Attachment of porcine blastocysts to fibroblast monolayers in vitro

The objective of this study was to compare the ability of porcine blastocysts to attach to various cellular and non-cellular substrates $\text{in vitro}$. One hundred twenty-two hatched blastocysts were collected from 17 handmated gilts and sows at slaughter. Blastocysts were randomly assigned to one of four treatments: Minimal Essential Medium (MEM) supplemented with 10% (v/v) heat treated fetal calf serum (HTFCS), monolayers of bovine uterine fibroblasts in MEM + 10% HTFCS (Buf), monolayers of bovine testicular fibroblasts in MEM + 10% HTFCS (Btes), and MEM + 10% HTFCS exposed to uterine fibroblasts for 24 hr to condition the medium (cMEM). Embryos were cultured individually in 24 well Linbro culture plates at 37 C in a humidified gas atmosphere of 5% CO₂ in air. Embryos were observed at 24 hr intervals by phase contrast microscopy (100X) and measured with an ocular micrometer. Blastocyst attachment was greater (P < .01) in Buf ($\text{21}\text{35}$) compared to MEM ($\text{7}\text{32}$), cMEM ($\text{9}\text{28}$), and Btes ($\text{1}\text{27}$). Embryo diameter was greater (P < .05) 24 hr prior to attachment in Buf compared to the other treatments. In addition, trophoblast monolayers continued to proliferate for 20 days when cocultured with uterine fibroblasts. These observations suggest that uterine fibroblasts provide a superior substratum for blastocyst attachment and the maintenance of swine trophoblast cells $\text{in vitro}$.     

Interferon inhibition of hexose monophosphate shunt activity as the mechanism of blocking differentiation of a preadipose cell line

Mouse L cell interferon (IFN $\text{α}\text{β}$) inhibited the differentiation of mouse 3T3-Li fibroblasts into adipocytes. IFN $\text{α}\text{β}$ also inhibited hexose monophosphate shunt (HMP) activity in these cells. HMP activity is required for the reducing power necessary for the conversion of 3T3-Li fibroblasts to adipocytes. Both IFN blockage of differentiation and HMP activity were reversed by the reducing agent 2-mercaptoethanol, probably through interaction with membrane receptors and not through direct inactivation of IFN. Several non-antiviral effects of IFN $\text{α}\text{β}$ on cellular function, including differentiation and immunoregulation, may be mediated at the biochemical level through blockage of HMP activity.     

The defect in the cblB class of human methylmalonic acidemia: Deficiency of cob(I)alamin adenosyltransferase activity in extracts of cultured fibroblasts

We show that the reductants present in the $\text{in}\text{vitro}$ assay used to measure the formation of adenosylcobalamin from cob(III)alamin by cell-free extracts of human fibroblasts result in the non-enzymatic reduction of cob(III)alamin to cob(I)alamin. Hence, the $\text{in}\text{vitro}$ assay uniquely estimates the activity of ATP:cob(I)alamin adenosyltransferase (EC 2.5.1.17). Based on additional studies with extracts of fibroblasts from patients in the $\text{cbl}\text{B}$ class of human methylmalonic acidemia and from their parents, we conclude that this mutant class results from a specific deficiency of adenosyltransferase activity which is inherited as an autosomal recessive trait.     

Accumulation of weak bases in relation to intralysosomal pH in cultured human skin fibroblasts

The volume of the lysosomal compartment in cultured human skin fibroblasts was estimated from the distribution between the cells and the medium of tracer amounts of labelled methylamine and chloroquine, which accumulate in the lysosomes, 2,2-dimethyloxazolidine-2,4-dione, which accumulates in the soluble cytoplasmic compartment relative to the lysosomes, and sucrose, which is excluded by the cells. In a foetal fibroblast line, the fractional volume of the lysosomal compartment was $\text{0.044 ± 0.007}$ ($\text{n}\text{= 8}$). In fibroblasts from a patient with the I-cell disease, the fractional volume was 0.15.The fractional volume of the lysosomal compartment was used to calculate the intralysosomal pH from the accumulation of the weak bases in the cells. The mean value obtained was $\text{5.29 ± 0.04}$ ($\text{n}\text{= 8}$).In fibroblasts incubated with various concentrations of chloroquine, the fractional volume of the lysosomal compartment and the accumulation of chloroquine in the cells were used to calculate the concentration of chloroquine in the lysosomes. The intralysosomal concentration increased from 3 to 114 mM as the extracellular concentration increased from 1 to 100 μM. Concomitantly, the intralysosomal pH increased from 5.3 in the absence of chloroquine to 5.9 in the presence of 100 μM chloroquine. A similar increase in intralysosomal pH could be calculated in fibroblasts incubated with different concentrations of ammonia.     

Interaction between Dolichos biflorus lectin and chick embryonic fibroblasts at different stages of development

The interaction between chick embryo fibroblasts and A1-specific blood group Dolichos biflorus lectin has been studied at various stages of embryo development. The site number (($\text{0.26±0.03) · 10}{}^6\text{sites/cell}$) remains the same during development whereas the affinity constant apparently decreases from 8-day cells onwards. The effects of cell number, temperature and time course on the Dolichos binding to fibroblasts were not age dependent. Competitive binding experiments revealed that Dolichos receptor sites were distinct from binding sites of Robina pseudoacacia lectin and concanavalin A, but partially related to binding sites of Ricinus lectin. Thymidine incorporation by fibroblasts in the presence of Dolichos lectin was age dependent. It was inhibited in 6-day cells and weakly stimulated in 16-day cells, but not modified in 12-day cells. Dolichos lectin effects on embryo fibroblasts were very specific because both binding to cells and effect on thymidine incorporation were blocked by $\text{N-}\text{acetylgalactosamine}$, the determinant of Dolichos lectin, as well as by Dolichos antiserum.     

Action of insulin analogues on cultured human fibroblasts reflects biological potency.

The biological sensitivity of cultured human skin-derived fibroblasts was examined in terms of the ability of insulin and insulin analogues to stimulate the uptake of alpha-aminoisobutyrate in these cells. The relative biological activity measured: insulin=desalanine insulin > proinsulin > desoctapeptide insulin parallels exactly the relative activity of these compounds on fat and muscle preparations both $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$. Inactive insulin analogues do not stimulate alpha-aminoisobutyrate uptake. It is concluded that the chemical specificity of human fibroblast insulin receptors is retained in culture and that the biological responsiveness (alpha-aminoisobutyrate uptake) of such cells to insulin should prove useful for comparative studies of receptors obtained from different individuals.     

Metabolic activation of 3-methylcholanthrene: mutagenic and transforming activities of the 9,10-dihydrodiol.

3-Methylcholanthrene and five related dihydrodiols have been tested for microsome-mediated mutagenicity towards $\text{Salmonella}\text{typhimurium}$ TA100 and for the induction of mutation to 8-azaguanine resistance in V79 Chinese hamster cells and malignant transformation in M2 mouse fibroblasts. In both mutagenicity test systems, the 9,10-diol was considerably more active than either the parent hydrocarbon, the related $\text{cis}$-2α,3-diol, the $\text{trans}$-4,5-, the $\text{trans}$-7,8- or the $\text{trans}$-11,12-dihydrodiols. At a non-toxic concentration (1μg/ml medium), the 9,10-diol induced the formation of more transformed malignant foci in cultures of M2 cells than 3-methylcholanthrene and the other diols were either inactive or only weakly active in this test system. The results obtained indicate that the 9,10-dihydrodiol derived from 3-methylcholanthrene is involved, presumably following conversion into the corresponding vicinal diol-epoxide, 9,10-dihydro-9,10-dihydroxy-3-methylcholanthrene 7,8-oxide, in the metabolic activation of this carcinogenic polycyclic hydrocarbon.     

Pharmacology of fibrosis: Definitions,limits and perspectives

Methods tested for inhibition of fibrotic lesion are classified as $\text{specific}$ - inhibiting exclusively collagen metabolism and $\text{non-specific}$ - controlling any step of the fibroproliferative inflammation preceding the activation of fibroblasts. A brief account is given on existing as well as perspective methods to present a framework for a concept on pharmacology of fibrosis.     

Activation of guanylate cyclase and inhibition of adenylate cyclase by cytotoxic preparations from marine sponges of Haliclona genus.

Of seven marine sponges tested only two, $\text{Haliclona}\text{viridis}$ and $\text{Haliclona}\text{rubens}$, yielded preparations that activated rat heart microsomal guanylate cyclase and exhibited direct hemolytic activity. These two preparations also inhibited basal and fluoride-activated adenylate cyclase in rat heart microsomes and glucagon-stimulated adenylate cyclase in rat liver plasma membranes. Hemolytic activity co-purified with nucleotide cyclase-modulating activity during a standard lipid fractionation procedure. This fraction was cytotoxic to 3T3-4a Swiss mouse fibroblasts.     

In situ degradation of sphingomyelin by cultured normal fibroblasts and fibroblasts from patients with Niemann-Pick disease type A and C

Cultured normal human skin fibroblasts actively degraded sphingomyelin [choline-methyl-¹⁴C] introduced in ethanolic solution in the culture medium. After 17 h incubation, about 65 to 80 % of the cellular radioactivity was recovered in phosphatidylcholine. In fibroblasts from Niemann-Pick disease type A the $\text{in situ}$ degradation of sphingomyelin was less than 2 % of controls, which was in good agreement with the strong decrease of the sphingomyelinase activity measured in $\text{vitro}$ by conventional methods. In the two cases of Niemann-Pick disease type C studied, the in situ degradation of sphingomyelin was significantly but not dramatically decreased compared to controls.     

Release of low density lipoprotein receptors from human fibroblasts or HeLa cells by tryptic digestion.

Trypsin treatment of cultured normal human skin fibroblasts or Hela cells releases material which is retained on a low density lipoprotein (LDL)-Sepharose affinity column, may be eluted from it with 2.5 M KI and, after dialysis, agglutinates LDL or apo-B-coated formocells. Such agglutination is prevented by preincubation of the receptor with LDL in solution or with arginine-rich protamine. Trypsin treatment of “receptor defective” or “receptor negative” mutant fibroblasts releases material which is retained on LDL-Sepharose column but fails to agglutinate LDL-coated formocells. The receptor may be labeled with $\text{6-[}{}^3\text{H]-glucosamine·HCl}$ and [${}^3\text{H}$]-leucine, it is inactivated by heating at 80°C for 10 min and may be obtained from normal fibroblasts or Hela cells, whether they were cultured in presence or in absence of lipoprotein-containing fetal calf serum.     

Prostaglandin and thromboxane production by fibroblasts and vascular endothelial cells

We have compared the production of prostaglandins in fibroblast-like cells and endothelial cells in culture. Of the fibroblasts studied $\text{10T}\text{1}\text{2}$, SHE, BP6T and KD produce significant amounts of PGI₂, PGE₂ and PGF_2F2 under optimal culture conditions, but only 3T3 and BHK produce TxA₂ in addition to PGI₂. The adult bovine aortic endothelial cells (ABAE) and fetal bovine heart endothelium (FBHE) synthesise PGI₂ but not TxA₂, either from endogenous or exogenous substrates. Both cultured endothelial cells and fibroblasts apparently lack 15-hydroxyprostaglandin dehydrogenase pathway and the ability to convert 6-Keto PGF_1α into 6-Keto PGE₁. PGI₂ production by ABAE was 3–5 times that of FBHE, about twice that of SHE cells and 6–8 times that of $\text{10T}\text{1}\text{2}$ or BP6T cells. Supernatants or media obtained from these cells inhibited aggregation of human platelet-rich plasma, a known biological effect of PGI₂. This effect was abolished when cell monolayers were preincubated with indomethacin or tranylcypromine. RIA and chromatographic data of 6-Keto PGF_1α from these experiments confirmed that the inhibition of platelet aggregation was due to the formation of PGI₂. The production of all prostanoids by endothelial cells or fibroflasts was significantly higher during the exponential phase of growth as compared to confluent monolayers. We propose that fibroblasts $\text{10T}\text{1}\text{2}$ or SHE can serve as useful experimental models for the study of metabolism and transport of PGI₂ and/or TxA₂ in cells of nonendothelial nature.     

Specific versus nonspecific target cell destruction by T lymphocytes sensitized in vitro. II. Responsible factors for nonspecific destruction

Cell-free supernatants of thoracic duct lymphocyte cultures which were stimulated in vitro by horse serum on syngeneic fibroblast monolayers are demonstrated to be cytotoxic on syngeneic embryonic fibroblasts by means of a direct cell count using microtest plates. Experimental supernatants showed up to 100% suppression of fibroblast growth at $\text{1}\text{3}$ dilution and up to 96% suppression at $\text{1}\text{4}$ dilution as compared to the control supernatants. Evidence is presented indicating that lymphocytes cultured on mosaic monolayers, which were comprised of syngeneic and xenogeneic fibroblasts, were reacting both to xenogeneic cells and horse serum in the medium at the cellular level. A hapten-to-carrier type relationship is suggested between xenogeneic antigen and horse serum. Absence of horse serum in the test cultures using these lymphocytes resulted in the abrogation of nonspecific toxic activity of lymphocytes while the specific activity, though diminished, remained. This again indicates the difference in the mechanisms underlying the specific and nonspecific target cell destruction by T cells.     

Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann-Pick type C and type D patients

Regulation of intracellular cholesterol metabolism has been studied in Epstein-Barr virus-transformed lymphoblasts from patients with Niemann-Pick type C (NPC) and the Nova Scotia type D (NPD) disease. Addition of LDL to normal lymphoblasts cultured in lipoprotein-deficient medium increased cholesterol esterification 10-fold (to a maximum of 1.0 nmol/h/mg protein at 15 h), while little stimulation was seen in NPC cells. The response by NPD lymphoblasts was intermediate, reaching approximately half of normal values by 12–24 h. Lymphoblasts from both NPC and NPD obligate heterozygotes exhibited 50% of normal LDL-stimulated cholesterol esterification at 6 h, when activity was $\text{s}\text{1}\text{?}\text{0\%}$ of normal values in patient cells. Fluorescence staining with filipin indicated excessive intracellular accumulation of LDL-derived cholesterol in both NPC and NPD lymphoblasts. Downregulation of LDL receptor mRNA levels by LDL, measured by S1 nuclease protection assay, was also impaired in NP lymphoblasts and fibroblasts (NPC > NPD), although a similar rate of receptor protein down-regulation by LDL ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 10–15}\text{h}$) was observed in normal and NP lymphoblasts. In contrast, LDL down-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA did not appear to be affected in NP cells: LDL produced a 3-fold (lymphoblasts) of > 10-fold (fibroblasts) decrease by 12 h in both normal and affected cells. Thus, NPC and NPD lymphoblasts exhibit distinct defects in cholesterol esterification and storage, similar to those observed in mutant fibroblasts. Other regulatory responses are also impaired in NPC lymphoblasts but appear to be less affected in NPD cells. Lymphoblasts should provide a valuable immortalized cell line model for study of defective regulation of cholesterol esterification and transfort in Niemann-Pick type II disease, and may also suitable for diagnosis and carrier detection.     

An uncommon fucosyl linkage in surface membranes of human neuroblastoma cells

The surface membranes of human neuroblastoma cells contain a fucosyl linkage, defined by using an α-L-fucosidase from almond emulsin specific for the cleavage of Fucα1→3G1cNAc and Fucα1→4G1cNAc. These linkages are not found in significant amounts on the surface of mouse neuroblastoma cells, or human or hamster fibroblasts. The enzyme released fucose from glycoproteins as well as glycopeptides, making it particularly useful for $\text{in}$$\text{vivo}$ studies.     

Carnitine uptake into human heart cells in culture

The uptake of radiolabeled carnitine and butyrobetaine has been studied in human heart cells (CCL 27). The uptake of carnitine is 3–10-fold higher in heart cells than in fibroblasts ($\text{pmol}\text{· μ}\text{g DNA}{}^{\mathrm{?1}}$). The uptake of carnitine increases with temperature coefficient $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ of 1.6 in the interval 10–20° C and with a negligible uptake at 4 and 10° C. The uptake of carnitine follows Michaelis-Menten kinetics with a $\text{K}{}_{\mathrm{<mtext>M</mtext>}}$ of $\text{4.8 ± 2.2 μ}\text{M}$ and $\text{V = 8.7 ± 3.2}\text{pmol}\text{· μ}\text{g DNA}{}^{\mathrm{?1}}\text{·}\text{h}{}^{\mathrm{?1}}$. Carnitine uptake is suppressed 90% by NaF (24 mM). Butyrobetaine is taken up into heart cells to the same extent as carnitine with a $\text{K}{}_{\mathrm{<mtext>M</mtext>}}$ of 5.7–17.3 μM and $\text{V = 8.7–9.3}\text{pmol}\text{· μ}\text{g DNA}{}^{\mathrm{?1}}\text{·}\text{h}{}^{\mathrm{?1}}$. Butyrobetaine inhibits competitively the uptake of carnitine and carnitine inhibits the uptake of butyrobetaine to the same extent. No conversion of radiolabeled butyrobetaine to carnitine, or carnitine to methyl choline was observed intra- or extracellulary during incubation. These data are compatible with a selective transport mechanism for carnitine which is also responsible for the uptake of butyrobetaine.     

Comparative studies between microbiological culture and uptake of uridine/uracil to detect mycoplasmal infection of cell cultures.

Controlled, prospective studies were performed to compare detection of cell culture mycoplasmas by ratio of uptake of tritiated uridine (UdR) to tritiated uracil (U) and by microbiological culture. Culture was by standard agar and broth inoculation with aerobic and anaerobic incubation; immunofluorescent staining of indicator cell cultures was used to detect M. hyorhinis. The ratio of uptake of UdR to U ($\text{UdR}\text{U}$) and interpretation of test results were by standard published methods and performed in triplicate. 115 cell cultures were simultaneously assayed by the two techniques. 84 cultures (73.1%) yielded agreement between the 2 methods; 2 cultures (1.7%) yielded conflicting results, and 29 cultures (25.2%) yielded $\text{UdR}\text{U}$ results in the questionable range. Conflicting results consisted of two negative $\text{UdR}\text{U}$ tests in mouse cell cultures infected with M. orale. In separate studies, 3T-6 cultures freshly infected with M. orale yielded negative $\text{UdR}\text{U}$ results 3 days after infection, questionable results after 10 days and a positive $\text{UdR}\text{U}$ 17 days after infection. $\text{UdR}\text{U}$ detected infection in fibroblast, epithelial, and lymphocyte cell cultures. Highest $\text{UdR}\text{U}$ ratios were detected in human skin fibroblasts at early population doubling levels (PDLs), 4064 in one culture at PDL4. $\text{UdR}\text{U}$ was determined for IMR-90, a human diploid fibroblast at 12 different PDLs using the same lot of media. $\text{UdR}\text{U}$ gradually decreased throughout the life of the culture, from 2 125 at PDL6 to 340 at PDL36. Cultures in phase III and others exhibiting poor growth frequently yielded questionable or false-positive $\text{UdR}\text{U}$ results and were not included in tabulations of these results. $\text{UdR}\text{U}$ determined in endothelial cell cultures decreased as population density increased. In a representative experiment performed over a 4-day period, the $\text{UdR}\text{U}$ values were 1 808, 955 and 356 when the number of endothelial cells in culture were 5.3 × 10⁵, 6.6 × 10⁵ and 1.1 × 10⁶, respectively.     

Separation of plasma membrane markers by glycerol-induced blistering of muscle cells

Glycerol (50%, w/w) was found to cause blistering of chick primary myoblast and fibroblast plasma membranes and extensive blistering of 5–6-day-old-myotube plasma membranes in tissue culture. The tips of myoblasts and fibroblasts appeared to be the most sensitive portion of the plasma membrane to the blistering effect of glycerol. The glycerol-induced blistering of myotubes was reduced and delayed by brief EDTA pretreatment.Glycerol treatment (50, 15 and 8% sequentially) of myotubes was used to remove plasma membrane blisters and a plasma membrane-enriched fraction was isolated from these blisters using a modified Dextran T500-polyethylene-glycol 6000 aqueous two-phase polymer system. This fraction was found to be enriched 4.1-fold for 5′-nucleotidase activity, but not for other putative plasma membrane markers, ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity or $\text{α-[}{}^{125}\text{I}\text{]}\text{bungarotoxin}$ binding material. Autoradiographs of $\text{α-[}{}^{125}\text{I}\text{]}\text{bungarotoxin}$, glycerol-treated (50%, w/w) myotubes showed the plasma membrane blisters to be devoid of reduced silver grains.5′-Nucleotidase was shown to be an ectoenzyme on myoblasts and 5-day-old myotubes and the total cellular activity was present on the cell surface. During the period of myoblast fusion and myotube formation, cell surface activity decreased to a low level while total cellular activity was elevated.     

N-acetylneuraminic acid and sialoglycoconjugate metabolism in fibroblasts from a patient with generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from a patient suffering from generalized N-acetylneuraminic acid storage disease were found to accumulate large amounts (approx. 4.0 μmol/g fresh weight) of free N-acetylneuraminic acid in a lysosome-enriched subcellular fraction. However, there were no detectable deficiencies in lysosomal hydrolase activities (including neuraminidase), and the activities of CMP-N-acetylneuraminic acid synthetase and N-acetylneuraminic acid aldolase were within normal limits. The cellular glycoconjugate composition was normal, and pathologic fibroblasts labeled with either [³H]glucosamine-HCl or $\text{N-[}{}^3\text{H]acetylmannosamine}$ showed a marked accumulation of labeled free N-acetylneuraminic acid, along with elevated incorporation into sialoglycoconjugates. Neither normal nor pathologic fibroblasts secreted labeled free N-acetylneuraminic acid into the culture medium. These results are consistent with an inherited defect in N-acetylneuraminic acid reutilization, resulting in the lysosomal accumulation of the free monosaccharide in generalized N-acetylneuraminic acid storage disease.     

Temporal variation of adenine ribonucleotides during the cell cycle of Chinese hamster fibroblasts in culture

Chinese hamster fibroblasts in monolayer culture (Don-C cell line) were synchronized by selective detachment of metaphase cells after brief treatment with colcemid. Replicate monolayer cultures were harvested at intervals after synchronization and ethanolic extracts were prepared for the determination of adenine ribonucleotides with the luciferin-luciferase assay. The level of ATP increased approx. 145% during the cell cycle, with the most rapid increase occurring during the G1 phase. One hour after synchronization (early G 1 phase), 1.3 nmoles of $\text{ATP}\text{10}{}^6$ cells were observed; a maximum of 3.2 nmoles of $\text{ATP}\text{10}{}^6$ cells was reached at 12 h (G 2 phase). The adenylate energy charge, $\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP}\text{)/(}\text{ATP}\text{+}\text{ADP}\text{+}\text{AMP}\text{)}$ was lowest during the G 1 phase (0.7) and increased to 0.9 during the late S and G 2 phase. A slight decrease of energy charge was observed during the second mitosis.