Effect of mycoplasma on interferon production and interferon assay in cell cultures

Armstrong, D. (The Children's Hospital of Philadelphia, Philadelphia, Pa.), and K. Paucker. Effect of mycoplasma on interferon production and interferon assay in cell cultures. J. Bacteriol. 92:97-101. 1966.-The influence of mycoplasma on the production and action of interferon was studied in cultures of both L and human embryonic kidney (HEK) cells. Mycoplasma hominis 1, the Negroni agent, and the F12 mycoplasma were used for infection of L cells, and M. hominis 1 and M. pneumoniae for inoculation of HEK cells. All strains were capable of multiplication in the culture systems employed. None produced detectable levels of interferon, and responsiveness of the cells to induction of interferon by virus remained unaltered. Infection with mycoplasma did not impair the sensitivity of the cells to the action of interferon, nor was the replication of vesicular stomatitis virus noticeably diminished.     

Isoperoxidases as markers of somatic embryogenesis in carrot cell suspension cultures

Growth, peroxidase activity and isoperoxidase pattern were studied during the growth cycle of 3 cell suspension lines of carrot ( Daucus carota L.), an embryogenic, a non-embryogenic and a habituated cell line. Isoelectric focusing of extracted proteins on agarose gels revealed the isoperoxidase pattern of the embryogenic line to include, among other differences, an isoperoxidase with a pl of pH 7.0 when grown under conditions stimulating embryogenesis. This isoperoxidase (P7.0: EC 1.11.1.7) was present between days 2 and 6 after subculturing, and this period correlates well with the early stages of somatic embryogenesis. This isoenzyme showed very low activity in the non-embryogenic and habituated cell suspension lines as well as in the embryogenic cell line in the presence of Daucus carota , 2,4–dichlorophenoxyacetic acid. P7.0 could probably be used as a biochemical marker of somatic embryogenesis.     

Bovine casein haplotypes: number, frequencies and applicability as genetic markers

Three casein loci, tightly linked on bovine chromosome 6, have been studied as haplotypes within families of bulls. The polymorphisms included in the study were CASAS1 (B, C), CASB (A1, A, A5, B), CASK (A, B, E) and a microsatellite in intron III of CASK. A new A5 variant of CASB, caused by a silent mutation in the triplet coding for amino acid 110, was detected by direct polymerase chain reaction sequencing. Our analysis of 306 sons and 15 sires revealed 10 different casein haplotypes with a cumulative polymorphic information content (PIC) of 0.78.     

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12-16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.     

Anthocyanin and proteins as biochemical markers in maize endosperm cultures

Endosperm maize cultures derived from a strain homozygous for all genes required for anthocyanin synthesis develop an intense pigmentation. Pigmenting ability is generally maintained in successive subcultures, altough colourless areas are frequently observed in pigmented cultures. The isolated colourless cell clusters show a growth rate higher than the coloured ones. These calli nevertheless do not lose the ability to synthesize anthocyanins, and in successive subcultures turn red again.The different growth rates associated with the ability of cells to accumulate pigments suggest the existence of different physiological states of the culture. To investigate this possibility we analyzed the polypeptide patterns of coloured and colourless cultures. SDS gel electrophoresis has demonstrated differences in soluble protein fractions, among which a 26 kD peptide, characteristic of pigmented tissues, has been evidenced. Zein, the major storage protein of maize endosperm is present, although at very low levels, both in pigmented and in unpigmented cultures, confirming that its synthesis occurs continuously in vitro.Abbreviations 2-4D 2,4-dichlorophenoxyacetic acid - SDS sodium dodecyl sulphate - PMSF Phenylmethyl sulphonyl fluoride - DAP days after pollination     

Use of iron from transferrin and microbial chelates as substrate for heme synthetase in transformed and primary erythroid cell cultures

The enzymatic heme production in cell-free extracts of virus-transformed Friend erythroleukemia cells and primary bone marrow cells from rabbits has been measured by determining the activity of heme synthetase after addition of iron sulfate, transferrin or microbial iron chelates. In transformed cells the amounts of heme formed did not show significant differences independent of which substrate was offered. In cell-free extracts of primary bone marrow cells no increase of heme production could be observed.     

Human hepatic cell cultures: in vitro and in vivo drug metabolism

Drug metabolism is the major determinant of drug clearance, and the factor most frequently responsible for inter-individual differences in drug pharmacokinetics. The expression of drug metabolising enzymes shows significant interspecies differences, and variability among human individuals (polymorphic or inducible enzymes) makes the accurate prediction of the metabolism of a new compound in humans difficult. Several key issues need to be addressed at the early stages of drug development to improve drug candidate selection: a) how fast the compound will be metabolised; b) what metabolites will be formed (metabolic profile); c) which enzymes are involved and to what extent; and d) whether drug metabolism will be affected directly (drug-drug interactions) or indirectly (enzyme induction) by the administered compound. Drug metabolism studies are routinely performed in laboratory animals, but they are not sufficiently accurate to predict the metabolic profiles of drugs in humans. Many of these issues can now be addressed by the use of relevant human in vitro models, which speed up the selection of new candidate drugs. Human hepatocytes are the closest in vitro model to the human liver, and they are the only model which can produce a metabolic profile of a drug which is very similar to that found in vivo. However, the use of human hepatocytes is restricted, because limited access to suitable tissue samples prevents their use in high throughput screening systems. The pharmaceutical industry has made great efforts to develop fast and reliable in vitro models to overcome these drawbacks. Comparative studies on liver microsomes and cells from animal species, including humans, are very useful for demonstrating species differences in the metabolic profile of given drug candidates, and are of great value in the judicious and justifiable selection of animal species for later pharmacokinetic and toxicological studies. Cytochrome P450 (CYP)-engineered cells (or microsomes from CYP-engineered cells, for example, Supersomes) have made the identification of the CYPs involved in the metabolism of a drug candidate more straightforward and much easier. However, the screening of compounds acting as potential CYP inducers can only be conducted in cellular systems fully capable of transcribing and translating CYP genes.     

Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium

We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo-like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.     

Fish cell cultures as in vitro models of pro-inflammatory responses elicited by immunostimulants

We have tested the elicitation of innate defence-related responses in two stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss) after they were exposed to different concentrations of lipopolysaccharide (LPS), levamisole, or polyinosinic polycytidylic acid (poly-I:C). For comparison, cultures of rainbow trout head kidney macrophages were also included in the study, and the effect of the immunostimulants on the phagocytic activity, the intracellular and extracellular reactive oxygen species and nitric oxide production were assayed. Although the responses varied depending upon the concentration of the immunostimulants and the particular cell line, our results demonstrate that those activities were enhanced in the TSS and TPS-2 cell lines after exposure to any of the immunostimulants. These results indicate that the stromal cells of the main lympho-haemopoietic organs of O. mykiss develop innate defence responses, which are enhanced by well-known immunostimulants. In addition, such enhancement of the defence responses in the TSS and TPS-2 cell lines could be also elicited when they were exposed to conditioned supernatants from levamisole- or poly I:C-stimulated HK macrophage cultures, thus demonstrating that the haemopoietic stromal cells respond to macrophage-derived factors. Moreover, we demonstrate that the stromal cell lines constitutively expressed the Toll-like receptors TLR3, TLR5 and TLR9 genes. The results are discussed considering the role of the lympho-haemopoietic stromal cells in the innate immune responses, and the possibility of using histiotypic cell cultures of non-leucocyte cells of the haemopoietic organs to develop in vitro methods to select new immunostimulant candidates for aquaculture.     

Use of iron from transferrin and microbial chelates as substrate for heme synthetase in transformed and primary erythroid cell cultures.

The enzymatic heme production in cell-free extracts of virus-transformed Friend erythroleukemia cells and primary bone marrow cells from rabbits has been measured by determining the activity of heme synthetase after addition of iron sulfate, transferrin or microbial iron chelates. In transformed cells the amounts of heme formed did not show significant difeerences independent of which substrate was offered. In cell-free extracts of primary bone marrow cells no increase of heme production could be observed.     

Human macrophage maturation in vitro: expression of functional transferrin binding sites of high affinity

Human blood monocytes (mo) when cultured in suspension on hydrophobic teflon membranes undergo terminal maturation to macrophages (MO). Together with the appearance of new lineage-restricted differentiation antigens, mature MO but not blood mo, express transferrin (TF) receptor molecules as detected by immunostaining methods. Here we report that radio- and fluorescein-labelled TF binds to a single class of high-affinity binding sites on MO but not on mo. As mo mature in vitro in the presence of human serum, their receptor numbers increase to about 10(6) per cell, showing an apparent Kd for Fe2TF of approximately 5 nM. These receptor numbers were comparable with our estimates for cultured K 562 human tumor cells, and about 20x greater than reported for human MO cultured in the presence of fetal calf serum. Our MO showed 58Fe uptake comparable with uptake by tumor cells and also exhibited TF-promoted uptakes of 61Ga. The possibility that MO might recycle stored iron through receptor-bound apoTF was not supported by experiments which showed that their Fe2TF receptors had much lower affinity for apoTF (Kd greater than 1 microM) and which could not detect separate high-affinity receptors specific for apoTF. Expression of TF receptors was not substantially altered by treatment with human recombinant interferon-gamma.     

Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures

The regulation of cell surface fibroblast growth factor (FGF) receptors during the differentiation of F9 teratocarcinoma cells was investigated. The capacity of F9 cells to bind ¹²⁵I-basic FGF (FGF-2) increased upon induction of differentiation with dibutyryl cAMP and retinoic acid. No change in binding capacity was observed in the first 24 h after addition of differentiating agents, but a sixfold increase in binding capacity was observed after 48 h and a fivefold increase after 72 h. Scatchard analysis of the binding data indicated that the increased binding of ¹²⁵I-FGF-2 was due to an increase in the number of receptors with no change in their affinity. When ¹²⁵I-FGF-2 was cross-linked to cell surface receptors, an increase in FGF-2-receptor complexes with molecular weights of 140,000–160,000 was also observed in the differentiated F9 cells. Undifferentiated F9 cells are known to secrete FGF-4 and cease expression of this molecule upon differentiation. To determine whether the low level of receptors in undifferentiated cells might be related to their production of FGF ligands, the ability of suramin, a drug that can disrupt FGF-receptor interactions, to modulate receptor number on F9 cells was investigated. Suramin treatment increased ¹²⁵I-FGF-2 binding capacity of undifferentiated F9 cells threefold but had little effect on the binding capacity of differentiated cells. In addition, antibodies to FGF-4 increased the ¹²⁵I-FGF-2 binding capacity of undifferentiated F9 cells by 58%. These results suggest that undifferentiated F9 cells might be responding in an autocrine manner to their own FGF ligands resulting in downregulation of cell surface FGF receptors. The increased number of receptors observed in differentiated cells may partly result from the decreased production of FGF ligands by these cells. © 1994 Wiley-Liss, Inc.     

Modeling tau polymerization in vitro: a review and synthesis

The major antigenic component of neurofibrillary pathology in a large number of neurodegenerative diseases consists of the microtubule-associated protein tau. It is currently unclear how tau protein makes the transition from an important component of the microtubule-based cytoskeleton to an insoluble polymerized state. In vitro techniques have been employed to study the polymerization of tau in an effort to understand the underlying molecular mechanisms responsible for this process. These efforts have resulted in the elucidation of roles played by the different parts of the molecule in the polymerization process. Here we discuss the advantages and disadvantages of the various techniques used to model tau polymerization and the discoveries arising from these techniques that have led to a better structural understanding of tau polymerization in relation to Alzheimer's disease and other tauopathies.     

Pregnancy and interferon tau regulate major histocompatibility complex class I and beta2-microglobulin expression in the ovine uterus

Major histocompatibility complex (MHC) class I molecules, consisting of an alpha chain and beta2-microglobulin (beta2MG), play an important role in immune rejection responses by discriminating self and nonself and are increased by type I interferons during antiviral responses. Interferon tau (IFNtau), the pregnancy-recognition signal in ruminants, is a type I interferon produced by the ovine conceptus between Days 11 and 21 of gestation. In study 1, expression of MHC class I alpha chain and beta2MG mRNA and protein was detected primarily in endometrial luminal epithelium (LE) and glandular epithelium (GE) on Days 10 and 12 of the estrous cycle and pregnancy. On Days 14-20 of pregnancy, MHC class I and beta2MG expression increased only in endometrial stroma and GE and, concurrently, was absent in LE and superficial ductal GE (sGE). Although neither MHC class I nor beta2MG proteins were detected in Day 20 trophectoderm, beta2MG mRNA was detected in conceptus trophectoderm. In study 2, cyclic ewes were ovariectomized on Day 5, treated daily with progesterone to Day 16, received intrauterine infusions between Days 11 and 16 of either control serum proteins or recombinant ovine IFNtau, and were hysterectomized on Day 17. The IFNtau increased MHC class I and beta2MG expression only in endometrial stroma and GE. During pregnancy, MHC class I and beta2MG gene expression is inhibited in endometrial LE and sGE but, paradoxically, is stimulated by IFNtau in the stroma and GE. The silencing of MHC class I alpha chain and beta2MG genes in the endometrial LE and sGE during pregnancy recognition and establishment may be a critical mechanism preventing immune rejection of the conceptus allograft.     

Gene expression in visceral endoderm: a comparison of mutant and wild- type F9 embryonal carcinoma cell differentiation

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.