Role of sphingosine kinase 2 in cell migration toward epidermal growth factor

Sphingosine 1-phosphate (S1P), produced by two sphingosine kinase isoenzymes, denoted SphK1 and SphK2, is the ligand for a family of five specific G protein-coupled receptors that regulate cytoskeletal rearrangements and cell motility. Whereas many growth factors stimulate SphK1, much less is known of the regulation of SphK2. Here we report that epidermal growth factor (EGF) stimulated SphK2 in HEK 293 cells. This is the first example of an agonist-dependent regulation of SphK2. Chemotaxis of HEK 293 cells toward EGF was inhibited by N,N-dimethylsphingosine, a competitive inhibitor of both SphKs, implicating S1P generation in this process. Down-regulating expression of SphK1 in HEK 293 cells with a specific siRNA abrogated migration toward EGF, whereas decreasing SphK2 expression had no effect. EGF contributes to the invasiveness of human breast cancer cells, and EGF receptor expression is associated with poor prognosis. EGF also stimulated SphK2 in MDA-MB-453 breast cancer cells. Surprisingly, however, down-regulation of SphK2 in these cells completely eliminated migration toward EGF without affecting fibronectin-induced haptotaxis. Our results suggest that SphK2 plays an important role in migration of MDA-MB-453 cells toward EGF.     

Activated K-ras and N-ras oncogenes in primary renal mesenchymal tumors induced in F344 rats by methyl(methoxymethyl)nitrosamine.

Administration of methyl(methoxymethyl)nitrosamine to newborn Fischer 344 rats results in the preferential induction of renal tumors arising from the mesenchymal component of the kidney. DNA from a significant proportion of these tumors was capable of transforming NIH/3T3 cells. This report describes the renal tumor model, the detection of two different ras transforming genes in the kidney tumors (the N-ras oncogene in 1 and K-ras oncogene in 10 kidney tumors) and the characterization of DNA sequences specifying the transformed phenotype.     

Community structure and demography of small-mammal populations in insular montane forests in southern India

bstract Small mammals were studied in insular montane forest patches in the Upper Nilgiris in southern India from February 1994 to September 1996. Nine patches were selected at two sites, one with a single large 600-ha patch, the other with several small patches ranging in size from 0.2 to 60 ha. The population characteristics and community structure of small mammals were studied in relation to patch size and habitat structure within the patches. The two most abundant species were Rattus rattus (2–36 individuals/ha) and Suncus montanus (0–11 individuals/ha), while the abundance of seven other species recorded were very low. The population characteristics of R. rattus examined were density, biomass, proportions of juveniles, sub-adults and adults, mean weight of all animals, mean weight of adults, sex ratio and persistence. The proportion of adults, sub-adults, mean weight of animals, and mean weight of adult males were correlated with patch size. Persistence of sub-adult females in the large patch was especially high. The density and biomass of other small-mammal species were also studied. Two indices of diversity, species richness and proportion of R.␣rattus were compared as measures of community structure. Seven habitat characteristics were measured; of these, canopy cover, canopy height and tree density were correlated with the size of the patch. Density and biomass of species other than R. rattus and proportion of R. rattus were correlated with canopy height. Density and biomass of species other than R. rattus were highest in smaller patches. While the population characteristics of R. rattus may be affected by patch size, the density of rare species may be influenced by factors related to lower canopy height. Migration between patches may be an important factor in maintaining populations in these patches. Received: 5 January 1998 / Accepted: 23 March 1998     

Animal models for breast cancer

Rodent mammary tumors induced by chemical carcinogens have proven to be very useful in the genetic analysis of initiation, promotion and progression of mammary carcinogenesis. We are studying rat mammary carcinomas induced by the chemical carcinogen, N-nitroso-N-methylurea. The earliest genetic event observed in the mammary gland is the activation of Ha-ras oncogenes, which is followed by promotion of the initiated cells by hormones involved in puberty. Preferential amplification of the mutated Ha-ras allele, of PRAD-1 and IGF2, loss of expression of the mitogenic growth factor gene, MK, and mutation in the tumor suppressor gene, p53, are seen in the mammary tumors during tumor progression.     

Parallel and antiparallel aggregation of alpha-helices. Crystal structures of two apolar decapeptides X-Trp-Ile-Ala-Aib-Ile-Val-Aib-Leu-Aib-Pro-OMe (X = Boc, Ac)

An apolar helical decapeptide with different end groups, Boc- or Ac-, crystallizes in a completely parallel fashion for the Boc-analog and in an antiparallel fashion for the Ac-analog. In both crystals, the packing motif consists of rows of parallel molecules. In the Boc-crystals, adjacent rows assemble with the helix axes pointed in the same direction. In the Ac-crystals, adjacent rows assemble with the helix axes pointed in opposite directions. The conformations of the molecules in both crystals are quite similar, predominantly alpha-helical, except for the tryptophanyl side chain where chi 1 congruent to 60 degrees in the Boc- analog and congruent to 180 degrees in the Ac-analog. As a result, there is one lateral hydrogen bond between helices, N(1 epsilon)...O(7), in the Ac-analog. The structures do not provide a ready rationalization of packing preference in terms of side-chain interactions and do not support a major role for helix dipole interactions in determining helix orientation in crystals. The crystal parameters are as follow. Boc-analog: C60H97N11O13.C3H7OH, space group Pl with a = 10.250(3) A, b = 12.451(4) A, c = 15.077(6) A, alpha = 96.55(3) degrees, beta = 92.31(3) degrees, gamma = 106.37(3) degrees, Z = 1, R = 5.5% for 5581 data ([F] greater than 3.0 sigma(F)), resolution 0.89 A. Ac-analog: C57H91N11O12, space group P2(1) with a = 9.965(1) A, b = 19.707(3) A, c = 16.648(3) A, beta = 94.08(1), Z = 2, R = 7.2% for 2530 data ([F] greater than 3.0 sigma(F)), resolution 1.00 A.     

Eradication of microscopic hepatic metastases by active specific immunization

Strain-2 guinea pigs, each with microscopic deposits of line 10 hepatocarcinoma in the liver, were treated by ID immunization with a mixture of irradiated tumor cells and an oil-in-water emulsion containing cell walls of Mycobacterium bovis strain Bacillus Calmette-Guérin (BCG CWE). Injection of line 10 hepatoma cells into the hepatic portal vein led to the development of tumor foci in the liver, metastasis in the hepatic lymph node, malignant ascites, and death. Active immunization using irradiated line 10 cells and BCG CWE was effective therapy when administered 1, 7, and 14 days after intraportal injection of line 10 cells. Effective immunization required both irradiated line 10 tumor cells and the BCG cell wall emulsion. Immunization with BCG CWE admixed with irradiated line 1 tumor cells, a hepatoma antigenically distinct from line 10, did not prevent outgrowth of line 10 deposits in the liver. Animals rendered free of disease could reject a challenge of line 10 tumor cells but not of line 1 tumor cells.     

Callus culture and plantlet production in Carica papaya (Var. Honey Dew)

Summary In order to develop techniques for efficient callus production and regeneration in Carica papaya (Var. Honey Dew), lamina, petiole, stem and root explants from in vitro plantlets were cultured in media supplemented with 2.0 mg/1 IBA and 0.5 mg/1 BAP. Use of in vitro-grown plantlets as an explant source helped to avoid contamination common in papaya tissue culture. Callusing was maximum in root explants cultured in a modified MS (half-strength) medium. Shoot reganeration was maxium in root-derived callus grown in full-strength modified MS medium supplemented with 0.5 mg/1 IBA and 1 to 2 mg/1 kinetin. A histological study indicated that shoot buds originated from peripheral cell layers of the callus. Each shoot regenerated from callus was subcultured using a multiplication medium. Root formation was induced in all shoots treated in half-strength of modified MS medium containing 2 mg/1 IBA and rooted shoots were transferred successfully to the field.Abbreviations MS Murashige and Skoog medium, 1962 - LS Linsmaier and Skoog medium, 1965 - BAP 6-Benzylaminopurine - FAA Formalin Acetic acid 30% Ethanol, 1110 - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA Napthaleneacetic acid - SH Schenk and Hilderbrandt medium, 1972     

The Bordetella Bps polysaccharide is critical for biofilm development in the mouse respiratory tract

Bordetellae are respiratory pathogens that infect both humans and animals. Bordetella bronchiseptica establishes asymptomatic and long-term to life-long infections of animal nasopharynges. While the human pathogen Bordetella pertussis is the etiological agent of the acute disease whooping cough in infants and young children, it is now being increasingly isolated from the nasopharynges of vaccinated adolescents and adults who sometimes show milder symptoms, such as prolonged cough illness. Although it has been shown that Bordetella can form biofilms in vitro, nothing is known about its biofilm mode of existence in mammalian hosts. Using indirect immunofluorescence and scanning electron microscopy, we examined nasal tissues from mice infected with B. bronchiseptica. Our results demonstrate that a wild-type strain formed robust biofilms that were adherent to the nasal epithelium and displayed architectural attributes characteristic of a number of bacterial biofilms formed on inert surfaces. We have previously shown that the Bordetella Bps polysaccharide encoded by the bpsABCD locus is critical for the stability and maintenance of three-dimensional structures of biofilms. We show here that Bps is essential for the formation of efficient nasal biofilms and is required for the colonization of the nose. Our results document a biofilm lifestyle for Bordetella in mammalian respiratory tracts and highlight the essential role of the Bps polysaccharide in this process and in persistence of the nares.     

Structural and functional analyses of the major outer membrane protein of Chlamydia trachomatis

Chlamydia trachomatis is a major pathogen throughout the world, and preventive measures have focused on the production of a vaccine using the major outer membrane protein (MOMP). Here, in elementary bodies and in preparations of the outer membrane, we identified native trimers of the MOMP. The trimers were stable under reducing conditions, although disulfide bonds appear to be present between the monomers of a trimer and between trimers. Cross-linking of the outer membrane complex demonstrated that the MOMP is most likely not in a close spatial relationship with the 60- and 12-kDa cysteine-rich proteins. Extraction of the MOMP from Chlamydia isolates under nondenaturing conditions yielded the trimeric conformation of this protein as shown by cross-linking and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with different concentrations of acrylamide. Using circular dichroism spectroscopy, we determined that the trimers were formed mainly of beta-pleated sheet structures in detergent micelles. Using a liposomal swelling assay, the MOMP was found to have porin activity, and the size of the pore was estimated to be approximately 2 nm in diameter. The trimers were found to be stable in SDS at temperatures ranging from 4 to 37 degrees C and over a pH range of 5.0 to 8.0. In addition, the trimers of MOMP were found to be resistant to digestion with trypsin. In conclusion, these results show that the native conformation of the MOMP of C. trachomatis is a trimer with predominantly a beta-sheet structure and porin function.