Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Involvement of protein kinase C in the mitogenic and chemotaxis effects of basic fibroblast growth factor on bovine cerebral cortex capillary endothelial cells

Basic fibroblast growth factor is increasingly implicated in cellular growth, differentiation, angiogenesis and oncogenesis. In culture, basic fibroblast growth factor greatly improved the growth rate of bovine brain cortex capillary endothelial cells. Down-regulation of protein kinase C by prolonged treatment with phorbol esters prevented the mitogenic effect of basic fibroblast growth factor on capillary endothelial cells. Furthermore, staurosporine, a potent protein kinase inhibitor, showed strong antiproliferative activity against basic fibroblast growth factor-induced endothelial cell growth. Similarly, the chemotaxis effect of basic fibroblast growth factor on capillary endothelial cells was abolished by down-regulation of protein kinase C or by staurosporine treatment. Therefore, it is suggested that protein kinase C could account for part of the angiogenic effect of basic fibroblast growth factor.     

Subpicosecond resonance Raman spectra of the early intermediates in the photocycle of bacteriorhodopsin

The resonance Raman spectra are presented for the species formed during the photocycle of bacteriorhodopsin (bR) on a timescale of 800-900 fs. In the ethylenic stretch region two intermediates were found with frequencies of 1,510 and 1,518 cm^-1, corresponding to species with optical absorption maxima at 660 and 625 nm, respectively. This leads to the assignment of the 1,518 cm^-1 band to the J₆₂₅ intermediate. In the fingerprint region, the appearance of a vibration at 1,195 cm^-1 strongly suggests that the isomerization indeed has taken place in a time less than the pulsewidth of our laser. This supports the previous proposals made on the basis of the optical spectra. The spectra are compared with those observed in tens of picoseconds up to nanoseconds.     

Origines du peuplement mammalien en Afrique du Nord durant le miocène terminal

The late Miocene North African mammalianassemblage is considered here from three viewpoints: survivals, extinctions, and immigrations. The Eurasiatic affinities of the large mammals slightly prevail over the Ethiopian affinities. Amongst the North African large mammals, 4 to 8 taxa are Eurasiatic immigrants, while 4 to 6 are of Subsaharian origin. Contrarily, the micromammalian fauna is highly endemic, with only one species, a murid (Paraethomys miocaenicus), considered here as being related to an Asiatic form (Karnimata darwini). Our study of Eurasian and African Miocene faunas reveals that during the late Astaracian-early Turolian interval, the Saharo-Arabic belt permitted very little latitudinal faunal exchanges. However, during the middle and late Turolian such faunal exchanges became frequent. The micromammal record unequivocally indicates that a brief period of faunal exchange occurred between Africa and western Europe at the end of the Miocene, corresponding with the Messinian Salinity Crisis. The increased intercontinental faunal exchange between Africa and Eurasia during the late Miocene coincides with, and counterbalances the extinction of more than 10 taxa at the Mio/Pliocene boundary.     

Cytofluorometric Determination of Nuclear DNA in Living and Preserved Algae

Three DNA-localizing fluorochromes used in conjunction with epi (incident) UV illumination were examined for sensitivity and selectivity for the cytofluorometric determination of nuclear DNA in ten species of six algal genera: Mougeotia, Oedogonium, Sirogonium, Spirogyra and Zygnema among the green algae, and the marine red alga Polysiphonia boldii. In comparison with absorption photometry for the determination of nuclear DNA, the cytofluorometric procedure proved to be simpler and considerably more sensitive. Following staining with 4',6-diamidino-2-phenylindole (DAPI), nuclei fluoresce blue-white, the fluorescence intensity of the DNA-DAPI complex being considerably greater than that of the unbound dye molecule. Algal strains stained with 2,5-bis[4'-aminopheny](1')]-1,3,4-oxadiazole (BAO) also showed brilliant blue-white nuclear fluorescence. Although the BAO schedule requires the use of freshly prepared dye and sulfite water, and careful control of hydrolysis, nuclear fluorescence of the stained specimens does not fade under irradiation of the UV beam as rapidly as it does with certain other fluorochrome procedures. A more useful fluorochrome was the fungal antibiotic mithramycin. Its staining schedule is simple and the bright orange-yellow fluorescence of the nuclei is associated with an exceptional degree of sensitivity and specificity for DNA. Forty-eight-year-old preserved filaments of Spirogyra jatobae, stained with either BAO or mithramycin, exhibited a fluorescence brilliance of nuclear and chloroplast DNA equal to that of fresh specimens of this species. The three schedules, but particularly the one with mithramycin, have proven useful in providing indirect evidence for variation in ploidy level in several of the above algal genera, and in verifying the assumed ploidy level of the gametophyte (haploid) and tetrasporophyte (diploid) of Polysiphonia boldii     

The effects of sediment type on growth rate and shell allometry in the soft shelled clam Mya arenaria L

Hatchery-reared juvenile Mya arenaria L. were grown for 11 wk in replicated gravel, sand, mud, and pearl net treatments under flow-through sea-water conditions in Maine. Analyses of variance showed significant differences between sediment treatments for final shell length, dry meat weight, chondrophore growth increment, and percent shell weight. Growth of juvenile M. arenaria was more rapid in fine sediments than in coarse sediments or nets.Regression slopes of shell length-shell height and shell length-shell depth varied significantly between sediment treatments. Slower-growing clams from nets and gravel were more globose than clams from sand or mud treatments. Clams grown in sand were longer and narrower than those from mud. Differences in growth rates and shell form were attributed primarily to the physical properties of the substrata, and their effects on the scope for growth of M. arenaria.     

Studies on the evolutionary relationships between hemoglobins inChironomus pallidivittatus andC. Tentans

Summary The monomeric hemoglobins ofChironomus tentans andC. pallidivittatus have been isolated and separated into their respective components by gel chromatography on Sephadex G-75 and ion-exchange chromatography on DEAE-Sephacel. The amino acid compositions of the purified components are given. The sequence of the 30 N-terminal amino acid residues of one of the monomeric components (Hb I fromC. pallidivittatus) was determined and found to be identical in almost all of its parts with the monomeric hemoglobins ofC. thummi (CTT III and CTT IV).Antibodies against the monomeric hemoglobins Hb I and Hb IIc and the dimeric fraction were highly specific and no cross reaction between dimeric and monomeric hemoglobins could be demonstrated. The antibodies against the monomers crossreact with the monomeric hemoglobins CTT III and CTT IV ofC. thummi. Taken together with genetic data, the immunological results indicate that divergence of monomeric from dimeric forms was an early event in the evolution of the various hemoglobins inChironomus.     

Critical fields in cell fusion and dielectrophoresis

It is increasingly recognized that exposure to electrical fields can reversibly increase the electrical permeability and conductivity of the cellular membrane. It is therefore worthwhile to determine under what circumstances the critical fields capable of affecting the cellular membrane can be reached. We have evaluated the field intensities E and the gradients of E² in the neighborhood of the more common field electrode shapes, the sphere-sphere and cylinder-cylinder electrodes, and put these in terms of the values useful for the practical electro-fusion and dielectrophoresis (DEP) of cells. The calculations were performed using the charge-image technique. From this it was observed that gross errors (underestimations of up to 1000-fold) of the field would be made if neglect of the electrode polarization were made as in simple electrostatic calculation. The dielectric forces (in terms of (E)²) show interesting spatial character.The electrical breakdown of cellular membranes affects the exchange of information and materials between the cell and its environment, and can be used to fuse cells of the same type or of differing types. It is moreover of importance in handling cells during DEP while obtaining their spectral characteristics or in cell-sorting. It is expected that the results here will aid in the more proper use of electrical fields for such ends.     

Membranes of the protoplast L-form of Proteus mirabilis

Isolated membranes of the cell wall-less stable protoplast L-form of Proteus mirabilis were characterized by density gradient centrifugation and by assay for their major chemical constituents, proteins, phospholipids and lipopolysaccharide, and for some specific marker enzymes of the cytoplasmic membrane. In most of the analyzed properties the L-form protoplast membrane resembled the bacterial cytoplasmic membrane, with some notable modifications. considerable amounts of lipopolysaccharide, normally an exclusive constituent of the outer membrane, were found. Furthermore, the L-form membranes contained the functions of the reduced nicotinamide adenine dinucleotide oxidase system, of d-lactate dehydrogenase (EC 1.1.1.28) and of succinate dehydrogenase (EC 1.3.99.1) at specific activities comparable to, or in some cases considerably higher than, those present in cytoplasmic membranes of the bacterial form. Of two peptidoglycan DD-carboxypetidase/transpeptidases (EC 3.4.17.8 and EC 2.3.2.10), which are normally present in the cytoplasmic membrane of the bacterial form of P. mirabilis, the membrane of the protoplast L-form contained only one. Electron microscopy of thin sectioned L-form protoplasts showed extensive heterogeneity of membraneous structures. In addition to the single membraneous integument, internal membrane-bounded vesicles and multiple stacks of membranes were present, as the result of unbalanced growth and membrane synthesis in the L-form state.