Biomedical subjects

G G Haraszthy

Publications and source records attributed to G G Haraszthy.

8 recordsLinked to original sources

PubMed2002

Molecular cloning of the fur gene from Actinobacillus actinomycetemcomitans.

In several bacterial species, iron availability in host tissues is coordinated with the expression of virulence determinants through the fur gene product. Initial experiments showed that a cloned Escherichia coli fur gene probe hybridized to Southern blots of Actinobacillus actinomycetemcomitans strain JP2 (serotype b) chromosomal DNA. The A. actinomycetemcomitans fur gene was then cloned utilizing partial functional complementation of the fur mutant in E. coli strain H1780. Analysis of the cloned DNA sequence revealed a 438-bp open reading frame with a deduced 146-amino-acid sequence exhibiting 80% identity to Haemophilus influenzae Fur and 62% identity to E. coli Fur. The pUC Aafur gene probe (generated from JP2 serotype b) hybridized to representatives from all five A. actinomycetemcomitans serotypes as well as to two strains derived from monkeys, suggesting that fur is widely distributed in A. actinomycetemcomitans. Open reading frames having >70% identity with the E. coli and H. influenzae flavodoxin and gyrase A genes, respectively, were found. Expression of the A. actinomycetemcomitans fur gene product repressed fiu expression and siderophore production in E. coli. A gel shift assay demonstrated that the expressed A. actinomycetemcomitans Fur protein bound the bacterial fur consensus sequence. Further characterization of the fur gene product in A. actinomycetemcomitans may improve our understanding of its role in the pathogenesis of periodontal disease and may lead to specific therapeutic modalities.

Aggregatibacter actinomycetemcomitans↗

PubMed1995

Identification of salivary proteins inhibiting herpes simplex virus 1 replication.

Salivary proteins play an important role in the maintenance of the oral ecology. Previous studies have indicated that human submandibular-sublingual and parotid salivas can selectively suppress the in vitro infectivity of herpes simplex virus 1. The purpose of this study was to identify the salivary components in human submandibular-sublingual saliva that modulate in vitro infectivity. Assessment of the interaction of viral particles with salivary components was accomplished using an in vitro solid-phase assay. These experiments revealed that herpes simplex virus particles selectively interact with the members of the salivary proline-rich protein and cystatin families. Subsequent yield reduction assays demonstrated the ability of proline-rich proteins and salivary cystatins to inhibit the viral replication, with basic proline-rich peptides being more effective. Subsequent assays suggest that basic proline-rich peptides reduced the virus titer by interfering with penetration and/or cellular processing of virus within the target cell. Collectively, these results further suggest that salivary proteins have an important role in the host defense mechanism against recurrent herpesvirus infection.

Animals↗

PubMed1993

Isolation and partial characterization of the Campylobacter rectus cytotoxin.

Previously, we reported the antigranulocytic activity of Campylobacter rectus media supernatants containing lipopolysaccharide (LPS) and a 104 kDa protein. Here, we monitored the release of protein and LPS through the growth cycle of C. rectus ATCC 33238 and identified the 104 kDa protein as the cytotoxin. LPS in media supernatants was quantitated by a KDO assay; the 104 kDa protein was detected on immunoblots with specific antibody (A104) and quantitated by amino acid analysis of membrane immobilized protein bands. C. rectus cell product release was independent of cell lysis. Over 24 h, the 104 kDa protein was released linearly while LPS was released in two plateaus; both increased in C. rectus culture supernatants 3 h after inoculation achieving maximum concentrations at 21 h of 3.1 micrograms/ml and 14.6 micrograms/ml, respectively. In 2 h, trypan blue viability assays, 37-47 micrograms of 12, 18 and 24 h supernatant protein killed 33-43% of HL-60 cells. Supernatant toxicity was heat sensitive and inhibited by A104. Sequencing the 16 N-terminal amino acids of the cytotoxin distinguished it from described C. rectus proteins. Similarities between epitopes and amino acid compositions of the Actinobacillus actinomycetemcomitans leukotoxin and C. rectus cytotoxin were observed. These data indicate that C. rectus secretes a 104 kDa cytotoxin.

Amino Acid Sequence↗

PubMed1993

Sialochemistry: a diagnostic tool?

Saliva has proven to be a discriminating element in forensic arenas, an effective indicator of acute diseases of salivary glands, and a promising probe for drug monitoring. With the advent of sensitive immunochemical assays, the compositional profile of human salivary secretions has been expanded considerably. Thus, the establishment of a range of "normal values" for a variety of "intrinsic" and "extrinsic" salivary components represented the initial step to use saliva as a diagnostic tool of oral health status. Unfortunately, numerous cross-sectional studies have shown a wide individual variation in the salivary composition of healthy populations, thus precluding its use as a diagnostic chair-side test for the screening of the most common chronic oral diseases (e.g. caries and periodontal disease). A possible explanation may arise from the wide functional versatility of salivary molecules. For instance, it has been recognized recently that in addition to its digestive properties, salivary amylase may modulate bacterial colonization, whereas histatins are not only antifungal but also bactericidal. Thus, low levels of already known antimicrobial salivary molecules (e.g., secretory IgA, lactoferrin, and lysozyme) could be compensated with higher concentrations of other molecules with antimicrobial activity, such as amylase and histatins. Consequently, for caries and periodontal diseases, longitudinal sialochemical studies may yield more insight than cross-sectional studies.

Aged↗

PubMed1992

Structural features of the low-molecular-mass human salivary mucin.

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

Amino Acid Sequence↗

PubMed1992

Characterization of an amylase-binding component of Streptococcus gordonii G9B.

The goal of the present study was to begin characterizing the amylase-binding component(s) on the surface of Streptococcus gordonii G9B. Alkali extracts but not phenol-water extracts of this bacterium inhibited 125I-amylase binding to S. gordonii G9B. To identify the bacterial components involved in amylase binding, the alkali extract was subjected to affinity chromatography on amylase-Sepharose. Immunoblotting with a rabbit antiserum against S. gordonii G9B revealed that a 20-kDa streptococcal component was eluted from the amylase-Sepharose with 1% sodium dodecyl sulfate (SDS), 2 M KSCN, or 0.1 M sodium citrate buffer, pH 4.5. Subsequently, the 20-kDa component was prepared from alkali extracts by electroelution from preparative SDS electrophoresis or by gel filtration chromatography. This component was trypsin sensitive, and an antibody raised against it inhibited the binding of 125I-amylase to S. gordonii G9B. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated that both bound amylase and the 20-kDa component were localized to the cell division septum on dividing cells or to polar zones on single cells. In addition, exponentially growing bacteria bound more 125I-amylase than stationary-phase cells did. Collectively, these results suggest that a 20-kDa amylase-binding component is present on the surface of the nascent streptococcal cell wall.

Amylases↗

PubMed1992

Inhibition of Porphyromonas gingivalis adhesion to Streptococcus gordonii by human submandibular-sublingual saliva.

Porphyromonas gingivalis W50 adheres in vitro to biofilms of Streptococcus gordonii G9B. This phenomenon is believed to facilitate the initial colonization of the oral cavity by P. gingivalis and to contribute to the maturation of dental plaque. In this report, we describe the modulating effects of human submandibular-sublingual saliva (HSMSL) on this in vitro model of intergeneric bacterial adhesion (coaggregation). HSMSL inhibited P. gingivalis adhesion to S. gordonii by 50% at a concentration of 57 micrograms of protein per ml. Maximum inhibitory activity was associated with a 43-kDa protein obtained by sequential Sephadex G200 gel filtration and CM52 ion-exchange chromatography of HSMSL. Pools of other column fractions of HSMSL showed no effect or were slightly stimulatory for bacterial adhesion. The binding of radioiodinated column fractions containing the 43-kDa protein by P. gingivalis was accompanied by their rapid enzymatic degradation. Treating P. gingivalis at 60 degrees C for 30 min or with protease inhibitors (phenylmethylsulfonyl fluoride and sodium iodoacetate) reduced adherence to streptococcal biofilms. These treatments did not prevent P. gingivalis from binding soluble HSMSL saliva components, although subsequent proteolysis was nearly eliminated. These observations indicate that surface-associated proteases of P. gingivalis, either independently or in concert with adjacent surface adhesins, interact with surfaces of oral streptococci to facilitate interbacterial adhesion. The adhesion-blocking properties of HSMSL, particularly the 43-kDa protein, may represent an important host defense mechanism in the oral cavity.

Bacterial Adhesion↗

PubMed1991

Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva.

The major components of human submandibular-sublingual saliva (HSMSL) are mucins, amylases, cystatins, proline-rich proteins and statherin. Structure-function studies of these molecules have been hampered by the small amounts of purified materials that can be isolated from human secretions. The present study describes an integrated purification protocol for the large-scale preparation of many of these molecules. To dissociate partially heterotypic complexes among salivary molecules, HSMSL was initially fractionated into four pools by gel filtration with 6 M-guanidine hydrochloride. Subsequent fractionation of these four pools by gel-filtration and ion-exchange chromatography resulted in the purification of high- and low-Mr mucins, neutral and acidic cystatins, acidic and basic proline-rich proteins and statherin. Many variants or isoforms of these salivary molecules have been identified and biochemically characterized. Biochemical studies indicated that the low-Mr mucin exists as two isoforms which vary in their sialic acid to fucose ratios. Three isoforms of acidic cystatin S were characterized which differ in their phosphate content. Two isoforms of a basic proline-rich peptide were identified; the smaller peptide was a truncated form missing the first seven amino acids.

Adult↗