In vitro characterization of biofilms formed by Kingella kingae.

The Gram-negative bacterium Kingella kingae is part of the normal oropharyngeal mucosal flora of children <4 years old. K. kingae can enter the submucosa and cause infections of the skeletal system in children, including septic arthritis and osteomyelitis. The organism is also associated with infective endocarditis in children and adults. Although biofilm formation has been coupled with pharyngeal colonization, osteoarticular infections, and infective endocarditis, no studies have investigated biofilm formation in K. kingae. In this study we measured biofilm formation by 79 K. kingae clinical isolates using a 96-well microtiter plate crystal violet binding assay. We found that 37 of 79 strains (47%) formed biofilms. All strains that formed biofilms produced corroding colonies on agar. Biofilm formation was inhibited by proteinase K and DNase I. DNase I also caused the detachment of pre-formed K. kingae biofilm colonies. A mutant strain carrying a deletion of the pilus gene cluster pilA1pilA2fimB did not produce corroding colonies on agar, autoaggregate in broth, or form biofilms. Biofilm forming strains have higher levels of pilA1 expression. The extracellular components of biofilms contained 490 µg cm-2 of protein, 0.68 µg cm-2 of DNA, and 0.4 µg cm-2 of total carbohydrates. We concluded that biofilm formation is common among K. kingae clinical isolates, and that biofilm formation is dependent on the production of proteinaceous pili and extracellular DNA. Biofilm development may have relevance to the colonization, transmission, and pathogenesis of this bacterium. Extracellular DNA production by K. kingae may facilitate horizontal gene transfer within the oral microbial community.

Environmental bacteria produce abundant and diverse antibiofilm compounds.

AIMS: The aim of this study was to isolate novel antibiofilm compounds produced by environmental bacteria. METHODS AND RESULTS: Cell-free extracts were prepared from lawns of bacteria cultured on agar. A total of 126 bacteria isolated from soil, cave and river habitats were employed. Extracts were tested for their ability to inhibit Staphylococcus aureus biofilm in a 96-well microtitre plate assay. A total of 55/126 extracts (44%) significantly inhibited Staph. aureus biofilm. Seven extracts were selected for further analysis. The antibiofilm activities in all seven extracts exhibited unique patterns of molecular mass, chemical polarity, heat stability and spectrum of activity against Staph. aureus, Staphylococcus epidermidis and Pseudomonas fluorescens, suggesting that these seven antibiofilm activities were mediated by unique chemical compounds with different mechanisms of action. CONCLUSIONS: Environmental bacteria produce abundant and diverse antibiofilm compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening cell-free extracts is a useful method for identifying secreted compounds that regulate biofilm formation. Such compounds may represent a novel source of antibiofilm agents for technological development.

Intestinal alkaline phosphatase preserves the normal homeostasis of gut microbiota.

BACKGROUND AND AIMS: The intestinal microbiota plays a critical role in maintaining human health; however, the mechanisms governing the normal homeostatic number and composition of these microbes are largely unknown. Previously it was shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, functions as a gut mucosal defence factor limiting the translocation of gut bacteria to mesenteric lymph nodes. In this study the role of IAP in the preservation of the normal homeostasis of the gut microbiota was investigated. METHODS: Bacterial culture was performed in aerobic and anaerobic conditions to quantify the number of bacteria in the stools of wild-type (WT) and IAP knockout (IAP-KO) C57BL/6 mice. Terminal restriction fragment length polymorphism, phylogenetic analyses and quantitative real-time PCR of subphylum-specific bacterial 16S rRNA genes were used to determine the compositional profiles of microbiotas. Oral supplementation of calf IAP (cIAP) was used to determine its effects on the recovery of commensal gut microbiota after antibiotic treatment and also on the colonisation of pathogenic bacteria. RESULTS: IAP-KO mice had dramatically fewer and also different types of aerobic and anaerobic microbes in their stools compared with WT mice. Oral supplementation of IAP favoured the growth of commensal bacteria, enhanced restoration of gut microbiota lost due to antibiotic treatment and inhibited the growth of a pathogenic bacterium (Salmonella typhimurium). CONCLUSIONS: IAP is involved in the maintenance of normal gut microbial homeostasis and may have therapeutic potential against dysbiosis and pathogenic infections.

Biofilm dispersal: mechanisms, clinical implications, and potential therapeutic uses.

Like all sessile organisms, surface-attached communities of bacteria known as biofilms must release and disperse cells into the environment to colonize new sites. For many pathogenic bacteria, biofilm dispersal plays an important role in the transmission of bacteria from environmental reservoirs to human hosts, in horizontal and vertical cross-host transmission, and in the exacerbation and spread of infection within a host. The molecular mechanisms of bacterial biofilm dispersal are only beginning to be elucidated. Biofilm dispersal is a promising area of research that may lead to the development of novel agents that inhibit biofilm formation or promote biofilm cell detachment. Such agents may be useful for the prevention and treatment of biofilms in a variety of industrial and clinical settings. This review describes the current status of research on biofilm dispersal, with an emphasis on studies aimed to characterize dispersal mechanisms, and to identify environmental cues and inter- and intracellular signals that regulate the dispersal process. The clinical implications of biofilm dispersal and the potential therapeutic applications of some of the most recent findings will also be discussed.

A survey of rodent-borne pathogens carried by wild-caught Norway rats: a potential threat to laboratory rodent colonies.

Unintentional infection of laboratory rodents can compromise scientific research as well as the health of the animals and animal handlers. The source of contamination often is unknown, but may be introduced by wild rats from surrounding environments. To determine whether rats in Baltimore, Maryland, USA carry infectious agents commonly found in laboratory rodent colonies, we live-trapped 162 rats during 2005 to 2006 and screened them for a panel of viruses, bacteria and parasites. Antibodies against rat coronavirus/sialodacryoadenitis virus (91.7%), Mycoplasma pulmonis (72.9%), cilia-associated respiratory bacillus (52.1%), rat parvovirus/rat minute virus (29.2%), Kilham rat virus (10.4%), Toolan's H-1 virus (10.4%), Sendai virus (4.2%) and Theiler's mouse encephalomyelitis virus (4.2%), were detected in wild-caught Norway rats. Antibodies against reovirus and pneumonia virus of mice were not detected in wild Norway rats. Endoparasites, including Nippostrongylus braziliensis (71.6%), Rodentolepis nana or Hymenolepis diminuta (34.4%), Hetarakis spumosa (24.1%) and Trichuris muris (14.8%), as well as ectoparasites (14.8%), were identified in wild-caught rats. The risk of pathogen transmission from wild-caught rats to laboratory colonies needs to be mitigated by minimizing exposures rather than assuming wild animals represent a minimal hazard.

Aggregatibacter actinomycetemcomitans serotype f O-polysaccharide mediates coaggregation with Fusobacterium nucleatum.

BACKGROUND/AIMS: Intergeneric bacterial coaggregation may play an important role in plaque development. METHODS: In this study we investigated the coaggregation reaction between two periodontal pathogens, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum. RESULTS: Previous studies showed that A. actinomycetemcomitans serotype b strains coaggregate with F. nucleatum strain PK1594, and that A. actinomycetemcomitans serotype b O-polysaccharide (O-PS) is the receptor responsible for coaggregation between A. actinomycetemcomitans and F. nucleatum. A. actinomycetemcomitans serotype f O-PS has been shown to be structurally and antigenically related to serotype b O-PS. In the present study we show that A. actinomycetemcomitans strain CU1060N, a serotype f strain, also coaggregated with F. nucleatum PK1594. Like coaggregation between serotype b strains and F. nucleatum, coaggregation between CU1060N and F. nucleatum was inhibited by galactose. An O-PS mutant of CU1060N failed to coaggregate with F. nucleatum. CONCLUSION: We concluded that A. actinomycetemcomitans serotype f O-PS, like serotype b O-PS, mediates coaggregation between A. actinomycetemcomitans and fusobacteria.

Modeling and biochemical analysis of the activity of antibiofilm agent Dispersin B.

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix (PGA), which is a linear polymer of beta(1,6)-linked N-acetylglucosamine (GlcNAc) residues. Dispersin B (DspB), a soluble glycoside hydrolase produced by the periodontal pathogen Actinobacillus actinomycetemcomitans degrades PGA. The enzyme DspB is an alpha/beta TIM-barrel protein and belongs to family 20 glycosyl hydrolases members. The enzyme activity of DspB with regard to its substrate specificity towards beta(1,6)-linked GlcNAc polymers and its endo/exo character was investigated through ligand docking and the hydrolysis of synthetic oligosaccharides. Ligand docking analysis suggested that beta(1,6)-linked GlcNAc oligosaccharide bound to the active site better that beta(1,4)-linked GlcNAc oligosaccharide. Our combined results indicate that DspB is an exo-acting enzyme that hydrolyzes beta(1,6)-linked N-acetylglucosamine oligomers.

Detachment and killing of Aggregatibacter actinomycetemcomitans biofilms by dispersin B and SDS.

The periodontopathogen Aggregatibacter actinomycetemcomitans forms tenacious biofilms on abiotic surfaces in vitro. The objective of the present study was to measure the susceptibility of A. actinomycetemcomitans biofilms to detachment and killing by the anionic surfactant sodium dodecyl sulfate (SDS). We found that biofilms formed by a wild-type strain were resistant to detachment by SDS. In contrast, biofilms formed by an isogenic mutant strain that was deficient in the production of PGA (poly-N-acetyl-glucosamine), a biofilm matrix polysaccharide, were sensitive to detachment by SDS. Pre-treatment of wild-type biofilms with dispersin B, a PGA-degrading enzyme, rendered them sensitive to detachment by SDS and resulted in a > 99% increase in SDS-mediated cell killing. We concluded that PGA protects A. actinomycetemcomitans cells from detachment and killing by SDS. Dispersin B and SDS may be useful agents for treating chronic infections caused by A. actinomycetemcomitans and other PGA-producing bacteria.

Synergistic activity of dispersin B and cefamandole nafate in inhibition of staphylococcal biofilm growth on polyurethanes.

Antibiotic therapies to eradicate medical device-associated infections often fail because of the ability of sessile bacteria, encased in their exopolysaccharide matrix, to be more drug resistant than planktonic organisms. In the last two decades, several strategies to prevent microbial adhesion and biofilm formation on the surfaces of medical devices, based mainly on the use of antiadhesive, antiseptic, and antibiotic coatings on polymer surfaces, have been developed. More recent alternative approaches are based on molecules able to interfere with quorum-sensing phenomena or to dissolve biofilms. Interestingly, a newly purified beta-N-acetylglucosaminidase, dispersin B, produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans, is able to dissolve mature biofilms produced by Staphylococcus epidermidis as well as some other bacterial species. Therefore, in this study, we developed new polymeric matrices able to bind dispersin B either alone or in combination with an antibiotic molecule, cefamandole nafate (CEF). We showed that our functionalized polyurethanes could adsorb a significant amount of dispersin B, which was able to exert its hydrolytic activity against the exopolysaccharide matrix produced by staphylococcal strains. When microbial biofilms were exposed to both dispersin B and CEF, a synergistic action became evident, thus characterizing these polymer-dispersin B-antibiotic systems as promising, highly effective tools for preventing bacterial colonization of medical devices.

Susceptibility of staphylococcal biofilms to enzymatic treatments depends on their chemical composition.

Bacterial infections are serious complications after orthopaedic implant surgery. Staphylococci, with Staphylococcus epidermidis as a leading species, are the prevalent and most important species involved in orthopaedic implant-related infections. The biofilm mode of growth of these bacteria on an implant surface protects the organisms from the host's immune system and from antibiotic therapy. Therapeutic agents that disintegrate the biofilm matrix would release planktonic cells into the environment and therefore allow antibiotics to eliminate the bacteria. An addition of a biofilm-degrading agent to a solution used for washing-draining procedures of infected orthopaedic implants would greatly improve the efficiency of the procedure and thus help to avoid the removal of the implant. We have previously shown that the extracellular staphylococcal matrix consists of a poly-N-acetylglucosamine (PNAG), extracellular teichoic acids (TAs) and protein components. In this study, we accessed the sensitivity of pre-formed biofilms of five clinical staphylococcal strains associated with orthopaedic prosthesis infections and with known compositions of the biofilm matrix to periodate, Pectinex Ultra SP, proteinase K, trypsin, pancreatin and dispersin B, an enzyme with a PNAG-hydrolysing activity. We also tested the effect of these agents on the purified carbohydrate components of staphylococcal biofilms, PNAG and TA. We found that the enzymatic detachment of staphylococcal biofilms depends on the nature of their constituents and varies between the clinical isolates. We suggest that a treatment with dispersin B followed by a protease (proteinase K or trypsin) could be capable to eradicate biofilms of a variety of staphylococcal strains on inert surfaces.

A survey of zoonotic pathogens carried by Norway rats in Baltimore, Maryland, USA.

Norway rats (Rattus norvegicus) carry several zoonotic pathogens and because rats and humans live in close proximity in urban environments, there exists potential for transmission. To identify zoonotic agents carried by rats in Baltimore, Maryland, USA, we live-trapped 201 rats during 2005-2006 and screened them for a panel of viruses, bacteria, and parasites. Antibodies against Seoul virus (57.7%), hepatitis E virus (HEV, 73.5%), Leptospira interrogans (65.3%), Bartonella elizabethae (34.1%), and Rickettsia typhi (7.0%) were detected in Norway rats. Endoparasites, including Calodium hepatica (87.9%) and Hymenolepis sp. (34.4%), and ectoparasites (13.9%, primarily Laelaps echidninus) also were present. The risk of human exposure to these pathogens is a significant public health concern. Because these pathogens cause non-specific and often self-limiting symptoms in humans, infection in human populations is probably underdiagnosed.

Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans.

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix that holds the bacteria together in a mass and firmly attaches the bacterial mass to the underlying surface. A major component of the extracellular polysaccharide matrix in several phylogenetically diverse bacteria is PGA, a linear polymer of N-acetylglucosamine residues in beta(1,6)-linkage. PGA is produced by the Gram-negative periodontopathogen Actinobacillus actinomycetemcomitans as well as by the Gram-positive device-associated pathogen Staphylococcus epidermidis. We recently reported that A.actinomycetemcomitans produces a soluble glycoside hydrolase named dispersin B, which degrades PGA. Here, we present the crystal structure of dispersin B at 2.0A in complex with a glycerol and an acetate ion at the active site. The enzyme crystallizes in the orthorhombic space group C222(1) with cell dimensions a=41.02A, b=86.13A, c=185.77A. The core of the enzyme consists a (beta/alpha)(8) barrel topology similar to other beta-hexosaminidases but significant differences exist in the arrangement of loops hovering in the vicinity of the active site. The location and interactions of the glycerol and acetate moieties in conjunction with the sequence analysis suggest that dispersin B cleaves beta(1,6)-linked N-acetylglucosamine polymer using a catalytic machinery similar to other family 20 hexosaminidases which cleave beta(1,4)-linked N-acetylglucosamine residues.

Sequence diversity in the major fimbrial subunit gene (flp-1) of Actinobacillus actinomycetemcomitans.

Cells of the periodontal pathogen Actinobacillus actinomycetemcomitans exhibit tight adherence to surfaces such as glass, plastic and hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize teeth and other surfaces. Tight adherence is mediated by long fibrils of bundled pili (fimbriae) that form on the surface of the cell. The flp-1 gene encodes the major pilin protein component of A. actinomycetemcomitans fimbriae. In this study we compared flp-1 DNA sequences from 43 strains of A. actinomycetemcomitans isolated in Europe, Japan and the United States and identified seven distinct flp-1 allelic classes. DNA and predicted protein sequences were almost completely conserved within each flp-1 class but were highly divergent between classes. Most amino acid substitutions occurred in the C-terminus of the pilin protein, a region that has been shown to be important for the bundling and adhesive properties of the pili. flp-1 classes correlated with serotypes and 16S rRNA genotypes in most strains. At least five strains showed evidence of horizontal transfer of flp-1 between strains of different serotypes and 16S rRNA genotypes. Four of the seven flp-1 classes were present in geographically diverse isolates. Strains representing all seven flp-1 classes, but not a strain carrying a transposon insertion in flp-1, bound avidly to polystyrene in an in vitro adherence assay. Strains representing six of the seven flp-1 classes were isolated from localized juvenile periodontitis patients, suggesting that phylogenetically diverse strains carry pathogenic potential. Our findings provide a framework for future biochemical, immunological and genetic studies of A. actinomycetemcomitans fimbriae.

Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f.

The oral bacterium Actinobacillus actinomycetemcomitans is implicated as a causative agent of localized juvenile periodontitis (LJP). A. actinomycetemcomitans is classified into five serotypes (a to e) corresponding to five structurally and antigenically distinct O polysaccharide (O-PS) components of their respective lipopolysaccharide molecules. Serotype b has been reported to be the dominant serotype isolated from LJP patients. We determined the lipopolysaccharide O-PS structure from A. actinomycetemcomitans CU1000, a strain isolated from a 13-year-old African-American female with LJP which had previously been classified as serotype b. The O-PS of strain CU1000 consisted of a trisaccharide repeating unit composed of L-rhamnose and 2-acetamido-2-deoxy-D-galactose (molar ratio, 2:1) with the structure -->2)-alpha-L-Rhap-(1-3)-2-O-(beta-D-GalpNAc)-alpha-L-Rhap-(1-->* O-PS from strain CU1000 was structurally and antigenically distinct from the O-PS molecules of the five known A. actinomycetemcomitans serotypes. Strain CU1000 was mutagenized with transposon IS903phikan, and three mutants that were deficient in O-PS synthesis were isolated. All three transposon insertions mapped to a single 1-kb region on the chromosome. The DNA sequence of a 13.1-kb region surrounding these transposon insertions contained a cluster of 14 open reading frames that was homologous to gene clusters responsible for the synthesis of A. actinomycetemcomitans serotype b, c, and e O-PS antigens. The CU1000 gene cluster contained two genes that were not present in serotype-specific O-PS antigen clusters of the other five known A. actinomycetemcomitans serotypes. These data indicate that strain CU1000 should be assigned to a new A. actinomycetemcomitans serotype, designated serotype f. A PCR assay using serotype-specific PCR primers showed that 3 out of 20 LJP patients surveyed (15%) harbored A. actinomycetemcomitans strains carrying the serotype f gene cluster. The finding of an A. actinomycetemcomitans serotype showing serological cross-reactivity with anti-serotype b-specific antiserum suggests that a reevaluation of strains previously classified as serotype b may be warranted.

flp-1, the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans, a Gram-negative bacterium responsible for localized juvenile periodontitis and other infections such as endocarditis, produces long fibrils of bundled pili that are believed to mediate non-specific, tenacious adherence to surfaces. Previous investigations have implicated an abundant, small ( approximately 6.5 kDa), fibril-associated protein (Flp/Fap) as the primary fibril subunit. Here, we report studies on fibril structure and on the function and evolution of Flp. High-resolution electron microscopy of adherent clinical strain CU1000N revealed long bundles of 5- to 7-nm-diameter pili, whose subunits appear to be arranged in a helical array similar to that observed for type IV pili in other bacteria. Fibrils were found to be associated with the bacterial cell surface and smaller structures thought to be membrane vesicles. A modified version of the CU1000N Flp1 polypeptide with the T7-TAG epitope fused to its C-terminus was expressed in the wild-type strain, and the presence of the modified Flp1 in fibrils was confirmed by immunogold electron microscopy with monoclonal antibody to T7-TAG. To determine the importance of Flp1 in fibril formation and cell adherence, we used transposon IS903phikan to isolate insertion mutations in the flp-1 gene (formerly designated flp). Mutants with insertions early in flp-1 fail to produce fibrils and do not adhere to surfaces. Both fibril production and adherence were restored by cloned flp-1 in trans, thus providing the first evidence that flp-1 is required for fibril formation and tight, non-specific adherence. One mutant was found to have an insertion near the 3' end of flp-1 that results in the expression of a truncated and altered C-terminus of Flp1. This mutant produced short, unbundled pili, and its adherence to surfaces was significantly less than that of wild-type bacteria. These findings and related observations with the Flp1-T7-TAG protein indicate that the C-terminus of Flp1 is important for the bundling and adherence properties of pili. Extensive sequence comparisons and phylogenetic analysis of 61 predicted prepilin genes of bacteria revealed flp-1 to be a member of a novel and widespread subfamily of type IV prepilin genes. Thus, Flp pili are likely to be expressed by diverse bacterial species. Furthermore, we found that it is common for bacterial genomes to contain multiple alleles of flp-like genes, including the open reading frame (flp-2, previously designated orfA) immediately downstream of flp-1 in A. actinomycetemcomitans. The duplication and divergence of flp genes in bacteria may be important to the diversification of the colonization properties of these organisms.

Percutaneous core biopsy of nonpalpable breast lesions: utility and impact on cost of diagnosis.

Percutaneous image-guided core breast biopsy is faster, less invasive, less deforming, and less expensive than surgical biopsy for diagnosing nonpalpable breast lesions. Percutaneous biopsy may be performed using different imaging guidance modalities (e.g., stereotaxis or ultrasound) and different tissue acquisition devices (e.g., automated needles or vacuum-assisted biopsy probes). Stereotactic biopsy may be used for all lesion types (masses and calcifications). Stereotactic 14-gauge automated core biopsy spared a surgical procedure in 76-85% of cases, decreasing the cost of diagnosis by 40-56%. Annual national savings from use of stereotactic 14-gauge automated core biopsy rather than surgical biopsy for nonpalpable lesions would exceed 100 million dollars. Stereotactic 11-gauge vacuum-assisted biopsy yields significant improvement in diagnosis of calcifications and may be used in lesions that are not amenable to stereotactic 14-gauge automated core biopsy. Stereotactic 11-gauge vacuum-assisted biopsy spared a surgical procedure in 76% cases, decreasing cost of diagnosis by 20%. Use of stereotactic 11-gauge vacuum-assisted biopsy for calcifications and for nonpalpable masses not amenable to stereotactic 14-gauge automated core biopsy would yield annual national savings exceeding 50 million dollars. Ultrasound-guided biopsy, used primarily for masses, has several advantages including speed, comfort, lack of radiation exposure, and real-time needle visualization. Ultrasound-guided core biopsy spared a surgical procedure in 85% cases, decreasing the cost of diagnosis by 56%. Although both ultrasound-guided core biopsy and stereotactic biopsy are less expensive than surgery, cost savings are greater for ultrasound-guided core biopsy. An annual national savings over $50 million could be realized with the use of ultrasound-guided core biopsy for nonpalpable masses. Future work should include evaluating the emerging new technologies for percutaneous breast biopsy and optimizating the choice of biopsy method for different clinical scenarios. Society may benefit from cost reduction as women benefit from a faster, less invasive, and less deforming approach to diagnosis.

Codon usage in Actinobacillus actinomycetemcomitans.

The codon usage patterns of 21 genes encompassing 5800 codons from Actinobacillus actinomycetemcomitans were analyzed. A. actinomycetemcomitans genes could be divided into two groups based on their function and G + C content. One group included those genes encoding basic cellular functions. This group displayed an average G + C content of 48%. A second group comprised genes encoding the leukotoxin determinant, an insertion sequence and a plasmid. This group displayed an average G + C content of 36%. These findings suggest that portions of the A. actinomycetemcomitans genome may have been acquired by horizontal gene transfer from one or more distantly related species. We present a table of A. actinomycetemcomitans codon usage. These data may be used to establish standards for computer programs that predict A. actinomycetemcomitans protein coding regions and may be useful in designing degenerate oligonucleotide probes.

Characterization of a soluble vascular endothelial growth factor receptor-immunoglobulin chimera.

To investigate the interaction between vascular endothelial growth factor (VEGF) and its receptor, we have constructed a chimeric protein consisting of the extracellular ligand-binding domain of the human VEGF receptor subtype KDR fused to a human IgG1 Fc domain (KDR-Fc). KDR-Fc was expressed in human 293 kidney epithelial cells as a 300-kDa secreted, dimeric glycoprotein that bound 125I-VEGF165 with high affinity (Kd = 150 pM). Unlike the full length cellular receptor, KDR-Fc did not require heparin for 125I-VEGF165 binding, although heparin did stimulate 125I-VEGF165 binding approximately 50 to 100%. Similar results were observed for KDR-Fc expressed in yeast cells. Since yeast do not synthesize heparan sulfate proteoglycans, we conclude that cellular heparan sulfates do not account for the lack of a heparin requirement for 125I-VEGF165 binding to KDR-Fc. The polycationic protein protamine, which inhibits (IC50 = 1 microgram/ml) 125I-VEGF165 binding to bovine aortic endothelial cells and other KDR-expressing cells by blocking heparin interactions, had no effect on the heparin independent component of 125I-VEGF165 binding to KDR-Fc. Protamine does inhibit (IC50 = 1 microgram/ml) the heparin dependent component of 125I-VEGF165 binding to KDR-Fc. KDR-Fc bound VEGF121 with the same affinity as VEGF165. Heparin had no effect on 125I-VEGF121 binding to KDR-Fc, indicating that heparin interaction with the 44 amino acids contained in VEGF165 but not VEGF121 allow for maximal VEGF165 binding. Deletion analysis of KDR-Fc demonstrated that the determinants required for high affinity VEGF binding are located in the three aminoterminal Ig-domains of the protein. Heparin had no effect on 125I-VEGF165 binding to the three Ig-domain receptor, suggesting that there are heparin binding determinants located in KDR Ig-domains 4 to 7.

Biological activity of human N-ras and K-ras genes containing the Asn17 dominant negative mutation.

Substitution of asparagine for serine at position 17 of human H-ras results in an impaired GTP-binding activity, causing the mutant Ras protein to be locked in a constitutively inactive GDP-bound state. Expression of this mutant in NIH 3T3 cells inhibits cell proliferation by blocking endogenous ras function. Plasmids that encode the analogous dominant negative mutation at position 17 in human N- and K-ras were constructed. These mutant ras genes, driven by a heavy metal-inducible sheep metallothionein promoter, were introduced by transfection into a variety of animal cell lines. All three mutant ras genes displayed an inhibitory phenotype when expressed in NIH 3T3 cells. This inhibition could be overcome by cotransfection with either activated H-ras or v-raf. These data indicate that the three human Ras proteins probably act through the same signal transduction pathway in NIH 3T3 cells and suggest that these mutations may confer similar phenotypes to other GTP/GDP-binding proteins.

Influence of season on seminal characteristics, testis size and serum testosterone in the western spotted skunk (Spilogale gracilis).

The western spotted skunk is a seasonally breeding mammal: most copulations occur in late September and early October. This study was performed to characterize the seasonal changes in concentrations of testosterone and in ejaculate quality. Captive males (n = 22) were maintained on a natural photoperiod for 15 months. Semen samples were collected by electroejaculation; testis size was measured; and blood samples were collected. Of 110 electroejaculation attempts, 104 (95%) resulted in successful fluid collection and 101 (97%) samples contained spermatozoa. Significant increases (P < 0.05) in serum testosterone concentration, testis size and ejaculate volume were observed from August to November. Mean concentration of testosterone in serum ranged from 0.15 +/- 0.05 ng ml-1 in mid-January to 6.42 +/- 1.79 ng ml-1 in early October. Mean testis size ranged from 1.22 +/- 0.25 cm2 in February to 2.68 +/- 0.08 cm2 in October. Mean ejaculate volume ranged from 11 +/- 3 microliters in March to 129 +/- 22 microliters in October. Seasonal changes in the number of spermatozoa per ejaculate or motility of spermatozoa were not observed. Mean number of spermatozoa per ejaculate was 8.14 +/- 0.85 x 10(6) spermatozoa (n = 97); motility was 56 +/- 2.4% (n = 93); semen pH was 7.76 +/- 0.20 (n = 6); osmolarity was 394 +/- 13 mmol kg-1 (n = 10); and 70.3 +/- 1.5% of the spermatozoa were morphologically normal (n = 47).(ABSTRACT TRUNCATED AT 250 WORDS)