Direct visualization of the structure of the "20 S" aggregate of coat protein of tobacco mosaic virus. The "disk" is the major structure at pH 7.0 and the Proto-helix at lower pH.

We have employed the rapid-freeze technique to prepare specimens for electron microscopy of a coat protein solution of tobacco mosaic virus at equilibrium at pH 7.0 and 6.8, ionic strength 0.1 M and 20 degrees C. The former are the conditions for the most rapid assembly of the virus from its isolated protein and RNA. At both pH values, the equilibrium mixture contains approximately 80% of a "20 S" aggregate and 20% of a "4 S" aggregate (the so-called A-protein). The specimens were prepared either totally unstained or positively stained with methyl mercury nitrate, which binds to an amino acid residue (Cys27) internally located within the subunit, which we show not to affect the virus assembly. The images in the electron microscope are compatible only with the major structure for the "20 S" aggregate at pH 7.0 containing two rings of subunits and these aggregates display the same binding contacts as those seen between the aggregate that forms the asymmetric unit in the crystal, which has been shown by X-ray crystallography to be a disk containing two rings, each of 17 subunits, oriented in the same direction. In contrast, the images from specimens prepared at pH 6.8 show the major structure to be a proto-helix at this slightly lower pH, demonstrating that the technique of cryo-electron microscopy is capable of distinguishing between these aggregates of tobacco mosaic virus coat protein. The main structure in solution at pH 7.0 must therefore be very similar to that in the crystal, although slight differences could occur and there are probably other, minor, components in a mixture of species sedimenting around 20 S under these conditions. The equilibrium between aggregates is extremely sensitive to conditions, with a drop of 0.2 pH unit tipping the disk to proto-helix ratio from approximately 10:1 at pH 7.0 to 1:10 at pH 6.8. This direct determination of the structure of the "20 S" aggregate in solution, under conditions for virus assembly, contradicts some recent speculation that it must be helical, and establishes that, at pH 7.0, it is in fact predominantly a two-layer disk as it had been modelled before.     

Development of a Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA) to Unravel the Intra-Pathovar Structure of Pseudomonas syringae pv. actinidiae Populations Worldwide

The bacterial canker of kiwifruit by Pseudomonas syringae pv. actinidiae is an emblematic example of a catastrophic disease of fruit crops. In 2008 a new, extremely virulent form of the pathogen emerged and rapidly devastated many Actinidia spp. orchards all over the world. In order to understand differences in populations within this pathovar and to elucidate their diffusion and movements on world scale, it is necessary to be able to quickly and on a routine basis compare new isolates with previous records. In this report a worldwide collection of 142 strains was analyzed by MLVA, chosen as investigative technique for its efficacy, reproducibility, simplicity and low cost. A panel of 13 Variable Number of Tandem Repeats (VNTR) loci was identified and used to describe the pathogen population. The MLVA clustering is highly congruent with the population structure as previously established by other molecular approaches including whole genome sequencing and correlates with geographic origin, time of isolation and virulence. For convenience, we divided the VNTR loci in two panels. Panel 1 assay, using six loci, recognizes 23 different haplotypes, clustered into ten complexes with highest congruence with previous classifications. Panel 2, with seven VNTR loci, provides discriminatory power. Using the total set of 13 VNTR loci, 58 haplotypes can be distinguished. The recent hypervirulent type shows very limited diversity and includes, beside the strains from Europe, New Zealand and Chile, a few strains from Shaanxi, China. A broad genetic variability is observed in China, but different types are also retrievable in Japan and Korea. The low virulent strains cluster together and are very different from the other MLVA genotypes. Data were used to generate a public database in MLVAbank. MLVA represents a very promising first-line assay for large-scale routine genotyping, prior to whole genome sequencing of only the most relevant samples.     

Haemogregarine specificity in two communities of Florida snakes, with descriptions of six new species of Hepatozoon (Apicomplexa: Hepatozoidae) and a possible species of Haemogregarina (Apicomplexa: Haemogregarinidae)

Five species of snakes in Florida, from Palm Beach County in the south and Alachua County 450 km to the north, occur in similar habitat but have distinctive Hepatozoon species characteristic of each host species. In Palm Beach County, Diadophis punctatus is host to Hepatozoon punctatus n. sp., Thamnophis sauritus sackenii to Hepatozoon sauritus n. sp., and Nerodia fasciata pictiventris to Hepatozoon pictiventris n. sp. In Alachua County, N. fasciata pictiventris is parasitized by Hepatozoon fasciatae n. sp., Seminatrix p. pygaea by Hepatozoon seminatrici n. sp., and Thamnophis s. sirtalis by Hepatozoon sirtalis n. sp. Each Hepatozoon sp. has distinctive gamonts and sporogonic characters and, in the 4 species where known, meronts. Nerodia floridana is host to Haemogregarina floridana n. sp. in both localities, with generic identification tentative, based upon presence of erythrocytic meronts. The presence of sporocysts in the proboscis of 31% of Aedes aegypti infected by H. pictiventris is the first report of infective stages of a reptilian Hepatozoon species within the mouthparts of a dipteran vector. This study suggests that in Florida, at least, the diversity of the Hepatozoon community not only equals but probably exceeds the diversity of the snake communities present, and that host specificity in nature may be much greater than that postulated from previous studies.     

Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Specific cleavage of the native type III collagen molecule with trypsin. Similarity of the cleavage products to collagenase-produced fragments and primary structure at the cleavage site.

Solutions of native Type III collagen (chain composition, [α1(III)]₃) exhibit a rapid and dramatic decrease in relative viscosity when incubated with trypsin. Cleavage products of the reaction were precipitated with ammonium sulfate and isolated in denatured form by molecular sieve chromatography. They were found to be comprised of: α1(III)-T1 (molecular weight, 71,000) derived from the NH₂-terminal portion of the Type III molecule; and α1(III)-T2 (molecular weight, 24,000) from the COOH-terminal portion of the molecule. Determination of the amino acid sequence at the NH₂-terminal portion of α1(III)-T2 as well as at the COOH-terminus of α(III)-T1 demonstrated that the products arose from specific cleavage of the type III molecule at an arginine-glycine bond corresponding to residues 780–781 in the repetitive triplet sequence of the α1(III) chain. The results suggest that the trypsin-susceptible bond in the native Type III collagen molecule resides in a region characterized by a relative lack of the normal collagen helicity.     

The radiation chemistry of some platinum-containing radiosensitizers and related compounds

Oxidation and reduction of cis- and trans-dichlorodiammine platinum II (cis- and trans-PDD), cis-dichlorobis(1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole-N3)-p latinum II (cis-Flap), and cis-dichlorobis(isopropylamine)-trans-dihydroxyplatinum IV (Chip) have been studied using pulse radiolysis. Spectra corresponding to platinum in various oxidation states have been observed and several rate constants have been obtained. Reduction of all the compounds, except cis-Flap, produces species of a lower oxidation state of platinum which subsequently have both chloride ligands replaced. Ultimately, these products disproportionate. In the case of cis-Flap, reduction occurred on the nitroimidazole ligand. This was verified by the absence of platinum metal after disproportionation. Oxidation of all four compounds consists of production of a higher oxidation state of platinum followed by replacement of chloride ligands and finally disproportionation of the products. Only cis-Flap and Chip could be reduced by oxidized DNA bases. The one-electron reduction potential of cis-Flap was found to be -370 +/- 10 mV. trans-Flap had almost the same value. It was not possible to measure the potentials of the other compounds since their ligands were replaced rapidly but it is estimated that the one-electron reduction potentials decrease in the order cis- or trans-Flap greater than Chip greater than cis-PDD greater than trans-PDD.     

Nonhistone chromosomal protein HMG 1 interactions with DNA. Fluorescence and thermal denaturation studies

The interaction of high mobility group protein 1 (HMG 1) isolated from chicken erythrocytes with DNA has been characterized using the intrinsic tryptophan fluorescence of the protein as a probe. It was found that the fluorescence is quenched approximately 30% upon binding to either single- or double-stranded DNA. Fluorescent titrations indicate that the physical site size for HMG 1 binding on native DNA is approximately 14 base pairs (or 14 bases for binding to single-stranded DNA). Binding to single-stranded poly(dA) is only slightly dependent on ionic strength, although the affinity for double-stranded DNA is strongly ionic strength-dependent and has an optimum at approximately 100-120 mM Na+. Above this range, binding to native DNA is virtually all electrostatic in nature. Although the affinity of HMG 1 for single-stranded DNA is higher than that for double-stranded DNA at the extremes of the ionic range studied, no clear evidence for a helix-destabilizing activity was obtained. At low ionic strength, the protein actually stabilized DNA against thermal denaturation, while at high ionic strength, HMG 1 appears to undergo denaturation below the Tm of the DNA. Studies of the environment of the tryptophan fluorophores using collisional quenchers iodide, cesium, and acrylamide suggest that the predominant fluorophore is relatively exposed but constrained in a rigid, positively charged environment.     

Antibody repertoire development in fetal and neonatal piglets: XIX. Undiversified B cells with hydrophobic HCDR3s preferentially proliferate in the porcine reproductive and respiratory syndrome

Porcine respiratory and reproductive syndrome virus (PRRSV) causes an extraordinary increase in the proportion of B cells resulting in lymphoid hyperplasia, hypergammaglobulinemia, and autoimmunity in neonatal piglets. Spectratypic analysis of B cells from neonatal isolator piglets show a non-Gaussian pattern with preferential expansion of clones bearing certain H chain third complementary region (HCDR3) lengths. However, only in PRRSV-infected isolator piglets was nearly the identical spectratype observed for all lymphoid tissues. This result suggests dissemination of the same dominant B cell clones throughout the body. B cell expansion in PRRS was not associated with preferential VH gene usage or repertoire diversification and these cells appeared to bear a naive phenotype. The B cell population observed during infection comprised those with hydrophobic HCDR3s, especially sequences encoded by reading frame 3 of DHA that generates the AMVLV motif. Thus, the hydropathicity profile of B cells after infection was skewed to favor those with hydrophobic binding sites, whereas the normally dominant region of the hydropathicity profile containing neutral HCDR3s was absent. We believe that the hypergammaglobulinemia results from the products of these cells. We speculate that PRRSV infection generates a product that engages the BCR of naive B cells, displaying the AMVLV and similar motifs in HCDR3 and resulting in their T-independent proliferation without repertoire diversification.     

Hsp90: Still a viable target in prostate cancer

Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the maturation, activation and stability of critical signaling proteins that drive the development and progression of prostate cancer, including the androgen receptor. Despite robust preclinical data demonstrating anti-tumor activity of first-generation Hsp90 inhibitors in prostate cancer, poor clinical responses initially cast doubt over the clinical utility of this class of agent. Recent advances in compound design and development, use of novel preclinical models and further biological insights into Hsp90 structure and function have now stimulated a resurgence in enthusiasm for these drugs as a therapeutic option. This review highlights how the development of new-generation Hsp90 inhibitors with improved physical and pharmacological properties is unfolding, and discusses the potential contexts for their use either as single agents or in combination, for men with metastatic prostate cancer.