Site-specific frameshift mutagenesis by a propanodeoxyguanosine adduct positioned in the (CpG)4 hot-spot of Salmonella typhimurium hisD3052 carried on an M13 vector.

Malondialdehyde induces frameshift mutations in Salmonella typhimurium strain hisD3052. The ability of propanodeoxyguanosine (PdG), a structural analog of the major malondialdehyde-deoxyguanosine adduct, to induce site-specific frameshift mutations was tested in the (CpG)4 hot-spot of hisD3052 carried on an M13 vector (M13MB102). PdG was introduced at position 6248 of duplex M13MB102 by ligation of the oligonucleotide 5'-CGC(PdG)CGGCATG-3' into a heteroduplex containing an 11-nucleotide gap in the (-)-strand between the SphI and BssHII restriction sites and deoxyuridine in place of thymidine in the (+)-strand. Ligation proceeded with 70% efficiency, and closed circular duplex DNA molecules were isolated in 40% yield. The adducted genome was sensitive to cleavage by SphI but resistant to cleavage by BssHII. Transformation of Escherichia coli strain JM105 with adducted M13MB102 led to 25% reduced survival relative to unadducted M13MB102 and produced frameshift mutations in 2.5% of the progeny phage. All of the mutations were deletions, and 70% occurred by deletion of CpG. Unadducted genomes exhibited a 40-fold lower mutation frequency, and all the mutations were single-base deletions at the sites of ligation of the 11-mer. These results illustrate that PdG, a structural analog of the major malondialdehyde-deoxyguanosine adduct, induces frameshift mutations in M13MB102 and that single-stranded nicks are efficient premutagenic lesions in this recombinant bacteriophage.     

Calcium regulation of growth and differentiation in Streptococcus pneumoniae.

Streptococcus pneumoniae requires 0.15 mM-Ca2+ in the medium for optimal growth. Increasing the Ca2+ concentration to 1 mM triggers either a differentiative state, competence for genetic transformation during exponential growth, or partial lysis as soon as the cultures enter stationary phase. Genetic and physiological data both suggest that these responses are under the control of activator(s), excreted in the presence of high Ca2+ concentrations. 45Ca2+ transport is also stimulated by the activator(s). The amiloride derivative 2',4'-dimethylbenzamil (DMB) inhibits 45Ca2+ transport and prevents lysis and competence development. This provides evidence in favour of the involvement of Ca2+ transport in competence and culture lysis. On the other hand, addition of DNA to a competent culture prevents lysis of wild-type bacteria while a mutant, defective for DNA uptake, is not protected from lysis by exogenous DNA. An hypothesis is proposed for competence induction as a global metabolic response to Ca2+, under the control of competence factor.     

Ovarian development, spawning frequency and batch fecundity in Encrasicholina heteroloba (Ruppell, 1858)

A histological and gravimetric analysis of oocyte development in Encrasicholina heteroloba (Ruppell, 1858) indicated that this species spawns serially and has a group-synchronous mode of ovarian development. A six stage maturity scale, based on both external morphology and oocyte composition, was proposed to classify ovarian development in E. heteroloba . The incidence of females with hydrated oocytes and post-ovulatory follicles in samples from two regions; the south Java Sea and Roviana lagoon, in the Solomon Islands were used to estimate spawning frequency. Estimates of mean inter-spawning intervals ranged from around 2 days in fish from Roviana lagoon to up to 16.7 days in fish from the south Java Sea. Batch fecundity was determined from the number of oocytes in the largest oocyte size class in ripe stage ovaries. Batch fecundity was related to size and was significantly greater for a given size in the Roviana lagoon population ( F = 0.081 × L ^4.89, Roviana population; F = 1.682 × L ^2.83, Jepara population).     

Effect of pH on MHC class II-peptide interactions.

The effect of pH on class II-peptide interactions has been analyzed using several mouse (IAd, IAk, IEd, IEk) and human (DR1, DR5, DR7) MHC specificities, and eight different class II-restricted determinants. In direct binding assays, acidic conditions led to increased binding capacity for many class II-peptide combinations. IE molecules seemed to bind optimally around pH 4.5, whereas IA molecules displayed binding optima in the 5.5 to 6.5 range. In contrast, the DR molecules studied were, in most cases, affected only marginally by pH changes in the 4.5 to 7.0 range. Despite these apparent isotype-specific trends, no general rule could be formulated, because even for the same class II molecules, the binding capacity could be increased for many peptides when the binding was performed under acidic conditions, was unaffected for some, and even decreased for others. The mechanisms responsible for this complex behavior were analyzed in more detail by kinetic and equilibrium analysis of three different class II-peptide combinations (IAd/OVA 323-339, IAk/HEL 46-61, and DR1/HA 307-319). It was found that acidic pH conditions could affect both on and off rates for class II-peptide complexes. Depending on the net balance of these effects, either increases, decreases, or no effect on overall affinities at equilibrium were detected. In the case of IAd/OVA 323-339, it was also found that acidic conditions influenced the binding capacity of class II molecules by increasing the fraction of sites available for peptide binding, presumably by favoring dissociation of endogenously bound, acid-sensitive peptides.     

Fibroblast stimulation in schistosomiasis. XII. Identification of CD4+ lymphocytes within schistosomal egg granulomas as a source of an apparently novel fibroblast growth factor (FsF-1).

Granulomas that form around Schistosoma mansoni eggs deposited in the liver secrete a variety of fibrogenic factors that may provide a molecular link between chronic inflammation and hepatic fibrogenesis in schistosomiasis. We recently isolated from conditioned medium of egg granuloma cultures a approximately equal to 60-kDa heparin-binding growth factor for fibroblasts. Because this protein is distinct from other defined heparin-binding growth factors, we designated it "fibroblast stimulating factor-1" (FsF-1). We now report that FsF-1 is a lymphokine. We prepared IgG antibody against purified FsF-1 and determined that it did not cross-react with a variety of growth factors or recombinant interleukins. Using two-color flow cytometry of dissociated granuloma cell suspensions, we observed that approximately 20% to 25% of granuloma CD4+ lymphocytes express surface FsF-1. We isolated CD4+ granuloma lymphocytes by FACS and observed that these cells spontaneously secrete into culture supernatant a fibroblast mitogen that is neutralized by anti-FsF-1 antibody. Furthermore, anti-FsF-1 can specifically immunoprecipitate a metabolically labeled protein produced by the granuloma CD4+ lymphocytes. The labeled protein has the same apparent molecular mass (approximately equal to 60 kDa) as FsF-1 purified from granuloma culture supernatants. These findings define CD4+ lymphocytes as a source of FsF-1. Because FsF-1 has biologic and chemical features distinct from most other defined lymphokines and from other heparin-binding growth factors, FsF-1 appears to be a novel lymphokine.     

Linear epitope mapping of an Sm B/B' polypeptide.

Autoantibodies binding the Sm B/B' peptides are commonly associated with SLE. IgG antibodies binding overlapping octapeptides of Sm B/B' have been evaluated in 10 patients with anti-Sm and anti-nRNP precipitins, 5 patients with other autoimmune serology, and 4 normal human sera. Neither normal controls nor patients without an anti-Sm precipitin significantly bind any of the Sm B/B' octapeptides. All sera tested containing an anti-Sm precipitin strongly bind octapeptides from eight regions of the Sm B/B' sequence. Three of these eight regions share the same octapeptide sequences (PPPGMRPP) that are consistently the most immunoreactive octapeptides from Sm B/B'. Binding of the similar PPPGIRGP, as well as binding to deletion and substitution peptides, suggest that the motif PPPG(I,M) (R,K) appears to best define this binding. Interestingly, PAPGMRPP in the nRNP C peptide is as antigenic as PPPGMRPP and may provide a partial explanation for the cross-reactivity shown between Sm and nRNP autoantibodies. However, the sequence, PPPGMIPP, from nRNP A is not antigenic. These data define the linear sequence autoantigenicity of the Sm B/B' protein. They also demonstrate that the predominant autoimmune epitope is a proline-rich sequence from which limited variance is permitted before antigenicity is destroyed.     

Homonuclear three-dimensional NOE-NOE nuclear magnetic resonance/spectra for structure determination of proteins in solution.

The solution structures of two proteins (CMTI-I, a trypsin inhibitor from Cucurbita maxima, and hisactophilin, an actin binding protein of 118 amino acids) have been determined based on the NOE data derived solely from the homonuclear 3D NOE-NOE magnetic resonance spectroscopy. Two different approaches for extraction of the structural information from the 3D NOE-NOE experiment were tested. One approach was based on the transformation of the 3D intensities into distance constraints. In the second, and more robust approach, the 3D NOE intensities were used directly in structure calculations, without the need to transform them into distance constraints. A new 2D potential function representing the 3D NOE-NOE intensity was developed and used in the simulated annealing protocol. For CMTI-I, a comparison between structures determined with the 3D NOE-NOE method and various 2D NOE approaches was carried out. The 3D data set allowed better definition of the structures than was previously possible with the 2D NOE procedures that used the isolated two-spin approximation to derive distance information.     

The NASA Specialized Center of Research and Training (NSCORT) in Gravitational Biology

The Life Sciences Division of NASA has initiated a NASA Specialized Centers of Research and Training (NSCORT) program. Three Centers were designated in late 1990, as the culmination of an in-depth peer review analysis of proposals from universities across the nation and around the world. Kansas State University was selected as the NSCORT in Gravitational Biology. This Center is headquartered in the KSU Division of Biology and has a research, training, and outreach function that focuses on cellular and developmental biology.     

Salmonellosis in gerbils induced by nonrelated experimental procedure.

An outbreak of salmonellosis in a gerbil colony was investigated. The clinical, bacteriologic, and pathologic findings are reported. Clinical signs included an occasional sudden death, depression, emaciation, dehydration, rough hair coat, and testicular enlargement. Not every sign was observed in every infected gerbil. At necropsy, 11 animals had lesions consistent with salmonellosis. Histopathologic lesions consisted of interstitial pneumonia, hepatic and splenic necrosis, meningitis, and suppurative orchitis. Splenic and intestinal amyloidosis were also noted. Salmonella, group D, was recovered from gerbil feces, a container in which adult mosquitos were reared, filarial inoculum, and a cockroach. An epizootiologic investigation led to salmonella-infected cockroaches as the possible source of animal contamination via mosquitos and the subsequent filarial inoculum.