Characteristics of two cell lines derived from a histologically undifferentiated bronchial carcinoma expressing neuroendocrine markers

Summary This study investigates the characteristics of two human cell lines—1PT and 1PT VARIANT A—both derived from the same histologically undifferentiated, neuroendocrine positive, non-small cell lung carcinoma (NSCLC) and capable of growth in unsupplemented serum-free minimum essential medium. In stationary culture, the cells of both lines grew both attached to a plastic substratum and in suspension; the 1PT VARIANT A line formed three-dimensional clusters of loosely adherent cells. The cell lines differed in their DNA content, the 1PT having 1.44 times and the 1PT VARIANT A having 2.39 times the normal human diploid DNA content. Chromosome counts supported this observation, the ploidy of the 1PT and VARIANT A lines being 1.11 and 1.64, respectively. On transmission electron microscopy the cells of both lines had dense core granules and immature desmosomes, whereas only the 1PT VARIANT A line had mucin granules. Both lines formed, in nude mice, tumors that, like the original tumor from which they were derived, were histologically undifferentiated and showed local invasion. The original tumor and both lines had demonstrable neuroendocrine markers. Cytokeratins were apparent in the tumor but not the cell lines, and neurofilaments were present in the cell lines only. Staining for epithelial membrane antigen, neural cell adhesion molecule, and desmoplakin differentiated between the two lines. These lines provide a useful model for the investigation of the biology of the neuroendocrine positive subgroup of NSCLC, which is clinically important because of the possible responsiveness of these tumors to chemotherapy.     

Simple electromyographic biofeedback treatment for chronic pediatric constipation/encopresis: Preliminary report

Pediatric constipation/encopresis is thought to be due, in part, to paradoxical constriction of the external anal sphincter (EAS) muscle during attempted defecation. This inappropriate contraction can lead to delayed, impacted, painful, and infrequent bowel movements. Standard Medical Care (SMC) involves disimpaction with enemas, followed by laxative therapy and diet modification, to maintain frequent soft stools. Using the case control method, the efficacy of SMC alone was compared with SMC plus EAS electromyographic biofeedback aimed at eliminating paradoxical contraction. Thirteen consecutive chronically constipated children received SMC plus biofeedback, and were compared with 13 age- and sex-matched children who received only SMC. Biofeedback subjects demonstrated post-treatment elimination of EAS paradoxical constriction. At 16 months follow-up parents of biofeedback children reported significantly greater improvement in constipation, encopresis, laxative use, and painful bowel movements compared to SMC.This research report was supported by the NIH under grant No. RO1 HD 28160.     

Mechanism of retinoid-induced activation of latent transforming growth factor-β in bovine endothelial cells

Cell-associated plasmin is a putative physiological activator of latent transforming growth factor-β (LTGF-β). Since retinoids enhance the production of plasminogen activator (PA) and thereby increase cell-associated plasmin activity, we tested the possibility that retinoids might induce the activation of LTGF-β using bovine endothelial cells (ECs) as a model system. ECs treated with physiological concentrations of retinol or retinoic acid formed active TGF-β in the culture media in a dose- and time-dependent fashion. Cells were treated with 2 μM retinol for 24 h, and the amount of TGF-β produced during a subsequent 12-h incubation period was measured. Out of a total of 14 pM LTGF-β secreted, 0.7 pM was converted to active TGF-β. Northern blot analyses showed that mRNA levels for TGF-β2 but not for TGF-β1 increased in cells treated with retinol. Inclusion of either inhibitors of PA or of plasmin or antibody against PA in the culture medium as well as depletion of plasminogen from the serum blocked the formation of TGF-β, suggesting that PA, plasminogen, and the resulting plasmin are essential for activation of LTGF-β in retinoid-stimulated cells. Antibody against the LTGF-β binding protein blocked activation implying that localization of LTGF-β through its binding protein may be important. However, inhibition of binding of LTGF-β to the cell surface mannose 6-phosphate receptor did not prevent activation. These data indicate that retinoids up-regulate the production of LTGF-β in ECs and induce activation of LTGF-β, perhaps, by increasing PA and plasmin levels. Thus, TGF-β might be a local mediator of some of the biological activities of retinoids both in vivo and in vitro. © 1993 Wiley-Liss, Inc.     

A τ Fragment Containing a Repetitive Sequence Induces Bundling of Actin Filaments

Abstract: Much indirect evidence suggests that the interconnections of actin microfilaments with the microtubule system are mediated by microtubule-associated proteins (MAPs). In this study we provide new data to support the interaction of a specific tubulin-binding domain on τ with actin in vitro. In actin polymerization assays, the synthetic peptide VRSKIGSTENLKHQPGGG, corresponding to the first repetitive sequence of τ protein, increased turbidity at 320 nm in a dose-dependent fashion. A salient feature of the τ peptide-induced assembly process is the formation of a large amount of actin filament bundles, as revealed by electron microscopic analysis. An increase in the τ peptide concentration resulted in a proportional increase in the bundling of actin filaments. It is interesting that a gradual decrease of pH within the range 7.6–4.7 resulted in a higher effect of τ peptide in promoting bundles of actin filaments. A similar pH-dependent effect was observed for τ protein-induced bundling. An analysis of the mechanisms that operate in the peptide induction of actin filament bundles suggests the involvement of electrostatic forces, because the neutralization of ɛ-aminolysyl residues by selective carbamoylation resulted in a complete loss of the peptide induction of actin bundles. The data suggest that a τ repetitive sequence (also found in MAP-2 and MAP-4) containing a common tubulin binding motif may constitute a functional domain on τ for the dynamics of the interconnections between actin filaments and microtu-bules.     

Familial mediterranean fever (FMF) in Moroccan Jews: Demonstration of a founder effect by extended haplotype analysis

Familial Mediterranean fever (FMF) is an autosomal recessive disease causing attacks of fever and serositis. The FMF gene (designated “MEF”) is on 16p, with the gene order 16cen–D16S80–MEF–D16S94–D16S283–D16S291–16pter. Here we report the association of FMF susceptibility with alleles at D16S94, D16S283, and D16S291 among 31 non-Ashkenazi Jewish families (14 Moroccan, 17 non-Moroccan). We observed highly significant associations at D16S283 and D16S291 among the Moroccan families. For the non-Moroccans, only the allelic association at D16S94 approached statistical significance. Haplotype analysis showed that 18/25 Moroccan FMF chromosomes, versus 0/21 noncarrier chromosomes, bore a specific haplotype for D16S94–D16S283–D16S291. Among non-Moroccans this haplotype was present in 6/26 FMF chromosomes versus 1/28 controls. Both groups of families are largely descended from Jews who fled the Spanish Inquisition. The strong haplotype association seen among the Moroccans is most likely a founder effect, given the recent origin and genetic isolation of the Moroccan Jewish community. The lower haplotype frequency among non-Moroccan carriers may reflect differences both in history and in population genetics.     

Extra- and Intracellular Laccases of the Chestnut Blight Fungus, Cryphonectria parasitica

A double-stranded RNA virus of the chestnut blight pathogen, Cryphonectria parasitica, has been shown previously to reduce accumulation of mRNAs of extracellular laccase (laccase A) produced by this fungus. Both extra- and intracellular laccases have been detected after growth of the fungus in liquid culture. In addition to cellular localization, the two laccases are distinguishable by time of appearance during growth and electrophoretic mobility. Laccase A was purified from the culture filtrate by standard protein purification procedures. The enzyme was characterized as a glycoprotein with a molecular mass of approximately 77 kDa. Both laccase A and laccase B activities were significantly reduced in the hypovirulent (double-stranded RNA-infected) strain UEP1 compared with the isogenic virulent (double-stranded RNA-free) strain EP155/2.     

Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method.

pFL1 is a pUC9 derivative that contains a 572-bp EcoRI insert cloned from plasmid DNA of Xanthomonas campestris pv. citri XC62. The nucleotide sequence of pFL1 was determined, and the sequence information was used to design primers for application of the polymerase chain reaction (PCR) to the detection of X. campestris pv. citri, the causal agent of citrus bacterial canker disease. Seven 18-bp oligonucleotide primers were designed and tested with DNA from X. campestris pv. citri strains and other strains of X. campestris associated with Citrus spp. as templates in the PCR. Four primer pairs directed the amplification of target DNA from X. campestris pv. citri strains but not from strains of X. campestris associated with a different disease, citrus bacterial spot. Primer pair 2-3 directed the specific amplification of target DNA from pathotype A but not other pathotypes of X. campestris pv. citri. A pH 9.0 buffer that contained 1% Triton X-100 and 0.1% gelatin was absolutely required for the successful amplification of the target DNA, which was 61% G+C. Limits of detection after amplification and gel electrophoresis were 25 pg of purified target DNA and about 10 cells when Southern blots were made after gel electrophoresis and probed with biotinylated pFL1. This level of detection represents an increase in sensitivity of about 100-fold over that of dot blotting with the same hybridization probe. PCR products of the expected sizes were amplified from DNA extracted from 7-month-old lesions from which viable bacteria could not be isolated. These products were confirmed to be specific for X. campestris pv. citri by Southern blotting. This PCR-based detection protocol will be a useful addition to current methods of detection of this pathogen, which is currently the target of international quarantine measures.