrier. Mol Mar Biol Biotechnol, 1997 Dec, 6(4), 279 - 88 Characterization of cell cultures derived from Fugu, the Japanese pufferfish; Bradford CS et al.; The Japanese pufferfish (genus Fugu), which possesses a highly compact genome, is becoming a popular model among those interested in sequencing and mapping the genomes of higher vertebrates . Although genomic libraries have been derived and used to study the molecular biology of Fugu, biological material derived from the living organism is difficult to obtain for laboratories distant from the Asian Pacific . We have established cell cultures from two Fugu species: kusafugu, Fugu niphobles, and torafugu, F . rubripes . Cultures derived from F . niphobles fry and F . rubripes eye have been passaged more than 60 times over the course of one year, representing approximately 180 population doublings . Proliferating cultures were also initiated from F . rubripes brain, liver, fin, spleen, kidney, swimbladder, and muscle . Karyotype analyses indicated that F . rubripes eye-derived cells possessed a chromosome number in the diploid range; F . niphobles fry cells were slightly hyperploid . Flow cytometry confirmed that the relative amounts of DNA present in cultured cells from both Fugu species were similar to that measured in blood cells collected from F . rubripes, and approximately one-seventh of that measured in diploid human cells . Telomerase activity was easily detectable in lysates prepared from F . niphobles fry cells and F . rubripes eye cells, consistent with the notion that these cultures are capable of indefinite proliferation. Mycoses, 1997, 40 Suppl 1, 64 - 72 {Involvement of secretory Candida proteinases in the adherence of C . tropicalis blastoconidia in a cell culture model}; Borg-von Zepelin M et al.; The influence of the heterologous acid secretory Candida proteinases on the adherence of the non-proteinase secreting strain of C . tropicalis DSM 4959 to epitheloid cells (vero line) was examined . The proteinases of the following Candida strains were used: C . albicans ATCC 10261 (serotype A), C . albicans ATCC 48867 (serotype B), C . tropicalis DSM 4238 . The assays were performed with the previously described in-vitro-adherence test {1} using the following principle steps: Candida proteinases and C . tropicalis blastoconidia were incubated with verocells in microtest plates in phosphate-buffer in the range of pH 4.0 to pH 7.0 . Adherent Candida cells were detected according to Filler et al . {2} with anti-Candida-mannoprotein antibodies and a secondary anti-rabbit-peroxidase conjugate . Compared to controls with denaturated proteinases, the photometric evaluation of adherent C . tropicalis cells showed, under optimal conditions, an augmentation of the adherence due to the Candida proteinases of about 50% . The optimum of this adherence augmentation was in the range of pH 5.5 which is outside the general activity optimum of Candida proteinases (pH 3) . The degree of purity of these proteinases had no marked influence on the adherence . The specificity of the proteinase dependent adherence augmentation could be demonstrated with the enzyme inhibitor Pepstatin A . C . tropicalis blastoconidia supplemented by pepstatin A and active Candida proteinase adhered in the same range as with denaturated proteinases in control tests . Our results suggest a function of Candida proteinases in the adherence process of blastoconidia to epithelia. Vet Med (Praha), 1997 Oct, 42(10), 281 - 7 Isolation and identification of porcine reproductive and respiratory syndrome virus in cell cultures; Valicek L et al.; Three strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in porcine lung macrophage (PLM) cultures from three swine herds . This has been the first successful isolation of PRRSV in the Czech Republic and the strains received the designations CAPM V-501, CAPM V-502 and CAPM V-503, respectively . All the three isolates in PLM were identified by immunofluorescence and immunoperoxidase tests and the strain CAPM V-502 also by electron microscopy using the ultrathin section technique . The strain CAPM V-502 has been adapted to the cell line MARC-145 . Viral RNA in PLM cultures infected with any of the isolated PRRSV strains was demonstrated by RT-PCR targeted to the more conserved ORF 7 genomic region encoding the nucleocapsid protein . The assessment of PCR products in agarose gel revealed a uniform size of 394 bp in all the three isolates and the European prototype strain Lelystad used as positive control. J Exp Ther Oncol, 1996 Jan, 1(1), 30 - 8 Rates of development of methotrexate resistance in heterogeneous mouse mammary tumor cell cultures; Miller BE et al.; Our previous studies have indicated that the expression by tumor cells of sensitivity to chemotherapeutic drugs such as methotrexate can be affected by the presence of other tumor cells; thus, otherwise methotrexate-resistant cells may respond to that drug in the presence of methotrexate-sensitive cells . In order to determine whether tumor heterogeneity also affects the emergence of drug resistance, we measured the rate of development of methotrexate resistance in mixed monolayer cultures of three mammary tumor subpopulation lines (66, 168TFAR, 4T07) that differ in degree of sensitivity to methotrexate . Cultures were treated weekly with 80 nM or 200 nM methotrexate . Each individual cell line was re-isolated from the mixture by passage in selective medium and then assayed for methotrexate sensitivity . Cultures of each of the three lines were treated and assayed in parallel . Few differences in the rate of development of methotrexate resistance were seen among cells from mixtures and cells cultured alone; line 4T07 appeared to become resistant somewhat more rapidly in mixtures . In untreated mixed cultures, line 66, the line least sensitive to methotrexate, gradually became dominant; this process was accelerated in treated cultures . One methotrexate-resistant subline from each parent cell line was tested to determine the mechanisms by which methotrexate resistance was increased . Two lines appeared to have increased levels of dihydrofolate reductase, and one exhibited decreased methotrexate transport as well . The third cell line had neither mechanism . Others have shown that tumor heterogeneity can act as a brake on the rate of development of new metastatic or immunogenic variants . Our data indicate that, at least in the model system we have tested, the rate of development of extrinsic drug resistance is substantially independent of pre-existing clonal diversity. Anticancer Res, 1997 Sep-Oct, 17(5A), 3225 - 31 Oncogene and HSP-70 expression in primary tumor cell cultures of renal cell carcinoma compared to their corresponding cell line; Kohler G et al.; BACKGROUND: Tumor progression in renal cell carcinoma (RCC) can be explained by a multistep model, in which the activation of certain oncogenes such as c-neu and c-fos appear to be early events in tumorigenesis, while the expression of p53 and pan-ras are found in advanced stages . MATERIAL AND METHODS: The expression of oncogenes and growth factors was examined in 29 primary tumor cell cultures (PTCC) of RCC using immunocytochemistry . RESULTS: In PTCC high expression of c-neu and c-fos was present in all tumors, whereas mdr, TGF-alpha, EGF, c-myc, pan-ras, p53 and HSP-70 was detected at low expression levels . In 27% (8/29) of PTCC, cell lines (CL) were established . Oncogene expression was increased in CL compared to PTCC . CONCLUSION: The pattern of oncogene expressions found in CL is similar to findings described in highly malignant tumors in vivo . Therefore, the establishment of CL seems to depend on a selective recruitment of tumor cells with an upregulated oncogene expression. Vaccine, 1997 Dec, 15(17-18), 1816 - 9 An immunogenicity and efficacy study of purified chick embryo cell culture rabies vaccine manufactured in Japan; Benjavongkulchai M et al.; Purified chick embryo cell rabies vaccine manufactured by the Chemo-Sero-Therapeutic Institute(Kaketsuken) at Kumamoto, Japan (Kaketsuken) was submitted to an immunogenicity and efficacy study . 52 severely rabies exposed patients were treated with the conventional five doses intramuscular WHO approved ('Essen') postexposure schedule . This included the administration of 40 IU kg-1 of equine rabies immune globulin on Day 0 . A control group of equally severely exposed subjects were treated with human diploid cell rabies vaccine manufactured by the Swiss Serum and Vaccine Institute as well as human rabies immune globulin . There were no deaths in either group in the more than 2 years follow-up period . Subjects treated with the chick embryo vaccine showed greater suppression of the neutralizing antibody response by the equine rabies immune globulin than those given the human diploid cell vaccine and human rabies immune globulin . A group of 20 less severely rabies exposed patients who received only the chick embryo vaccine without immune globulin all had antibody titers greater than the WHO minimal acceptable level on Day 14, 30, 90 and 180 . Fourteen subjects among the severely exposed vaccine and immune globulin study group were given vaccine boosters on Day 180 because of low antibody titers . It is concluded that chick embryo rabies vaccine manufactured by Kaketsuken is an immunogenic and effective rabies vaccine, but that the potency of future batches must be increased to provide a greater safety margin. Am J Respir Cell Mol Biol, 1997 Dec, 17(6), 672 - 82 Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses; Alcorn JL et al.; Studies of the regulation of surfactant lipoprotein metabolism and secretion and surfactant protein gene expression have been hampered by the lack of a cell culture system in which the phenotypic properties of type II cells are maintained . We have developed a primary culture system that facilitates the maintenance of a number of morphologic and biochemical properties of type II pneumonocytes for up to 2 wk . Cells were isolated by collagenase digestion of midgestation human fetal lung tissue that had been maintained in organ culture in the presence of dibutyryl cyclic AMP (Bt2cAMP) for 5 days . The isolated cells were enriched for epithelial components by treatment with DEAE-dextran, plated on an extracellular matrix (ECM) derived from Madin-Darby canine kidney (MDCK) cells, and incubated at an air/liquid interface in a minimal amount of culture medium containing Bt2cAMP . The cell cultures were comprised of islands of round epithelial-like cells containing numerous dense osmiophilic granules, surrounded by sparse spindle-shaped cells with the appearance of fibroblasts . Ultrastructural examination revealed that the osmiophilic granules had the appearance of lamellar bodies, the distinguishing feature of type II pneumonocytes . Additionally, the cultures maintained elevated levels of SP-A gene expression for up to 2 wk . The expression of mRNAs encoding SP-A, SP-B, and SP-C were regulated in the cultured cells by glucocorticoids and cyclic AMP in a manner similar to that observed in fetal lung tissue in organ culture . The differentiated phenotype was most apparent when the cells were cultured at an air/liquid interface . In order to utilize the cultured type II cells for study of the effects of overexpression of various proteins and for promoter analysis, it is of essence to transfect DNA constructs into these cells with high efficiency . Unfortunately, we found the cells to be refractory to efficient transfer of DNA using conventional methods (i.e., lipofection, electroporation, or calcium phosphate-mediated transfection) . However, replication-defective recombinant human adenoviruses were found to provide a highly efficient means of introducing DNA into the type II pneumonocytes . Furthermore, we observed in type II cell-enriched cultures infected with recombinant adenoviruses containing the lacZ gene under control of a cytomegalovirus promoter, that beta-galactosidase was expressed uniformly in the islands of type II cells and surrounding fibroblasts . By contrast, in cultures infected with recombinant adenoviruses containing the human growth hormone (hGH) gene under control of the SP-A gene promoter and 5'-flanking region, hGH was expressed only in the type II cells . Thus, this culture system provides an excellent means for identifying genomic elements that mediate type II cell-specific gene expression. Brain Res Brain Res Protoc, 1997 Oct, 1(4), 344 - 6 An in vitro cell culture system for the aggregation of embryonic chick central nervous system neurons; Monnerie H et al.; The use of dissociated neuronal cell cultures is a very widespread technique . It is useful to study specific interactions between cells and resolve molecular mechanisms underlying neural development and function . For instance, the extended family of neurotrophic factors was identified and further studied especially using such techniques . Several growth factors have also been studied in this way as well as other developmental molecules . In this paper we describe a method of culturing chick cortical cells, in the complete absence of serum, which results in an enhanced aggregation of neurons by few days in culture . This cell culture system is particularly convenient to perform functional analyses of various molecules involved in neuronal cell adhesion mechanisms, such as extracellular matrix proteins or cell adhesion molecules, that require the establishment of in vitro paradigms in order to analyze their influence on cell-substratum and cell-cell interactions, as previously reported . We have successfully studied the effect of specific glycoproteins from the subcommissural organ on neuronal cell adhesion using this cell culture system. Invert Neurosci, 1996 Jun, 2(1), 41 - 9 Aplysia hemolymph promotes neurite outgrowth and synaptogenesis of identified Helix neurons in cell culture; Ghirardi M et al.; Hemolymph of adult Aplysia californica significantly affects neurite outgrowth of identified neurons of the land snail Helix pomatia . The metacerebral giant cell (MGC) and the motoneuron C3 from the cerebral ganglion and the neuron B2 from the buccal ganglion of H . pomatia were isolated by enzymatic and mechanical dissociation and plated onto poly-L-lysine-coated dishes either containing culture medium conditioned by Helix ganglia, or pre-treated with Aplysia hemolymph . To determine the extent of neuronal growth we measured the neurite elongation and the neuritic field of cultured neurons at different time points . Aplysia hemolymph enhances the extent and rate of linear outgrowth and the branching domain of Helix neurons . With the hemolymph treatment the MGC neuron more consistently forms specific chemical synapses with its follower cell B2, and these connections are more effective than those established in the presence of the conditioned medium. Arch Biochem Biophys, 1997 Nov 15, 347(2), 249 - 55 4-Coumaroyl coenzyme A 3-hydroxylase activity from cell cultures of Lithospermum erythrorhizon and its relationship to polyphenol oxidase; Wang ZX et al.; A 4-coumaroyl-CoA 3-hydroxylase activity was purified 4600-fold from cell cultures of Lithospermum erythrorhizon . The enzyme showed a molecular mass of 42,400 +/- 1700 Da in gel chromatography and required ascorbate, NADH, or NADPH as cofactors . 4-Coumaroyl-CoA, 4-coumarate, p-cresol, and several other phenolic substances, but not tyrosine, were accepted as substrates for the hydroxylation . Besides hydroxylase activity, the enzyme showed diphenol oxidase activity . Both activities were inhibited by diethyldithiocarbamate or beta-mercaptoethanol, although at different concentrations . The enzyme showed striking similarity to a 4-coumaroyl-glucose 3-hydroxylase from sweet potato (Ipomoe batatas) roots, which has reportedly been purified to homogeneity and identified as a specific enzyme of chlorogenic acid biosynthesis . Close examination and comparison to a commercially available polyphenol oxidase, however, suggest that the enzyme activities purified from both Lithospermum and sweet potato are polyphenol oxidases rather than specific enzymes of secondary metabolism . Mol Cell Endocrinol, 1997 Oct 20, 133(2), 81 - 8 Leukocytes modulate 11beta-hydroxysteroid dehydrogenase (11beta-HSD) activity in human granulosa-lutein cell cultures; Evagelatou M et al.; It is well established that there are interactions between the immune and reproductive systems . The ovary contains indigenous macrophages, as well as other classes of leukocytes in smaller numbers . Cytokines secreted by these cells have been shown to have the ability to regulate ovarian steroidogenesis . In the present study, the effect of leukocytes on 11beta-hydroxysteroid dehydrogenase (11beta-HSD) in human granulosa-lutein cells was examined . In addition, individual cytokines were also tested for their ability to regulate this enzyme . The follicular aspirates of patients undergoing IVF treatment were used as a source of granulosa cells . Cells isolated from these aspirates were found to contain between 15 and 60% leukocytes as assessed by flow cytometry (FACS) . Leukocytes were removed from the sample preparations by the use of immunomagnetic beads coated with CD45 antibody, which recognises a surface antigen on all classes of leukocyte . Removal of leukocytes significantly decreased the 11beta-HSD activity in the granulosa cells, assayed after 3 days of culture, from 7.3 (2-20) to 3.5 (1-10) pmol cortisone formed/50000 cells/4 h (medians and ranges, n = 15) . Addition of IL-5 and IL-6 significantly increased the 11beta-HSD activity in granulosa cell cultures both in the presence and absence of leukocytes . Addition of IL-4 and IFN-gamma increased 11beta-HSD activity only in the leukocyte-depleted granulosa cell cultures, whereas IL-2 had no effect on either of the cultures . The data suggests that leukocytes interact with the ovarian cells through cytokine secretion and/or cell-cell contact to increase the 11beta-HSD activity in human granulosa cells. Biosci Biotechnol Biochem, 1997 Nov, 61(11), 1844 - 7 Growth factors for a primary chick muscle cell culture from shochu distillery by-products; Mahfudz LD et al.; An unidentified growth factor (UGF) was separated from shochu distillery by-products (SDBP) and its effect on the growth of a primary chick muscle cell culture was investigated . Chick muscle cells were isolated from fertile eggs (13-day-old embryos) of commercial broilers . UGF was separated on Sephadex LH-20 with a solvent system of water-methanol-ethylene dichloride (10:90:20, v/v), and the fraction eluted between 136 and 164 min was collected (fraction I) . Fraction I was further purified by HPLC with an Inertsil ODS-2 column using a solvent system of methanol-butanol (80:20, v/v) . Three fractions having retention times of 3.76, 4.57, and 5.12 min were collected and are referred to as fraction A, B, and C, respectively . In experiment 1, chick muscle cells were cultured in an m-199 medium containing 0.001, 0.01, or 0.1% of fraction I . In experiment 2, chick muscle cells were cultured with 0.01 or 0.005% of each fraction A, B, and C . Creatine kinase (CK) activity, protein and DNA contents were measured as indices of myotube growth, cell growth and cell proliferation, respectively . N tau-methylhistidine (N tau-MH) release from the muscle cell was also measured to observe the effect on proteolysis . In experiment 1, the protein content was significantly (p < 0.05) increased by fraction I, despite the low dose level . CK activity was significantly (p < 0.05) higher than the control when 0.001% of fraction I was added to the medium . However, increasing the level beyond 0.01% did not further increase the CK activity . The DNA content was not significantly changed . In experiment 2, the protein content, CK activity, and DNA content were significantly (p < 0.05) higher when fractions A and B were added to the medium . However, this was not the case when fraction C was added . N tau-MH release was significantly (p < 0.05) higher when fraction A was added, but, was significantly (p < 0.05) lower when fraction B was added, while fraction C had no effect on N tau-MH release . The present results show that SDBP contained two growth-promoting factors for a primary chick muscle cell culture, although their modes of action may be different. Virology, 1997 Nov 24, 238(2), 424 - 31 A point mutation in the Sendai virus accessory C proteins attenuates virulence for mice, but not virus growth in cell culture; Garcin D et al.; A mutant Sendai virus (SevMVC), which grows much better than its progenitor virus (SeVM) in cell culture, but, in strong contrast to SeVM, is totally avirulent for mice, has been described . SeVMVC contains two amino acid substitutions relative to SeVM, namely, F170S in the C protein and E2050A in the L protein . We have examined which substitutions were responsible for the above phenotypes by exchanging the C gene of our reference strain Z with those of SeVH (another reference strain), SeVM, and SeVMVC, in turn . We have found that the F170S mutation in the CMVC protein is responsible both for enhanced replication in cell culture and for avirulence in mice . Avirulence appeared to be due to restricted viral replication primarily after day 1, implicating some aspect of innate immunity in this process . The SeV C proteins thus appear to be required for multiple cycles of replication in mice. Spine, 1997 Nov 15, 22(22), 2598 - 601; discussion 2602 Murine nucleus pulposus-derived cells secrete interleukins-1-beta, -6, and -10 and granulocyte-macrophage colony-stimulating factor in cell culture; Rand N et al.; STUDY DESIGN: Cultures established from murine disc-derived cells were stimulated by lipopolysaccharide . The cells' capacity to secrete proinflammatory cytokines and interleukin-10 with and without lipopolysaccharide stimulation was determined using enzyme-linked immunosorbent assays . OBJECTIVES: To determine the capacity of disc-derived cells to secrete proinflammatory cytokines, and the effect of lipopolysaccharide stimulation on such secretion . SUMMARY OF BACKGROUND DATA: The pathophysiology of compressive radiculopathy is unclear . Inflammation is a possible explanation . Proinflammatory cytokine secretion was demonstrated in herniated nucleus pulposus . It is unknown whether these cytokines are secreted from disc-derived cells or from infiltrating inflammatory cells in the herniated nucleus pulposus . METHODS: Discs were microsurgically harvested from inbred mice and cut to allow the nucleus pulposus to establish cell culture . A study group was exposed to lipopolysaccharide stimulation . Media were harvested from the study and control groups 24 hours later . Secretion of interleukins-1-, -6, and -10, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha were determined using enzyme-linked immunosorbent assays . RESULTS: Basal secretion of interleukins-6 and -10, but no basal secretion of interleukin-1-, granulocyte-macrophage colony-stimulating factor, or tumor necrosis factor-alpha was detected . Secretion of interleukin-1- rose from zero to 27.69 pg/10(5) cells, and granulocyte-macrophage colony-stimulating factor secretion rose from zero to 9.77 pg/10(5) cells after lipopolysaccharide stimulation . A 75-fold increase in interleukin-6 secretion and a 150-fold increase in interleukin-10 secretion were detected after stimulation with lipopolysaccharide . No tumor necrosis factor-alpha secretion was detectable . All result had high statistical significance (all P < 0.001) . CONCLUSIONS: Cultured murine disc-derived cells have the capacity to secrete proinflammatory cytokines and interleukin-10 in the absence of inflammatory cells . This finding supports the hypothesis that disc-derived cells are capable of initiating or amplifying an inflammatory process. Am J Clin Nutr, 1997 Dec, 66(6 Suppl), 1506S - 1512S Methodologic issues, theoretical considerations, and design criteria for experimental animal and cell culture experiments; Birt DF; This article provides background information that is important when evaluating the relevance to humans of particular animal or in vitro experiments designed to assess the relations between fatty acids and cancer . Considerations in designing carcinogenesis studies to assess the relation between dietary fatty acids and human cancer include selection of the animal model and design of the experimental diets . Animal carcinogenesis models are generally best for evaluating the early phases of cancer development: the initiation and promotion of cancer . Transplantation protocols have been developed for evaluating the effect of diet on the growth and metastasis of partially or fully transformed cells . The variables that are important in such models are the origin and biology of the cell line, the animal host used for the implantation, the site of transplantation, whether the primary tumor is excised after a period of time to allow for metastasis, and when the diets are fed relative to the different phases of tumor growth and metastasis . Studies in cultured cells have been particularly useful for assessing the mechanisms by which fatty acids affect cancer . Considerations in designing studies with cultured cells include selection of the cell line, cell culture conditions, selection of biological endpoints that are relevant to human cancer, and in vivo confirmation of the mechanisms observed in vitro . Design considerations for each of these experimental approaches are discussed and the contributions of each approach are summarized. Plant Physiol, 1997 Nov, 115(3), 1057 - 64 Induction of 12-oxo-phytodienoic acid in wounded plants and elicited plant cell cultures; Parchmann S et al.; Jasmonic acid (JA) is rapidly biosynthesized from alpha-linolenic acid in plants upon contact with pathogens or wounding, and triggers gene activation, leading to the synthesis of defensive secondary metabolites and proteins . Despite the recent finding that its precursor, 12-oxo-phytodienoic acid (PDA), is a more powerful inducer of gene activation, interest has focused so far almost exclusively on JA . A validated negative chemical ionization-gas chromatography-mass spectrometry method has been developed that allows the simultaneous quantification of endogenous 12-oxo-PDA and JA in plant tissues . In six out of eight plant species tested maximal levels of 12-oxo-PDA exceeded peak levels of JA by approximately 3- to 5-fold after elicitation with a yeast cell wall preparation or when plants were wounded . These experiments support the hypothesis that 12-oxo-PDA acts as the predominant jasmonate signal in most plants, whereas JA remains an active metabolite of its precursor . Furthermore, JA but not 12-oxo-PDA was shown to be secreted into the medium from cultured plant cells, suggesting that JA may also act as an intercellular signal. Eur J Cancer, 1997 Sep, 33(10), 1661 - 7 Tumour-derived, endocrine, exogenous and therapeutic factors differentially modulate cytokine secretion in whole blood cell culture; Fischer JR et al.; Following our previous results which showed that TGF-beta 1 suppressed the secretion of certain cytokines, we investigated the effects of different endogenous and exogenous factors on cytokine secretion in whole blood cell culture by using an enzyme-linked immunosorbent assay (ELISA) for measurement of cytokine concentrations . Several molecules including dexamethasone, noradrenaline (NA) and ethanol differentially inhibited mitogen-induced cytokine secretion . Dexamethasone and noradrenaline suppressed secretion of IL-2, IFN alpha, IFN gamma, TNF alpha, IL-1 alpha and IL-1 beta . beta-Endorphin and Leu-Enkephalin had no significant influence on cytokine secretion . Suppression of cytokine secretion by TGF-beta 1 was further intensified significantly and dose dependently by addition of noradrenaline . GM-CSF stimulated the secretion of IL-1 alpha, IL-1 beta and TNF gamma, but had no influence on the secretion of IL-2, IFN alpha and IFN gamma . G-CSF, IL-3 and SCF did not significantly influence secretion of all cytokines tested . Thus, endogenous and exogenous factors differentially influence cytokine secretion by immunocompetent cells. Acta Neuropathol (Berl), 1997 Nov, 94(5), 425 - 35 Central neurocytoma: an immunohistochemical, ultrastructural and cell culture study; Ishiuchi S et al.; To clarify the histogenesis and differentiation potential of central neurocytoma, a pathological investigation of seven tumors from three patients was conducted using immunohistochemistry and ultrastructural analysis in addition to systematic in vitro studies . Six tumors were studied immunohistochemically and five were examined ultrastructurally . All cases that were immunostained were positive for synaptophysin in nuclear-free neuropil islands . In five tumors, a few tumor cells, in addition to reactive astrocytes, were positive for glial fibrillary acidic protein (GFAP) . Vimentin staining was also positive in a few tumor cells of five specimens . Neurofilament staining was always negative . All cases for which ultrastructure was examined showed various synaptic abnormalities . Cultured cells were subdivided into three distinct tumor cell types: neuronal cells which stained for neurofilament proteins with neurosecretory granules; small flat undifferentiated cells with a high nuclear-cytoplasmic ratio and scant cytoplasmic organelles; and small round or multipolar astrocytic cells with 10-nm intermediate filaments which stained for GFAP . Our tissue culture studies disclosed that cultured neurocytoma cells form a cellular mosaic similar to subependymal plate layers that are composed of mitotically active cells, neurons and glia. J Virol, 1997 Dec, 71(12), 9515 - 23 Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment; Lundkvist A et al.; This paper reports the establishment of a model for hantavirus host adaptation . Wild-type (wt) (bank vole-passaged) and Vero E6 cell-cultured variants of Puumala virus strain Kazan were analyzed for their virologic and genetic properties . The wt variant was well adapted for reproduction in bank voles but not in cell culture, while the Vero E6 strains replicated to much higher efficiency in cell culture but did not reproducibly infect bank voles . Comparison of the consensus sequences of the respective viral genomes revealed no differences in the coding region of the S gene . However, the noncoding regions of the S gene were found to be different at positions 26 and 1577 . In one additional and independent adaptation experiment, all analyzed cDNA clones from the Vero E6-adapted variant were found to carry substitutions at position 1580 of the S segment, just 3 nucleotides downstream of the mutation observed in the first adaptation . No differences were found in the consensus sequences of the entire M segments from the wt and the Vero E6-adapted variants . The results indicated different impacts of the S and the M genomic segments for the adaptation process and selective advantages for the variants that carried altered noncoding sequences of the S segment . We conclude that the isolation in cell culture resulted in a phenotypically and genotypically altered hantavirus. J Virol, 1997 Dec, 71(12), 8973 - 82 Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse; Bukreyev A et al.; The small hydrophobic protein SH of human respiratory syncytial virus (RSV) is a short transmembrane surface protein of unknown function . A full-length cDNA of RSV strain A2 (subgroup A) antigenomic RNA was modified such that the entire SH gene, including the transcription signals and the complete mRNA-encoding sequence, was deleted and replaced by a synthetic intergenic region . This reduced the length of the antigenome by 398 nucleotides and ablated expression of 1 of the 10 RSV mRNAs . Recombinant virus containing this engineered deletion was recovered, and the absence of the SH gene was confirmed by reverse transcription in conjunction with PCR . Northern blot analysis of intracellular RNAs and gel electrophoresis of labeled intracellular proteins confirmed the lack of expression of the SH mRNA and protein . The absence of the SH gene did not noticeably affect RNA replication, but two effects on transcription were noted . First, synthesis of the G, F, and M2 mRNAs was increased, presumably due to their being one position closer to the promoter in the gene order . Second, transcription of genes downstream of the engineered site exhibited a steeper gradient of polarity . On monolayers of HEp-2 cells, the SH-minus virus produced syncytia which were at least equivalent in size to those of the wild type and produced plaques which were 70% larger . Furthermore, the SH-minus virus grew somewhat better (up to 12.6-fold) than wild-type recombinant RSV in certain cell lines . While the function of the SH protein remains to be determined, it seems to be completely dispensable for growth in tissue culture and fusion function . When inoculated intranasally into mice, the SH-minus virus resembled the wild-type recombinant virus in its efficiency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract . In mice, the SH-minus and wild-type recombinant viruses were similarly immunogenic and effective in inducing resistance to virus challenge. Biochem Biophys Res Commun, 1997 Nov 7, 240(1), 122 - 7 Expression of alpha 2 macroglobulin receptor/low density lipoprotein receptor-related protein is cell culture density-dependent in human breast cancer cell line BT-20; Li Y et al.; alpha 2Macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) is a multifunctional cell plasma membrane receptor that binds and facilitates the endocytosis of a number of ligands involved in protease regulation and lipoprotein metabolism . In the invasive breast cancer cell line BT-20 we show that the expression of alpha 2MR/LRP can be strongly affected by cell culture density . By comparing measurements of mRNA, total cellular alpha 2MR/LRP antigen, and cell surface alpha 2MR/LRP expression we have demonstrated a marked cell density-dependent regulation of this receptor expression . Increasing the cell density results in a 3.4-fold increase in cell surface alpha 2MR/LRP expression . This corresponds to a marked increase in alpha 2MR/LRP mRNA in Northern blots of total RNA from cells cultured at high density and to consistent increases in alpha 2MR/LRP heavy chain in Western blots of cell lysates from high density cultures . These studies together demonstrate the significant up-regulation of alpha 2MR/LRP expression in BT-20 by increased cell density. Carcinogenesis, 1997 Oct, 18(10), 1889 - 95 O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures . Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro; Liu Y et al.; Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein . An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively . The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity . Exposure of buccal cell cultures to various organic or water-based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts) . Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff . An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect . The well-established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM . Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro . Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53 . Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers. Biomaterials, 1997 Oct, 18(20), 1355 - 9 Hydrogels in endovascular embolization . VI . Toxicity tests of poly(2-hydroxyethyl methacrylate) particles on cell cultures; Horak D et al.; Cytotoxicity of poly(2-hydroxyethyl methacrylate) {poly(HEMA)} hydrogel spherical particles, prepared by radical suspension polymerization and designed for endovascular occlusion, was studied in vitro on cell cultures . Testing methods included a direct contact test and extraction test . No inhibition of growth of cells surrounding the poly(HEMA) beads and a very low inhibition of cell viability, only in concentrated extracts in long-term contact, were observed . As a result, poly(HEMA) beads can be considered non-toxic. Microsc Res Tech, 1997 Oct 15, 39(2), 150 - 6 Paracrine communication in the anterior pituitary as studied in reaggregate cell cultures; Vankelecom H et al.; The classical image of the endocrine system is that secretory function of a gland is regulated from outside that gland by other organs . Focused on the pituitary gland, hormone secretion by the anterior lobe is under control of peptides and biogenic amines produced by the hypothalamus . About a decade ago, our group launched the new idea that functioning of the anterior pituitary (AP) is also regulated from within, i.e., that the constituent cell types inter-communicate to control hormone secretion . Extensive in vitro research has now provided a body of evidence that paracrine communication plays an important role, not only in regulation of hormone secretion but also in development, growth, and differentiation of the AP {reviewed in Denef (1994) The Pituitary Gland, pp . 351-378} . It further revealed that crosstalk between the cells is mediated by local, paracrine, factors . The main objective of our research is to identify those factors, their actions and the producing and target cell type(s) in order to unravel the paracrine communication network that is functional in the AP . Equally important, we set the step towards in vivo examination of the results obtained in vitro using transgenic mice . In the present article, we will review the technology used, three examples of AP cell-to-cell interactions studied, and we will discuss the value of transgenic animal models in the study of AP paracrine communication. Brain Res, 1997 Aug 22, 766(1-2), 153 - 61 Nicotinic acetylcholine sensitivity of rat petrosal sensory neurons in dissociated cell culture; Zhong H et al.; Using whole-cell, patch-clamp techniques we investigated acetylcholine (ACh) sensitivity of dissociated sensory neurons from rat petrosal ganglia after 4 h-14 days in vitro . In approx . 68% of petrosal neurons (PN; n = 109) ACh, applied by fast perfusion or pressure ejection from a 'puffer' pipette, caused a rapid depolarization associated with a conductance increase . Under voltage clamp near the resting potential (approx . - 60 mV), ACh induced a hexamethonium-sensitive, inward current (IACh), mimicked by nicotine application, suggesting the presence of neuronal nicotinic acetylcholine receptors (nAChR) . The reversal potential of IACh occurred near 0 mV (n = 4), a region where the I-V curve displayed a prominent rectification . The dose-response relation for IACh versus ACh concentration was fitted by the Hill equation with EC50 = approx . 33.9 microM and Hill coefficient = approx . 1.6 . The activation phase of IACh was well fitted by a single exponential with mean (+/- S.E.M.) time constant of 102 +/- 82 ms (n = 6); the desensitization phase of IACh was best fitted by the sum of two exponentials, with time constant of 870 +/- 210 ms (n = 6) and 8576 +/- 1435 ms (at -70 mV) . Fluctuation analysis yielded an apparent single-channel conductance of 21.6 +/- 10 pS (mean +/- S.E.M.; n = 4) . These data indicate that a major subpopulation of sensory neurons in visceral petrosal ganglia of the rat express nAChR . Thus, if similar receptors are present on corresponding nerve terminals, they could mediate fast afferent excitation in response to ACh released at peripheral targets, e.g., the chemosensory carotid body. Mol Cell Endocrinol, 1997 Sep 30, 133(1), 41 - 8 Interleukin-1 beta inhibits progesterone accumulation in rat corpora luteal cell cultures in a mechanism dissociated from its effects on nitric oxide and prostaglandin E accumulation; Hurwitz A et al.; The objective of this study was to examine the effect of interleukin-1 beta (IL-1) on progesterone (P) biosynthesis and the potential intermediary involvement of prostaglandin (PG) E and nitric oxide (NO) in P accumulation in PMSG/hCG-primed rat corpora luteal (CL) cell cultures . Exposure of primed CL cells to IL-1 (10 ng/ml) for 48 h resulted in a 65-86% reduction (P < 0.01) in P accumulation concurrent with a 2-3.4-fold increase in PGE content, a 70% increase in PGF2 alpha content and a 1.9-3.3-fold increase in nitrite generation . These effects were abolished by the IL-1 receptor antagonist, suggesting specific IL-1 receptor-mediated effects . Indomethacin, a cyclooxygenase inhibitor, abolished PGE and PGF2 alpha production and attenuated the basal (but not IL-1-stimulated) accumulation of P . N(G)-Nitro-L-arginine (NNLA), a competitive inhibitor of nitrite synthesis, slightly reduced basal P accumulation but had no effect on IL-1-induced suppression of P accumulation . NNLA reduced basal PGE accumulation and IL-1-stimulated PGE accumulation (55 and 61%, respectively) . Transforming growth factor beta 1 (TGF-beta 1; 10 ng/ml) significantly attenuated the IL-1-stimulated PGE and NO production (61 and 42%, respectively), but did not affect the ability of IL-1 to suppress P accumulation . Thus, NO, PGF2 alpha and PGE are not obligatory intermediaries of IL-1-mediated suppression of P accumulation in rat CL, but are involved in basal P biosynthesis and NO seems to have a regulatory role in the biosynthesis of PGE . The present observations suggest a pleiotropic response of PMSG/hCG-primed CL cells to IL-1, characterized by an independent suppression of P accumulation and a concomitant increase in NO, PGF2 alpha and PGE generation . Since IL-1 attenuates P accumulation, these findings may imply a direct autocrine/paracrine function for IL-1 in the maintenance or the demise of rat CL. J Immunoassay, 1997 Nov, 18(4), 371 - 88 Enhanced cytokine detection by a novel cell culture-based ELISA; Shankar G et al.; Production of some cytokines, such as IL-4 and IL-10, often occurs at low levels and is difficult to detect by standard ELISA techniques . In many cases the level of detection is at or near to the limits of sensitivity of the assay due either to minimal synthesis and/or cytokine consumption . In an effort to enhance the quantitation of weakly detected cytokines we have developed a unique cell culture-capture ELISA . Lymphocytes are incubated in an anti-cytokine antibody coated ELISA plate for the last 6 hours of a 24 hour in vitro activation period . Use of this cell culture capture method consistently enhanced detection of several T cell cytokines compared to conventional ELISA techniques . Moreover, this technique was found to enhance detection without altering the rate of cytokine secretion which occurred prior to the cell culture capture period . Thus, the cell culture capture ELISA may be useful for detection of a variety of cytokines which are produced at low levels and have traditionally been difficult to quantify. Am J Physiol, 1997 Oct, 273(4 Pt 1), C1109 - 23 Three-dimensional cell cultures: from molecular mechanisms to clinical applications; Mueller-Klieser W; This article reviews actual advances in the development and application of three-dimensional (3-D) cell culture systems . Recent therapeutically oriented studies include characterization of multicellular-mediated drug resistance, novel ways of quantifying hypoxia, and new approaches to more efficient immunotherapy . Recent progress toward understanding the development of necrosis in tumor spheroids has been made using novel spheroid models . 3-D cultures have been used for studies on molecular mechanisms involved in invasion and metastasis, with a major focus on the role of E-cadherin . Similarly, tumor angiogenesis and the significance of vascular endothelial growth factor have been investigated in a variety of 3-D culture systems . There are many ongoing developments in tissue modeling or remodeling that promise significant progress toward the development of bioartificial liver support and artificial blood . Perhaps one of the most interesting areas of basic research with 3-D cultures is the characterization of embryoid bodies obtained from stable embryonic stem cells . These models have greatly increased the understanding of embryonic development, in particular through the notable exceptional advances in cardiogenesis. Proc Natl Acad Sci U S A, 1997 Nov 11, 94(23), 12644 - 8 Cerebral cortical astroglia from the trisomy 16 mouse, a model for down syndrome, produce neuronal cholinergic deficits in cell culture; Nelson PG et al.; Trisomy 21 (Down syndrome) is associated with a high incidence of Alzheimer disease and with deficits in cholinergic function in humans . We used the trisomy 16 (Ts16) mouse model for Down syndrome to identify the cellular basis for the cholinergic dysfunction . Cholinergic neurons and cerebral cortical astroglia, obtained separately from Ts16 mouse fetuses and their euploid littermates, were cultured in various combinations . Choline acetyltransferase activity and cholinergic neuron number were both depressed in cultures in which both neurons and glia were derived from Ts16 fetuses . Cholinergic function of normal neurons was significantly down-regulated by coculture with Ts16 glia . Conversely, neurons from Ts16 animals could express normal cholinergic function when grown with normal glia . These observations indicate that astroglia may contribute strongly to the abnormal cholinergic function in the mouse Ts16 model for Down syndrome . The Ts16 glia could lack a cholinergic supporting factor present in normal glia or contain a factor that down-regulates cholinergic function. Toxicology, 1997 Dec 5, 123(3), 207 - 15 Cyanide induced DNA fragmentation in mammalian cell cultures; Bhattacharya R et al.; Cyanide is a mitochondrial poison and its toxicity is mediated through histotoxic hypoxia . Although cyanide is regarded as a neurotoxin, its other toxic manifestations are also well documented . Cyanide triggers all those events which can lead to DNA damage, but its genotoxic potential has not been established yet . The present investigation addresses the DNA damage induced by cyanide in rat thymocytes in vitro . Cell viability (eosin Y exclusion and LDH leakage) along with DNA strand breaks were measured in thymocytes exposed to 1.25-10 mM KCN for various time intervals . Cleavage into oligonucleosomal fragments of extracted DNA from cyanide treated thymocytes were visualized on gel electrophoresis . Cyanide produced both time and dose dependent DNA fragmentation accompanied by cytotoxicity . The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was minimal in Ca2+ free medium . The DNA fragmentation was attenuated by Zn2+ (modulator of Ca2+/Mg2+-dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker) . Cyanide induced DNA damage was further observed in baby hamster kidney cells (BHK-21), where unlike thymocytes, internucleosomal DNA fragmentation was not observed . Thymocytes were more sensitive to cyanide as compared to BHK-21 cells. J Neuroendocrinol, 1997 Sep, 9(9), 669 - 75 Primary cell culture of LHRH neurones from embryonic olfactory placode in the sheep (Ovis aries); Duittoz AH et al.; The aim of this study was to establish an in vitro model of ovine luteinizing hormone-releasing hormone (LHRH) neurones . Olfactory placodes from 26 day-old sheep embryos (E26) were used for explant culture . Cultures were maintained successfully up to 35 days, but were usually used at 17 days for immunocytochemistry . LHRH and neuronal markers such as neurofilament (NF) were detected by immunocytochemistry within and/or outside the explant . Three main types of LHRH positive cells are described: (1) neuroblastic LHRH and NF immunoreactive cells with round cell body and very short neurites found mainly within the explant, (2) migrating LHRH bipolar neurones with an fusiform cell body, found outside the explant, (3) network LHRH neuron, bipolar or multipolar with long neurites connecting other LHRH neurons . Cell morphology was very similar to that which has been described in the adult sheep brain . These results strongly suggest that LHRH neurones in the sheep originate from the olfactory placode . This mode may represent a useful tool to study LHRH neurones directly in the sheep. Microbiology, 1997 Oct, 143 ( Pt 10), 3349 - 56 Production of putative virulence factors by Renibacterium salmoninarum grown in cell culture; McIntosh D et al.; A cell culture system, employing the fish cell line Epithelioma papillosum cyprini (EPC), was developed to study the synthesis of intracellular antigen and the expression of putative virulence factors by Renibacterium salmoninarum . EPC cultures infected with R . salmoninarum could be maintained for 7 weeks, during which the pathogen multiplied intracellularly . Immunohistochemical examination of infected cultures revealed the production of the p57 antigen, haemolysin and cytolysin . The intracellular nature of the infection was confirmed by transmission electron microscopic examination of EPC monolayers . A comparison of the relative virulence of bacterial cells cultured in EPC cells and on agar plates revealed that the former were markedly more virulent in challenge experiments with juvenile rainbow trout (Oncorhynchus mykiss Walbaum) . The EPC cell culture model provided a system for the study of R . salmoninarum under more natural conditions than those achieved with plate culture techniques. Curr Opin Biotechnol, 1997 Oct, 8(5), 590 - 4 Stable insect cell cultures for recombinant protein production; McCarroll L et al.; Insect cells are relatively cheap to maintain and are capable of producing accurately translated and correctly processed heterologous proteins . Recent research has focused on the development of improved expression vectors for continuous, high-level production of foreign proteins, including a number of membrane-targeted receptors, in Drosophila and lepidopteran insect cells . Mosquito cells have also been employed for studies on the control of vector-borne diseases, such as malaria. Eur J Clin Chem Clin Biochem, 1997 Sep, 35(9), 661 - 7 Effect of cyclosporine A on the release of tissue factor pathway inhibitor from endothelial cells in heart transplant patients and cell culture; Tiemann C et al.; We investigated the influence of cyclosporine A on the concentration of tissue factor pathway inhibitor and von Willebrand factor antigen in plasma of heart transplant outpatients . Tissue factor pathway inhibitor was quantified in plasma of blood donors (n = 50) and heart transplant outpatients (n = 50) by a chromogenic substrate assay with a mean of 32.4 micrograms/l and 98.2 micrograms/l, respectively . Von Willebrand factor antigen was determined with an enzyme-linked immunoassay with a mean of 90.9% for blood donors and 184.5% in plasma of heart transplant recipients . In addition, we investigated the effect of cyclosporine A on endothelial cell cultures over an incubation period of four days . A dose-dependent effect of cyclosporine A on the release of endothelial tissue factor pathway inhibitor and von Willebrand factor antigen was determined in a concentration range from 100 to 200 micrograms/l cyclosporine A . The tissue factor pathway inhibitor and von Willebrand factor antigen concentrations in the cell culture supernatant increased during the incubation time according to the cyclosporine A concentration 2-3 fold and 2 fold, respectively . For a further elucidation of the cyclosporine A effect we investigated the influence of cremophor EL, the vehicle of cyclosporine A . Cremophor EL alone did not increase the tissue factor pathway inhibitor release . However, the release was enhanced 2-4 fold after co-stimulation with the calcium ionophore A 23187 (10(-4) mol/l) in a concentration-dependent mode . We conclude that a generalized endothelial damage or activation is most probably caused by cyclosporine A and its vehicle cremophor EL . This process probably depends upon the increase of cytosolic free calcium, as described for the liberation of von Willebrand factor by endothelial cells. Cell Biol Toxicol, 1997 Oct, 13(6), 399 - 403 Liver cell culture methods to measure DNA alterations produced by chemicals and radiation; Williams GM; Liver culture systems, both primary cultures of hepatocytes and replicating cell lines, can be used in a variety of ways to study the DNA-damaging effects of chemicals and radiation . The present report describes some of the available methods and their interpretation. FEBS Lett, 1997 Sep 29, 415(2), 160 - 2 Opposite effects of cell differentiation and apoptosis on Ap3A/Ap4A ratio in human cell cultures; Vartanian A et al.; The biological role of diadenosine oligophosphates (DAOP) remains obscure in spite of numerous attempts to solve this enigma . It is known that Ap3A contrary to Ap4A accumulates in human cultured cells treated with interferons (IFNs) alpha or gamma . Since IFNs are considered as antiproliferative regulators, we assumed that different cell status may be associated with varying intracellular levels of DAOP . Promyelocytic human cell line HL60 induced by phorbol ester (TPA) to differentiate to macrophage-like cells in culture exhibits a profound loss of proliferative potential . Here we have shown a 4-5-fold increase in Ap3A concentration in HL60 cells induced by TPA, similar to the effect of IFN, while the Ap4A concentration remained unchanged . On the contrary, in cells undergoing apoptosis induced by VP16, a topoisomerase II inhibitor, the Ap3A concentration considerably decreased, while the Ap4A concentration increased . These findings combined with earlier results suggest an involvement of the Ap3A/Ap4A ratio in signal transduction pathways controlling the cell status. Neurosci Lett, 1997 Sep 19, 233(2-3), 148 - 50 Fast blue, a fluorescent tracer in glioma cell culture, affects cell proliferation and motility; Vince GH et al.; The azo-dye, Fast Blue (FB), initially employed for retrograde neuronal tracing is increasingly used in cell invasion and migration studies to detect living cells in monolayer and glioma tumor cell spheroid models . As yet, it is assumed that a cell stained with a tracker dye retains the characteristics of the original cell . The following experiments compared the adhesion, migration and proliferation properties of the cell lines U373 and GaMG with and without FB staining . FB staining reduced cell adhesion (P < 0.01) and proliferative activity (P < 0.01) and also had a significant inhibitory effect on cell migration (P < 0.001) . From the results presented it follows that FB staining markedly influences basic cell characteristics. Microsc Res Tech, 1997 Oct 1, 39(1), 71 - 84 Monolayer and three-dimensional cell culture and living tissue culture of gallbladder epithelium; Nakanuma Y et al.; Several models for preparing and isolating human and animal gallbladder epithelial cells, including low-grade gallbladder carcinoma cells, as well as proposed systems for culturing these isolated epithelial cells are reviewed here . Several reports concerning tissue culture of the gallbladder are also reviewed . The cell culture systems are divided into monolayer cell culture on collagen-coated or uncoated culture dishes or other culture substrate and three-dimensional cell culture in collagen gel . To prepare and isolate gallbladder epithelial cells, digestion of the gallbladder mucosa, abrasion of the mucosal epithelial cells, and excision of epithelial outgrowth of mucosal explants are applied . In monolayer cell culture, most of the specific biological features of isolated and cultured cells characteristic to the gallbladder are gradually lost after several passages, though quantitative and objective analyses of the pathophysiology of cultured cells and their secretory substances can be performed . Tissue culture using explants of the gallbladder has mainly been used for physiological studies of the gallbladder, such as investigating the transport of water and electrolytes . In this tissue culture system, quantitative assessment is difficult, though the original and specific biological and histological characteristics of the gallbladder are retained . Three-dimensional collagen gel culture could be an ideal model combining monolayer cell culture and tissue culture systems, and create controllable conditions or environments when several biologically active substances, such as growth factors, proinflammatory cytokines and adhesion molecules, are added to the culture medium . Advantages and shortcomings of individual cultivation models are discussed, and selecting the culture model most appropriate to the purpose of the study will facilitate investigations of the biology and pathogenetic mechanisms of gallbladder diseases such as cholelithiasis. Ann N Y Acad Sci, 1997 Sep 26, 826, 416 - 21 Effects of cholinesterase inhibitors on the secretion of beta-amyloid precursor protein in cell cultures; Lahiri DK et al.; One of the main characteristics of Alzheimer's disease (AD) is the cerebrovascular deposition of the amyloid beta-peptide (A beta), which is derived from a larger beta-amyloid precursor protein (beta APP) . The majority of beta APP is processed by either a secretory of lysosomal/endosomal pathway . Carboxyl-truncated soluble derivatives of beta APP (sAPP) are generated by the proteolytic processing of full-length beta APP by either alpha- or beta-secretase enzyme . Our objective is to determine whether the processing of beta APP can be regulated by cholinesterase inhibitors, some of which were shown to produce a moderate improvement in memory and cognitive functions in patients with Alzheimer's disease . Here we have analyzed the levels of sAPP derivatives in cultured cells treated with different drugs by immunoblotting samples of conditioned media . The immunoreactive protein bands were developed by probing with the monoclonal antibody 22C11 . Treating neuroblastoma, pheochromocytoma and fibroblast cells with high dose of either 3,4-diaminopyridine, metrifonate, or physostigmine did not inhibit the secretion of sAPP . Treating glioblastoma with either 3,4-diaminopyridine or metrifonate showed an increase in secretion of sAPP . However, treatment of cells with tacrine reduced release of sAPP in conditioned media of cell lines studied . The difference in action of metrifonate, physostigmine, and tacrine on beta APP is independent of their anticholinesterase activities . Our results suggests that noncatalytic functions of cholinesterase inhibitors can be utilized to alter the metabolism of beta APP, which might in turn affect the process of deposition of A beta, a key component of the cerebrovascular amyloid detected in AD. J Clin Endocrinol Metab, 1997 Oct, 82(10), 3464 - 70 Natural antiestrogen receptor autoantibodies in man with estrogenic activity in mammary carcinoma cell culture: study of their mechanism of action; evidence for involvement of estrogen-like epitopes; Tassignon J et al.; We previously reported that human natural autoantibodies enriched in antiestrogen receptor Ig (IgGs) display estrogenic activity in MCF-7 mammary carcinoma cells . In this study, we investigated IgGs' mechanism of action . We showed that: 1) IgGs Fab fragments (which contain only one antigen binding site) induced an estrogenic response in MCF-7 cells, producing estrogen receptor (ER) down-regulation and an increase in progesterone receptor concentration; 2) IgGs specifically inhibited MCF-7 cell surface labeling with fluorescent estradiol (E2)-BSA conjugates; 3) this inhibition of E2-BSA binding to membrane estrogen binding sites was largely caused by natural anti-E2-BSA antibodies (Ab) selectively associated with the natural anti-ER Ab within IgGs; 4) furthermore, these natural anti-E2-BSA Ab accounted for most of IgGs estrogenic activity in cell culture; 5) however, when incubated with cytosolic ER, they did not behave like estrogens, but they decreased ER hormone binding capacity; and 6) although IgGs stimulated cAMP production, their anti-E2-BSA Ab subpopulation did not . In conclusion, the estrogenic activity of IgGs does not involve Ab mimicking E2 molecular configuration or ligand-independent cAMP mediated pathways, membrane Fc receptors, and membrane receptor cross-linking mechanisms . On the contrary, IgGs seem to function by neutralizing estrogen-like epitopes, associated with ER-related peptides, which might inhibit ER activation. Pharm Res, 1997 Sep, 14(9), 1210 - 5 Estimation of the relative contribution of the transcellular and paracellular pathway to the transport of passively absorbed drugs in the Caco-2 cell culture model; Pade V et al.; PURPOSE: The objective of this investigation was to determine, using the Caco-2 cell culture model, the extent to which the paracellular and transcellular routes contributed to the transport of passively absorbed drugs . An effort was also made to determine the controlling factors in this process . METHODS: We selected a heterologous series of drugs with varying physicochemical parameters for the investigation . Effective permeability coefficients of the model drugs (naproxen, phenytoin, salicylic acid, chlorothiazide, furosemide, propranolol, diltiazem, ephedrine, and cimetidine), at pH 7.2 and pH 5.4, were estimated using confluent monolayers of Caco-2 cells . The biophysical model approach, based on molecular size restricted diffusion within an electrostatic field of force, used by Adson et al . (1,2), was employed to estimate the permeability coefficients of the ionized and unionized forms of the drugs for the paracellular and transcellular route . RESULTS AND CONCLUSIONS: The permeability coefficients of the acidic drugs was greater at pH 5.4, whereas that of the basic drugs was greater at pH 7.2 and the transcellular pathway was the favored pathway for most drugs, probably due to its larger accessible surface area . The paracellular permeability of the drugs was size and charge dependent . The permeability of the drugs through the tight junctions decreased with increasing molecular size . Further, the pathway also appeared to be cation-selective, with the positively charged cations of weak bases permeating the aqueous pores of the paracellular pathway at a faster rate than the negatively charged anions of weak acids . Thus, the extent to which the paracellular and transcellular routes are utilized in drug transport is influenced by the fraction of ionized and unionized species (which in turn depends upon the pKa of the drug and the pH of the solution), the intrinsic partition coefficient of the drug, the size of the molecule and its charge. Bioconjug Chem, 1997 Sep-Oct, 8(5), 708 - 13 Expression and characterization of bryodin 1 and a bryodin 1-based single-chain immunotoxin from tobacco cell culture; Francisco JA et al.; Bryodin 1 (BD1) is a potent ribosome-inactivating protein (RIP) isolated from the plant Bryonia dioica . It is relatively nontoxic in rodents (LD50 > 40 mg/kg) and represents a potential improvement over other RIPs and bacterial toxins that have been used in immunotoxins . Recombinant BD1, expressed in Escherichia coli, localizes to insoluble inclusion bodies necessitating denaturation and refolding steps to generate active protein . In this report, BD1 was expressed as a soluble recombinant protein in tobacco cell culture (ntBD1) and purified to near homogeneity with yields of up to 30 mg/(L of culture) . The protein synthesis inhibition activity of ntBD1 was identical to that of both native BD1 isolated from the roots of B . dioica and recombinant BD1 expressed in E . coli . Toxicology analysis showed that ntBD1 was well tolerated in rats at doses that cannot be achieved with most other toxin components of immunotoxins . Additionally, a single-chain immunotoxin composed of BD1 fused to the single-chain Fv region of the anti-CD40 antibody G28-5 (ntBD1-G28-5 sFv) was expressed in tobacco tissue culture as a soluble protein and was specifically cytotoxic toward CD40 expressing non-Hodgkin's lymphoma cells in vitro . These data indicate that tobacco tissue culture is a viable system for soluble expression of BD1 and BD1-containing immunotoxins. Ophthal Plast Reconstr Surg, 1997 Sep, 13(3), 168 - 73 An immortalized cell culture from a malignant mixed tumor of the lacrimal gland; Lauer SA et al.; Tumor cells from a malignant mixed tumor of the lacrimal gland were maintained in tissue culture for more than 55 generations . Comparative immunohistochemical analysis was performed on whole tumor sections and on the tumor cell culture to define the origin of the cells in culture . The cultured cells expressed cytokeratin, smooth-muscle actin, S-100 protein, and vimentin and were negative for glial fibrillary acidic protein . Tumor sections expressed cytokeratin but were negative for muscle-specific actin, vimentin, and glial fibrillary acidic protein . Through tissue culture studies of salivary gland epithelial neoplasias, which are very similar to lacrimal gland epithelial neoplasias, pluripotential stem cells have been identified . Similar tissue culture analysis of lacrimal gland epithelial neoplasms can be a valuable tool for studying the origin of these uncommon tumors. Planta Med, 1997 Oct, 63(5), 429 - 32 Antiviral activity of natural sulphated galactans on herpes virus multiplication in cell culture; Carlucci MJ et al.; A sulphated galactan (SG) with low molecular weight (app . 2800) was isolated from extracts of Cryptopleura ramosa, a red seaweed from the South American coasts . The compound was a selective inhibitor of HSV-1 and HSV-2 replication in Vero cells with 50% inhibitory concentrations (IC50) in the range 1.6-4.2 micrograms/ml and a 50% cytotoxic concentration (CC50) of 476 micrograms/ml . SG was also effective against HSV-1 in cells of neural origin such as murine astrocytes . The mode of action of SG could be ascribed to an inhibitory action on virus adsorption . Furthermore, SG did not inhibit the blood coagulation process at concentrations highly exceeding the IC50. Neurochem Res, 1997 Oct, 22(10), 1299 - 307 Activities of enzymes involved in the metabolism of platelet-activating factor in neural cell cultures during proliferation and differentiation; Francescangeli E et al.; Platelet-Activating Factor (PAF) is a potent lipid mediator involved in physiological and pathological events in the nervous tissue where it can be synthesized by two distinct pathways . The last reaction of the de novo pathway utilizes CDPcholine and alkylacetylglycerol and is catalyzed by a specific phosphocholinetransferase (PAF-PCT) whereas the remodelling pathway ends with the reaction catalyzed by lyso-PAF acetyltransferase (lyso-PAF AcT) utilizing lyso-PAF, a product of phospholipase A2 activity, and acetyl-CoA . The levels of PAF in the nervous tissue are also regulated by PAF acetylhydrolase that inactivates this mediator . We have studied the activities of these enzymes during cell proliferation and differentiation in two experimental models: 1) neuronal and glial primary cell cultures from chick embryo and 2) LA-N-1 neuroblastoma cells induced to differentiate by retinoic acid (RA) . In undifferentiated neuronal cells from 8-days chick embryos the activity of PAF-PCT was much higher than that of lyso-PAF AcT but it decreased during the period of cellular proliferation up to the arrest of mitosis (day 1-3) . During this period no significant changes of lyso-PAF AcT activity was observed . Both enzyme activities increased during the period of neuronal maturation and the formation of cellular contacts and synaptic-like junctions . The activity of PAF acetylhydrolase was unchanged during the development of the neuronal cultures . PAF-PCT activity did not change during the development of chick embryo glial cultures but lyso-PAF AcT activity increased up to the 12th day . RA treatment of LA-N-1 cell culture in proliferation decreased PAF-PCT activity and had no significant effect on lyso-PAF AcT and PAF acetylhydrolase indicating that the synthesis of PAF by the enzyme catalyzing the last step of the de novo pathway is inhibited when the LA-N-1 cells are induced to differentiate . These data suggest that: 1) in chick embryo primary cultures, both pathways are potentially able to contribute to PAF synthesis during development of neuronal cells particularly when they form synaptic-like junctions whereas, during development of glial cells, only the remodelling pathway might be particularly active on synthesizing PAF; 2) in LA-N-1 neuroblastoma cells PAF-synthesizing enzymes coexist and, when cells start to differentiate the contribution of the de novo pathway to PAF biosynthesis might be reduced. Neurochem Res, 1997 Oct, 22(10), 1205 - 13 Effects of maturation on the phospholipid and phospholipid fatty acid compositions in primary rat cortical astrocyte cell cultures; Murphy EJ et al.; Phospholipid and phospholipid fatty acid compositional changes were studied in rat cortical astrocytes during dibutyryl cyclic adenosine monophosphate (dBcAMP, 0.25 mM) treatment starting after 14 days in culture (DIC) . After 15 DIC, ethanolamine- and choline glycerophospholipid levels were increased 1.2- and 1.3-fold, respectively in treated compared to control cells . However, after 21 and 28 DIC, these levels were not significantly different between groups . Both groups had an increase in phosphatidylserine levels with increasing time in culture . Similarly, ethanolamine plasmalogen levels were transiently elevated after 21 DIC, but returned to previous levels after 28 DIC . The phospholipid fatty acid compositions for the acid stable and labile ethanolamine- and choline glycerophospholipids indicated that in dBcAMP treated cells, 20:4 n-6 and 22:6 n-3 proportions were elevated with increasing time in culture relative to control cells . As 20:4 n-6 proportions increased, there was a concomitant decrease in 20:3 n-9 proportions, suggesting an up regulation of n-6 series elongation and desaturation . In contrast, in control cells, the 20:4 n-6 proportions decreased with a corresponding increase in the 20:3 n-9 proportions . Thus, in treated cells, the cellular phospholipid fatty acid composition was dramatically different than control cells, suggesting that dBcAMP treatment may act to increase fatty acid elongation and desaturation. Anat Anz, 1997 Oct, 179(5), 453 - 60 Diversity of pituitary cells in primary cell culture . An immunocytochemical study; Orgnero de Gaisan E et al.; In cell cultures of dispersed rat anterior pituitary, the specific identification of each cell type based on their staining properties and the ultrastructural features of secretory granules has proved to be unreliable . The existence of pituitary cell subtypes and the striking remodeling of the cell surface and intracellular organelles, further complicate the specific identification of pituitary cell populations . An immunocytochemical study of dissociated pituitary cells in culture was carried out to identify the cellular hormonal content by applying specific antibodies against prolactin (PRL), and growth (GH), luteinizing (LH beta), adrenocorticotrophic (ACTH) and thyrotrophic (TSH) hormones . Specifically bound IgG was exposed by the electron microscope with protein A-gold complex . Typical lactotrophs, somatotrophs and gonadotrophs are easily recognized because they retain the main features described in the pituitary tissue in situ . Other undefined groups of cells bearing small or medium round secretory granules can be identified by immunocytochemistry as PRL, GH or TSH producing cells . The latter technique was critical for the characterization of the hormonal content of secretory granules, the shape, size, electron density and cytoplasmic distribution of which differ substantially from those described in the intact gland . Cells displaying rare small oval or sharp pointed secretory granules were identified as gonadotrophs with anti-LH beta, while corticotrophs showed gra