Phenotype modulation in non-adherent and adherent sublines of Walker carcinosarcoma cells: the role of cell-substratum contacts and microtubules in controlling cell shape, locomotion and cytoskeletal structure

We characterised two sublines of Walker carcinosarcoma cells generated by epigenetic changes. Subline 1 cells were mostly polarised and made no or only non-adhesive cell-substratum contacts. Subline 2 cells were spread, adhesive and mainly non-polar. Subline 1 cells migrate in a non-adhesive mode which is very efficient but operates only in a 3D environment, whereas subline 2 cells migrate in an adhesive mode, which is less efficient but works on 2D and 3D substrata. Nocodazole had little or no effect on shape, polarity and locomotion of subline 1 cells. In glass-adherent subline 2 cells, 10(-6)M nocodazole increased the proportion of polarised cells migrating in an adhesive mode and decreased adhesion to the substratum, whereas 10(-5)M nocodazole further reduced the contacts and the cells reverted to a non-adhesive mode of locomotion. When non-polar subline 2 cells were detached mechanically or by nocodazole, they became polarised and morphologically indistinguishable from non-adherent subline 1 cells. On more adhesive plastic substrata, subline 2 cells produced heterogeneous responses to nocodazole including loss of polarity. The phenotypes of Walker carcinosarcoma sublines have similarities with a broad range of cell types ranging from leucocytes to fibroblast-like cells, suggesting that these phenotypic differences can be controlled by the adhesive and contractile state rather than the cell type. Adhesion modulates contractility (isometric or isotonic contraction) and vice versa and this determines morphology (shape, F-actin, myosin and alpha-actinin), locomotion and responses to microtubule-disassembly. The model may be applied to analyse the mechanisms controlling the phenotype of cells in general.     

Variance in LDH isozyme patterns in a Chinese hamster (Cricetulus griseus) colony.

1. LDH activity and isozyme pattern were examined in the liver and epididymal fat pad of animals in 12 different sublines of the Upjohn Chinese hamster colony, which was established to produce animals with spontaneous diabetes. 2. Considerable divergence was observed and the animals could be divided into 3 groups according to LDH-H activity. Each group was significantly different from the other in epididymal fat pad LDH-1, 2,3 and 5 and liver LDH-3, 4 and 5. 3. The variance in LDH isozyme pattern bore no relationship to the state of diabetes but appeared to arise from other genetic determinants. However, within a single subline, a significant correlation between blood sugar and epididymal fat pad LDH-5 was observed.     

The structure of tracheobronchial mucins from cystic fibrosis and control patients.

Tracheobronchial mucin samples from control and cystic fibrosis patients were purified by gel filtration chromatography on Sephacryl S-1000 and by density gradient centrifugation. Normal secretions contained high molecular weight (approximately 10(7] mucins, whereas the cystic fibrosis secretions contained relatively small amounts of high molecular weight mucin together with larger quantities of lower molecular weight mucin fragments. These probably represent products of protease digestion. Reducing the disulfide bonds in either the control or cystic fibrosis high molecular weight mucin fractions released subunits of approximately 2000 kDa. Treating these subunits with trypsin released glycopeptides of 300 kDa. Trypsin treatment of unreduced mucin also released fragments of 2000 kDa that could be converted into 300-kDa glycopeptides upon disulfide bond reduction. Thus, protease-susceptible linkages within these mucins must be cross-linked by disulfide bonds so that the full effects of proteolytic degradation of mucins remain cryptic until disulfide bonds are reduced. Since various combinations of protease treatment and disulfide bond reduction release either 2000- or 300-kDa fragments, these fragments must represent important elements of mucin structure. The high molecular weight fractions of cystic fibrosis mucins appear to be indistinguishable from control mucins. Their amino acid compositions are the same, and various combinations of disulfide bond reduction and protease treatment release products of identical size and amino acid composition. Sulfate and carbohydrate compositions did vary considerably from sample to sample, but the limited number of samples tested did not demonstrate a cystic fibrosis-specific pattern. Thus, tracheobronchial mucins from cystic fibrosis and control patients are very similar, and both share the same generalized structure previously determined for salivary, cervical, and intestinal mucins.     

Adenosine release from isolated rat adipocytes: influence of fat cell concentration and cell size

The release of adenosine by isolated rat adipocytes into the incubation medium was studied in relation to fat cell size and concentration. Incubations were carried out for 60 min at 37 degrees C in Krebs-Ringer bicarbonate-albumin medium containing 6 mM glucose. 2'-Deoxycoformycin was added to inhibit endogenous adenosine deaminase activity (maximal suppression was achieved at 0.8 microM concentration of the inhibitor). The data show that (a) the amount of adenosine released into the medium was similar for the first and second 30-min incubation periods; (b) increasing adipocyte concentration markedly inhibited adenosine release; and (c) large fat cells (volume greater than 300 pl) released significantly more adenosine (per fat cell) into the medium than smaller fat cells (volume less than 180 pl) when incubated at concentrations of less than or equal to 350,000 cells/ml. Above this cell concentration, differences between adenosine release and cell size were not noted. Adenosine release by isolated rat adipocytes appears to be a precisely regulated process which is exquisitly sensitive to the number of fat cells in the incubation medium and, to a certain extent, to the adipocyte size.     

Saturation analysis of cellular retinoid binding proteins: application to retinoic acid resistant human neuroblastoma cells and to human tumors

A method for saturation analysis of cellular retinoic acid and retinol binding proteins, CRABP and CRBP, respectively, in cultured cells and human tumor samples, and its application to a retinoic acid resistant subline of the human neuroblastoma LA-N-5 cell line is described. Assessment of retinoid binding was accomplished by incubation of cytosols with increasing concentrations of [3H]retinoid (28-43 Ci/mmol; 1 Ci = 37 GBq) for 24 h. Bound retinoid was separated from free retinoid by adsorption with dextran-coated charcoal. Nonspecific binding was quantitated in parallel incubations which had been treated with p-chloromercuribenzene sulfonate (PCMBS), resulting in selective elimination of sulfhydryl-dependent ligand binding to both CRABP and CRBP. Quantitation was accomplished by Scatchard analysis of specific (PCMBS sensitive) binding. Employing this technique, specific retinoid binding was attributed to the presence of 2S macromolecules which displayed the known properties of CRABP and CRBP, namely ligand specificity, saturability, high ligand affinity, and PCMBS sensitivity. The apparent dissociation constants (Kd) for retinoic acid binding in cytosols prepared from murine 3T6 fibroblasts, rat testes, and a human ovarian tumor were 7, 11, and 35 nM, respectively. These preparations also bound retinol with high affinity, exhibiting Kds of 12, 26, and 48 nM, respectively. A retinoic acid resistant subline of LA-N-5 cells designated LA-N-5-R9 was established by long-term culture in the presence of 10(-6) M retinoic acid. This subline is resistant to the effects of retinoic acid in that it requires a 10-fold higher concentration of retinoic acid for 50% inhibition of growth than the parent line and displays no retinoic acid induced morphologic differentiation. Saturation analysis of CRABP in the parent and resistant subline reveal no significant alteration in either CRABP content or affinity. These results indicate that resistance to retinoic acid induced differentiation in LA-N-5-R9 occurs distal to CRABP binding or that CRABP does not mediate this response to retinoic acid.     

Genetic evaluation of ovulatory disorders in Austrian Fleckvieh cows: a comparison between linear models and survival analysis

The objective of this study was to compare linear models and survival analysis for genetic evaluation of ovulatory disorders, which included veterinary treatments of silent heat/anestrus and cystic ovaries. Data of 23 450 daughters of 274 Austrian Fleckvieh sires were analyzed. For linear model analyses, ovulatory disorders were defined as a binary response (presence or absence) in the time periods from calving to 150 days after calving and from calving to 300 days after calving. For survival analysis, ovulatory disorders were defined either as the number of days from calving to the day of the first treatment for an ovulatory disorder (uncensored record) or from calving to the day of culling, or the last day of the period under investigation (until 150 or 300 days after calving; censored record). Estimates of heritability were very similar (0.016 to 0.020) across methods and periods. Correlations between sire estimated breeding value from linear model and survival analysis were 0.98, whereas correlations between different time periods were somewhat lower (0.95 and 0.96). The results showed that the length of time period had a larger effect on genetic evaluation than methodology.     

Distinct phenotypic expression of immunogenicity and immunosensitivity in sublines of a tumor

Continuous in vitro or in vivo passage of a BALB/c leukemia has resulted in generation of 2 immunologically distinct sublines. The subline maintained by in vitro passage failed to stimulate an allogeneic response but was susceptible to lysis by alloreactive cytotoxic cells. Conversely, the subline maintained by in vivo passage induced an allogeneic response but was resistant to lysis by cytotoxic T lymphocytes (CTL) reactive to H-2d antigens. Resistance to lysis occurred despite expression of H-2d antigens in a form recognizable by differentiated alloreactive CTL, as determined by cold-target inhibition experiments. Moreover, resistance was immunologically specific, in that the subline was susceptible to immune lysis mediated through recognition of other determinants. The results imply that the display and/or orientation of antigen in the cell membrane of these sublines that is required for a lytic event is distinct from the antigen expression necessary for immunologic recognition.     

Ethyl methanesulfonate-induced mutations of major genes for quantitative phenotypes: mutant alleles for altered activity of brain or liver enzymes in C57BL/6J mice and their inheritance across several generations

We recently identified and confirmed 8 induced mutations in the N2 and N3 progeny of ethyl methanesulfonate (EMS) treated C57BL/6J mice. Each of these mutations altered specific enzyme activities. These separate mutant sublines have been maintained through several generations as heterozygous mutant carriers. The percent decrease of the specific enzyme activity from normal in each subline was calculated for each generation. Additionally, the percentage of breeders within each mutant subline producing abnormal progeny and the fraction of such breeders' total progeny possessing abnormal activity were determined. The aberrant activity values observed in progeny of a confirmed mutant carrier were all lower than normal. 4 of the mutant sublines had decreases in enzyme activities which were constant across the generations analyzed. 3 of the mutant sublines had decreases in activities which were consistent over early generations but changed significantly in later generations. Another subline with decreased enzyme activity was lost. For 7 of the sublines, the number of progeny having altered activity and the number of breeders producing mutant progeny approximated that expected for single gene inheritance. In the remaining subline, a change in the decrease in enzyme activity probably accounts for the deviation from expected inheritance. Although the phenotypes for these quantitative traits are considered to be quasi-continuous, the data indicate that the mutations are probably of major genes.     

Selection of Morphologically Normal Cell Lines from Polyoma-transformed BHK21/13 Hamster Fibroblasts

A selective method was devised for the isolation of "revertants" from polyoma-transformed sublines derived from BHK21/13 Syrian hamster fibroblasts. A hybrid, polyploid subline was obtained by growing together, in mixed culture in the presence of aminopterin, two variant BHK21/13 sublines lacking either inosinic acid pyrophosphorylase or thymidine kinase. Whereas these variant sublines were resistant to 6-thioguanine or to 5-bromodeoxyuridine, the hybrid had regained sensitivity to both analogues. By plating a polyoma-transformed subline derived from this hybrid in the presence of 6-thioguanine, resistant clones were obtained with a frequency of about 10(-4). All of these surviving clones had a reduced chromosome complement and some of them had regained a normal phenotype.     

Cell infiltration in various organ and dilatation of the urinary tubule in NON mice

The non-obese non-diabetic (NON) mouse, which shows no glycosuria, is a subline of the non-obese diabetic (NOD) mouse. Cellular infiltrations in various organs were observed by light and electron microscopy in both sexes from 30 to 300 days after birth. These infiltrations were found in the kidney, pancreas, mandibular gland, parotid gland, exorbital lacrimal gland, and thyroid gland, but not in the adrenal gland, sublingual gland, testis and ovary. The infiltrating cells were mononuclear cells, mostly small lymphocytes. The population and frequency of these cellular infiltrations were weak generally; especially the infiltration into the pancreatic islet, which was very weak compared with that in NOD mice. Dilation of the proximal tubule occurred only in the females at 60 days or more after birth and it gradually increased with age. Numerous acidophil bodies appeared in the epithelial cells and the lumen of these dilated urinary tubules. These bodies were PAS-positive and stained with MT, and They had electron-dense complex structures.     

Body composition and fat distribution among older Jat females: A rural–urban comparison

The aim of the present cross-sectional study is to describe and compare age related changes in body composition and fat patterning among rural and urban Jat females of Haryana State, India. A total of 600 females (rural = 300, urban = 300), ranging in age from 40 to 70 years were selected by the purposive sampling method. Body weight, height, two circumferences (waist and hip) and skinfold thickness at five different sites (biceps, triceps, calf, subscapular, and supra-iliac) were taken on each participant. To study total adiposity, indices such as body mass index (BMI), grand mean thickness (GMT), total body fat and percentage fat were analyzed statistically. The fat distribution pattern was studied using waist/hip ratio, subscapular/triceps ratio and responsiveness of five skinfold sites towards accumulation of fat at different sites with advancing age. Results indicate a decline in almost every dimension including level of fatness between the mid-fourth and mid-fifth decades of life in both rural and urban females. Urban Jat females were heavier (57.36 kg vs. 56.07 kg, p > 0.05) and significantly taller (1553.3 mm vs. 1534.5 mm, p < 0.001) than their rural counterparts. Urban females also exhibited higher mean values for both the circumferences, five skinfold thicknesses as well as for lean body mass, total fat and percentage fat than the rural females. This is also evident from their higher mean values for body mass index and grand mean thickness. Waist/hip ratio values in rural and urban females showed upper body fat predominance, with urban females having relatively more abdominal fat. Results of subscapular/triceps ratio showed that rural and urban females gained proportionally similar amounts of subcutaneous fat at trunk and extremity sites until 45 years of age. Subsequently trunk skinfolds increased relatively more in thickness. The magnitude of this increase was comparatively greater in rural females up to 55 years and among urban females from 55 to 70 years. The profiles of subcutaneous fat accumulation and sensitivity of each skinfold site also revealed more fat deposition in the trunk region compared to extremities in both rural and urban females. The present study demonstrated differential rates of fat redistribution among rural and urban females.     

Proposed structure of two defective viral DNA oligomers produced in 3T3 cells transformed by the ts-a mutant of polyoma virus.

The various oligomeric viral DNA species produced at 32 C by two related syblines of ts-a-transformed mouse 3T3 cells were characterized. Results from the analysis of the cleavage products observed after digestion with restriction endonucleases from Haemophilus parainfluenzae, Escherichia coli RI, and Haemophilus suis are consistent with the assumption that in both sublines, the major oligomeric component is a dimer from which a segment of different length is deleted. The major oligomeric (27S) component in subline 1 was estimated to be 1.77 times the size of the viral monomer, and the major (25.5S) component in subline 15 was estimated to be 1.54 times the size of the viral monomer. These size estimates were confirmed by electron micrograph measurements. The larger oligomers produced by both sublines were found to be multiples of the major oligomeric component of each subline.     

Trypanosoma cruzi: infectivity modulation of a clone after passages through different hosts

Although Trypanosoma cruzi virulence can be modified through passages in vivo or long-term in vitro culture, the mechanisms involved are poorly understood. Here we report modifications in the infectivity of a T. cruzi clone after passages in different hosts without detectable changes in parasite genetic patterns. A clone was obtained from a T. cruzi IIe isolate and showed to be less virulent than the original isolate (p<0.05). This clone was enzymatically similar to the original isolate as shown by multilocus enzyme electrophoresis. Infection of this clone was compared by successive passages in mice and guinea pigs. The mouse-passaged subline became more virulent for both host species compared to the guinea pig-passaged subline (p<0.05). The clone line displayed similar random amplified polymorphic DNA patterns before and after passages in different hosts suggesting that alterations in virulence could be a result of a differential expression of virulence factors.     

Chromosome changes in methotrexate-resistant cell lines of Drosophila melanogaster

Cells resistant to methotrexate (MTX) were obtained from two established cell lines of D. melanogaster and a karyological analysis was performed. No conspicuous changes in the frequencies of cell types were observed in MTX-resistant (MTX^R) subline 0.57, as compared with the original line, whose cells were mostly near-tetraploid. On the contrary, altered karyotypes were frequently noticed in the C1 82 MTX^R subline, as compared with the original line, whose cells were mostly diploid. The C1 82 MTX^R subline was characterized by mostly tetraploid cells and by the presence of chromosome fragments (in 48% of them). The mitotic segregation suggests the presence of a centromere in these fragments and the fluorescence pattern, after quinacrine staining, suggests that they may be derivatives of the X chromosome.     

Carbohydrates modulate opiate receptor mediated mechanisms during postprandial endocrine function

The present study was designed to determine the role of carbohydrates during naloxone-induced opiate receptor blockade upon the postprandial rise of plasma somatostatin (SLI), insulin and pancreatic polypeptide (PP) levels in response to protein and fat test meals in conscious dogs. Test meals consisting of 50 g liver extract + 50 g sucrose or 50 g corn oil + 50 g sucrose dissolved in 300 ml water were instilled intragastrically, respectively. Additionally, liver extract and fat meals were given with a concomitant intravenous infusion of glucose. To all test meals either naloxone (4 mg) or saline was added. The addition of sucrose to liver extract or the infusion of i.v. glucose during the liver meal abolished the inhibitory effect of naloxone on the rise of postprandial somatostatin levels which has been described recently. The addition of carbohydrate either orally or intravenously to the fat meal resulted in an even stimulatory effect of naloxone upon the rise of postprandial somatostatin levels. Insulin levels were not changed during liver extract + sucrose or i.v. glucose, respectively. When sucrose or i.v. glucose was administered together with the fat meal the addition of naloxone augmented postprandial insulin secretion. Pancreatic polypeptide (PP) release was augmented during the combination of sucrose or i.v. glucose with the fat and liver meal when naloxone was present in the meals. The present data demonstrate that the addition of carbohydrates either orally or intravenously to fat and protein meals modulates the effect of endogenous opiates in the regulation of postprandial somatostatin, insulin and pancreatic polypeptide release in dogs in a way that carbohydrates induce inhibitory mechanisms that are mediated via endogenous opiate receptors.     

Loss of target-cell plasma-membrane protein induced by sensitized allogeneic lymphocytes

Sensitized allogeneic aggressor lymphocytes caused a marked loss of plasma-membrane protein in target L cells of a subline sensitive to lymphocyte- or lymphotoxin-induced lysis. By sodium dodecyl sulfate-polyacrylamide electrophoretic analysis, this loss was shown to be general and not restricted to specific membrane fractions. Target L cells of a subline partially resistant to attack by lymphocytes or lymphotoxin showed little or no loss of membrane protein after incubation with sensitized lymphocytes. These observations suggest that target-cell plasma-membrane damage plays an important role in lymphocyte-mediated cytolysis.     

The synergistic effect of X-rays and deficiencies in DNA repair in P-M hybrid dysgenesis in Drosophila melanogaster.

X-rays and deficiencies in DNA repair had a synergistic effect on genetic damage associated with P-element mobility in Drosophila melanogaster. These interactions, using sterility and fecundity as endpoints, were tested in dysgenic males deficient in either excision or post-replication DNA repair. Three sublines of the Harwich P strain were used for the construction of hybrid males. These sublines differ in P-induction ability based on gonadal dysgenesis sterility (GD) and snw mutability tests, in P-element insertion site pattern, and in the types of defective P-elements, such as KP elements, they possess. A lower degree of gonadal dysgenesis was correlated with the presence of KP elements. GD sterility and snw mutability were not always correlated. Dysgenic hybrids originating from the standard reference subline, Harwich(white), were much more sensitive to the post-replication repair than the excision repair defect. In contrast, sterility of hybrids derived from the weak subline was least affected by, and that of hybrids of the strongest subline was most affected by either DNA repair deficiency. The exacerbation by X-rays of the effects of DNA repair deficiencies on genetic damage indicates that both repair mechanisms are required for processing DNA lesions induced by the combined effect of P activity and ionizing radiation.     

Actively induced experimental allergic orchitis in Lewis-resistant (Le-R) rats: reversibility of disease resistance by immunization with Bordetella pertussis

Differential susceptibility to the induction of experimental allergic orchitis (EAO) was examined in Lewis/NCr and Le-R subline rats. Lewis/NCr rats were found to be fully susceptible to the induction of EAO whereas Le-R subline rats were not. Disease resistance exhibited by Le-R rats could be overcome by including Bordetella pertussis in the immunization protocol. However, reversal of resistance with B. pertussis was dependent on the dose of rat testicular homogenate in the inoculum and found to be effective only at lower doses of antigen (10 mg/rat). Disease resistance in Le-R rats as well as B. pertussis-induced reversal of resistance did not appear to be associated with either (1) a significant difference in the number of mast cells in the ductus efferentes, the anatomic location of the earliest inflammatory infiltrates, or (2) an alteration in the phenotypic expression of either innate or B. pertussis-induced sensitivity to vasoactive amines. The results are discussed in the context of the role of B. pertussis in other animal models of organ-specific autoimmune diseases. It is proposed that the phenotypic expression of resistance to EAO in Le-R rats is a result of a mutation in a common regulatory locus affecting susceptibility to multiple autoimmune diseases and whose immunoregulatory action is normally exerted during the sensitization phase of the immune response.     

Mutagen-induced diploid human lymphoblast variants containing altered hypoxanthine guanine phosphoribosyl transferase

The human lymphoblast line MGL8 was treated with HAT and subsequently "mutagenized" with EMS (200 microgram/ml) to give 15% survival, and 6-thioguanine-resistant cells were selected by cloning in soft agarose containing the drug (1 microgram/ml). Eighteen sublines of independently derived resistant clones were isolated and studied in detail. One subline had a low residual HGPRT activity of about 1% of the parental cells. The HGPRT of this subline had a higher Km for PRPP, was more sensitive to heat, and was degraded faster by trypsin than the enzyme in extracts of MGL8 cells. This resistant subline and three others contained CRM levels of 1--38%, compared to the wild-type, so they probably represent true structural mutants of the HGPRT gene. All the variants maintained the karyotype of the parental line (46, XY, 6p-).