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E Kraig

Publications and source records attributed to E Kraig.

At least 37 records · Page 2Linked to original sources
PubMed1991

Regulation of leukotoxin in leukotoxic and nonleukotoxic strains of Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans is a gram-negative bacterium that has been implicated in the etiology of several forms of periodontitis, especially localized juvenile periodontitis. A potent leukotoxin (Lkt) is produced by most A. actinomycetemcomitans isolates from patients with periodontal disease, but some isolates are leukotoxin nonproducing (Lkt-). The molecular bases for the differences in leukotoxin expression are being explored to clarify the role of leukotoxin in pathogenesis. We have previously cloned the leukotoxin structural gene, lktA, from the leukotoxin-producing (Lkt+) strain JP2 and have shown that it is linked to three other genes, lktB, lktC, and lktD, whose gene products are thought to be required for activation and localization of the leukotoxin. These genes have now been used in Southern blot analysis to demonstrate that Lkt- strains, like Lkt+ strains, contain all four genes of the lkt gene cluster. While restriction fragment length polymorphisms were detected, they did not correlate with toxin phenotype. RNA blot analysis demonstrated that Lkt+ strains produced two transcripts, one 9.3 kb in length and the other 4.3 kb. They encode lktCABD and lktCA. respectively. Lkt- strains contained significantly lower levels of the 4.3-kb transcript with no discernible 9.3-kb message. The leukotoxic activity of the A. actinomycetemcomitans strains, measured by chromium release assays, correlated with the lkt RNA content. Therefore, a major component of leukotoxin regulation is at the level of RNA transcription or stability. Interestingly, the lkt RNAs in JP2 are regulated during growth phase, being greatly reduced in cells approaching stationary phase. Thus, the regulation of lkt RNA can be affected by both genotype and environment.

Actinobacillus↗
PubMed1990

Two ultraviolet tumor-specific suppressor cell clones. One expresses Ig RNA and the other expresses T cell receptor-alpha and -beta RNA.

Two splenic cell clones were derived from mice bearing UV-induced skin tumors; both clones are I-J+, Thy-1+, and J11D+. Cellular extracts, but not culture supernatants, from the two clones, designated T4 and T5, specifically suppress primary cytotoxic and proliferative responses to UV-induced tumor targets. Therefore, clones T4 and T5 would appear to function as putative I-J+ suppressor cells with specificity for an antigenic epitope common to several UV-induced tumors. To derive more information concerning the status of Ag-receptor genes in the suppressive clones, we analyzed the rearrangement and expression of Ig and TCR genes. The first clone, T4, expressed Thy-1, a T cell-surface marker, yet it had not rearranged its TCR-beta, -gamma, and -delta gene families, nor were TCR-alpha, -beta, -gamma, or -delta transcripts detectable. Instead, clone T4 contained rearranged Ig H chain genes and expressed mu RNA. Because neither lambda nor kappa L chain transcripts were detected, this clone could not utilize a complete Ig molecule as its Ag receptor. On the other hand, clone T5 contained exclusively TCR gene rearrangements; it expressed alpha and beta transcripts of the correct sizes. However, the level of RNA that could encode the delta-chain of the CD3 accessory molecule was below detection, so it is unlikely that this TCR was expressed on the cell surface. The rearrangement of Ig genes in one clone and TCR genes in the other clone does not necessarily indicate that these genes encode the Ag receptors used by these clones. However, it suggests either that suppressor-like cells can derive from different lymphocyte precursor lineages or that rearrangements of the known TCR genes or the Ig genes are irrelevant to the function of this cell lineage.

Animals↗
PubMed1990

Molecular approaches to leucotoxin as a virulence component in Actinobacillus actinomycetemcomitans.

A strategy has been developed to examine the hypothesis that leucotoxin is a critical virulence factor of Actinobacillus actinomycetemcomitans in a non-human primate (Macaca fascicularis). Firstly the leucotoxin gene from A. actinomycetemcomitans was cloned and sequenced. This DNA contained a functional leucotoxin gene, as protein extracts of Escherichia coli with the cloned sequences lysed appropriate human cell lines. The protein encoded by lktA shared at least 42% identity with P. haemolytica leucotoxin and with the alpha-haemolysins from E. coli and A. pleuropneumoniae. The lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be related to the LktC proteins from these other bacteria and with which it shared at least 49% amino acid identity. Despite the overall homology to the other leucotoxins/haemolysins, the LktA from A. actinomycetemcomitans has several unique properties including a very basic pI of 9.7, as compared to pIs approx. 6.2 for lktA proteins in other bacteria. Using the cloned genes as probes produced evidence that a TOX- strain contains the leucotoxin gene but fails to transcribe it at high levels. The second avenue of investigation was to develop methods for examining the humoral immune responses in the monkey to bacterial toxins such as lktA. A. actinomycetemcomitans was detected in subgingival plaque samples from approx. 40% of the animals. A. actinomycetemcomitans comprised less than 1% to 9% of the flora. Most A. actinomycetemcomitans isolates were serotype b and each of the monkeys had serum IgG antibody to A. actinomycetemcomitans serotype b (generally considered to be lktA-producing strains). An ELISA was developed to examine the isotype/subclass distribution, level and avidity of serum antibody in the monkey following parenteral immunization with a prototype bacterial exotoxin (tetanus toxoid). IgG1 and IgG3 antibody predominated over IgG2 and IgG4 after primary immunization. Secondary immunization elicited enriched IgG1 and IgG4 responses. Primary immunization increased avidity indices of IgG to tetanus toxoid from approx. 0.9 (baseline) to a mean of 1.72 and secondary immunization significantly increased the avidity index to 2.56.

Actinobacillus↗
PubMed1990

Na+, K(+)-ATPase of the photoreceptor: selective expression of alpha 3 and beta 2 isoforms.

In photoreceptors, Na+, K(+)-ATPase maintains the ion gradients which power the dark current that sustains the response to light. The enzyme is composed of at least two polypeptides: alpha (the catalytic subunit) and beta. Three different isoforms of the alpha subunit and two isoforms of the beta subunit have been identified in rat. In some tissues, the isoenzymes have been shown to be differentially expressed during development or in response to varying physiological conditions. RNAs prepared from isolated photoreceptors and from whole retina were analyzed on blots that were hybridized with cDNA probes for the alpha 1, alpha 2, alpha 3, beta 1 and beta 2 isoforms. The predominant alpha and beta subunit mRNAs present in the photoreceptor preparation were those encoding the alpha 3 and beta 2 isoforms, accounting for 85% of the total alpha signal and 79% of the total beta signal, respectively. Proportions of each mRNA were similar in retina, but very different from those observed in two control tissues, brain and kidney. To confirm that the alpha-subunit mRNA species detected were translated, membranes prepared from isolated photoreceptors and whole retina were examined by immunoblotting. The antibodies detected a pattern of alpha isoform distribution in these tissues and in kidney and brain controls that agreed remarkably well with the pattern of mRNA expression in the same tissues. Moreover, the alpha 3 isoform was detectable in the inner segment plasma membrane of the photoreceptor by electron microscopic immunocytochemistry. These results indicate that alpha 3, and beta 2 are the predominant isoforms of Na+, K(+)-ATPase expressed in photoreceptors and retina.

Animals↗
PubMed1990

Nucleotide sequence of the leukotoxin gene from Actinobacillus actinomycetemcomitans: homology to the alpha-hemolysin/leukotoxin gene family.

The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of localized juvenile periodontitis. To initiate a genetic analysis into the role of this protein in disease, we have cloned its gene, lktA. We now present the complete nucleotide sequence of the lktA gene from A. actinomycetemcomitans. When the deduced amino acid sequence of the leukotoxin protein was compared with those of other proteins, it was found to be homologous to the leukotoxin from Pasteurella haemolytica and to the alpha-hemolysins from Escherichia coli and Actinobacillus pleuropneumoniae. Each alignment showed at least 42% identity. As in the other organisms, the lktA gene of A. actinomycetemcomitans was linked to another gene, lktC, which is thought to be involved in the activation of the leukotoxin. The predicted LktC protein was related to the leukotoxin/hemolysin C proteins from the other bacteria, since they shared a minimum of 49% amino acid identity. Surprisingly, although actinobacillus species are more closely related to pasteurellae than to members of the family Enterobacteriaciae, LktA and LktC from A. actinomycetemcomitans shared significantly greater sequence identity with the E. coli alpha-hemolysin proteins than with the P. haemolytica leukotoxin proteins. Despite the overall homology to the other leukotoxin/hemolysin proteins, the LktA protein from A. actinomycetemcomitans has several unique properties. Most strikingly, it is a very basic protein with a calculated pI of 9.7; the other toxins have estimated pIs around 6.2. The unusual features of the A. actinomycetemcomitans protein are discussed in light of the different species and target-cell specificities of the hemolysins and the leukotoxins.

Actinobacillus↗
PubMed1989

Cloning and expression of the leukotoxin gene from Actinobacillus actinomycetemcomitans.

The leukotoxin produced by Actinobacillus actinomycetemcomitans has been implicated in the etiology of juvenile periodontitis. To initiate a genetic analysis of the role of this protein in disease, we have cloned the leukotoxin gene in Escherichia coli. Recombinant colonies carrying toxin gene sequences were isolated by screening a genomic A. actinomycetemcomitans library with a DNA probe for the leukotoxin gene from a related bacterium, Pasteurella haemolytica. To demonstrate that the cloned A. actinomycetemcomitans DNA contained a functional leukotoxin gene, protein extracts of E. coli containing the A. actinomycetemcomitans clone were tested directly for leukotoxic activity against human cell lines in chromium release assays. A construct containing the entire cloned region produced a functional toxin. No cytotoxicity was seen when extracts from cells containing plasmids with deletions in the putative coding region were used. Furthermore, the toxin produced by the cloned gene has the same target cell specificity as the leukotoxin extracted directly from A. actinomycetemcomitans. These results indicate that sequences encoding a functional leukotoxin have been cloned and are expressed in E. coli. Southern blot analysis of DNA from leukotoxin-producing (Lkt+) and non-leukotoxin-producing (Lkt-) strains indicated that the Lkt- strain also contained a copy of the gene.

Actinobacillus↗
PubMed1989

Dual functions of a cis-acting element within the rat prolactin gene promoter.

Within the promoter region of the rat prolactin gene lies a TA-rich imperfect palindrome. The possible functions of the 18-base-pair symmetrical sequence were investigated by using an in vitro transcription system. Prolactin templates with and without the palindrome were transcriptionally assayed in both pituitary and nonpituitary extracts. Our results indicated that the palindromic sequence has at least two functions in the regulation of prolactin transcription.

Animals↗
PubMed1988

Strain-dependent expression of VH gene families.

The tremendous diversity of the antibody specificity repertoire stems from the ability of each developing B cell to select one out of many possible variable, diversity, and joining gene segments by specific rearrangement of the DNA. The mechanism by which V region gene segments is selected is not known. Moreover, evidence for both random and nonrandom expression of VH genes in mature B cells has been presented previously. In this report, the technique of in situ hybridization is used to accurately measure at the single cell level VH gene family expression in LPS-induced cells from several strains. In this way, at least one-third of the B cells are stimulated and a large sampling of activated splenocytes from each strain analyzed. The use of in situ hybridization eliminates any potential biases resulting from transformation protocols. In addition, because all populations of cells are analyzed by both in situ hybridization and immunocytochemical staining with anti-IgM, the proportion of cells detected by in situ hybridization could be compared with the proportion of B cells, blasts, and plasma cells in the population. It was concluded from these comparisons that the cells being detected by in situ hybridization under the conditions described are plasmablasts and plasma cells. Therefore, an accurate measure of the functional and expressed VH gene repertoire could be made. The results clearly demonstrate strain-dependent variation in VH gene family expression, particularly VH 7183 and VH J558 with up to three-fold differences observed. Thus, either there is considerable strain variation in the number of functional VH gene family segments or the expression of VH genes is not entirely random.

Animals↗
PubMed1987

Interleukin 2, T cell receptor and sex hormone studies in autoimmune mice.

Systemic lupus erythematosus (SLE) is a disease of immune dysregulation in which B cell hyperactivity and T cell deficiency are important characteristics. Sex factors also play a major role in the pathogenesis based on the physiologic effects of estrogen in promoting immunologic hyperactivity. Our findings suggest that a posttranscriptional mechanism is responsible for the functional interleukin 2 (IL-2) defect since transcription of the IL-2 message occurs after mitogenic stimulation. The proliferating cell in the MRL/lpr mouse model of lupus may be an immature T cell. The T cell receptor in these mice has a lower molecular weight than normal. This aberrant T cell receptor might be explained by a defect in glycosylation. The administration of estrogen to pregnant mice late in gestation results in offspring with a permanently altered immune system. These mice develop features of autoimmunity similar to those that occur spontaneously in genetically susceptible autoimmune mice. This phenomenon may have etiopathological significance for familial SLE.

Animals↗
PubMed1983

T and B cells that recognize the same antigen do not transcribe similar heavy chain variable region gene segments.

We have attempted to determine whether T cells and B cells that have the same antigenic specificity and whose receptors share idiotypic determinants in fact express similar VH gene segments. To do this, we have obtained and characterized a cDNA clone containing the entire coding sequence for the VH gene from a glutamic acid60/alanine30/tyrosine10 (GAT)-binding immunoglobulin that carries the CGAT idiotype. The GAT-VH clone was hybridized to Northern blots of GAT-specific T cell RNAs; there was no evidence of a T cell transcript that hybridized to the GAT-VH probe. The T cells analyzed included: (a) 10 GAT-binding suppressor T cell hybridomas, 6 of which secreted factors with CGAT idiotypic determinants, (b) one GAT-specific helper T cell hybridoma, and (c) two GAT-specific helper T cell lines grown in the absence of feeder cells. The detection limit of the Northern blot analysis was 1-2 copies of a particular mRNA species per cell for the hybridomas and 5-10 copies per cell for the T cell lines. Therefore, we conclude that T and B lymphocytes responding to GAT do not utilize similar VH gene segments. Furthermore, the presence of idiotypic determinants on T lymphocytes does not necessarily imply close structural similarity between T and B cell antigen receptors.

Animals↗
PubMed1983

Three T cell hybridomas do not contain detectable heavy chain variable gene transcripts.

We attempted to determine whether T cells express any VH gene segments. cDNA libraries were constructed from one suppressor and two helper T cell hybridomas. Both the library construction and screening were designed to maximize detection of a wide range of VH gene segments. One screening method should detect about half of the sequenced VH genes, while the second should detect most of these genes. The probability of detecting a VH gene homologous to the probes and present at 10 copies per cell was 77% for one helper cell cDNA library, 88% for the second helper cell library, and greater than 99% for the suppressor cell library. No cDNA clones with VH gene segments were detected. From this result, we conclude that VH gene segments are not likely to encode the antigen-specific receptor in the cells we tested.

Animals↗
PubMed1983

RNA transcripts for I-J polypeptides are apparently not encoded between the I-A and I-E subregions of the murine major histocompatibility complex.

The I-J subregion of the mouse major histocompatibility complex has been reported to encode antigenic determinants expressed by suppressor T cells. Previously, cosmid clones were obtained from mouse sperm DNA that contain all of the sequences between the I-A and I-E subregions, where I-J has been mapped genetically. However, hybridization of these sequences to RNA prepared from several I-J-positive suppressor T-cell hybridomas did not reveal the presence of a transcript. In addition, no rearrangements in this DNA were detected in the suppressor T cells that we have analyzed. Our results indicate that the I-J polypeptides are not encoded between the I-A and I-E subregions of the major histocompatibility complex. We discuss several hypotheses concerning the possible location and expression of I-J genes.

Animals↗
PubMed1982

Sporulation and rna2 lower ribosomal protein mRNA levels by different mechanisms in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the levels of ribosomal protein mRNAs are regulated coordinately. Vegetative strains carrying the temperature-sensitive rna2 mutation exhibit a dramatic decrease in the levels of most ribosomal protein mRNAs at the restrictive temperature. Similarly, in wild-type cells induced to sporulate by nitrogen starvation, there is a fivefold reduction in the relative synthesis rate of ribosomal proteins. Using Northern gel analysis and cloned ribosomal protein genes, we compared the way in which ribosomal protein mRNA is affected under these two conditions. In vegetative rna2 cells, incubation at 34 degrees C led to the disappearance of ribosomal protein mRNAs and the accumulation of higher-molecular-weight precursor RNAs. A different phenotype was observed during sporulation. Although sporulating conditions led to a significant reduction in the relative abundance of ribosomal protein mRNA, there was no detectable accumulation of precursor RNAs even in rna2/rna2 diploids at 34 degrees C. A suppressor of rna2 and of other rna mutations, SRN1, at least partially relieved the block in the splicing of the ribosomal protein 51 intron in vegetative rna2 cells but did not detectably affect the level of ribosomal protein mRNA in sporulating cells. We concluded that the rna2 mutation and sporulation conditions affected ribosomal protein mRNA metabolism in two quite different ways. In vegetative cells the mutant rna2 effected a block which occurred primarily in post-transcriptional processing, whereas in sporulating cells the ribosomal protein mRNA levels were decreased by some other mechanism, presumably a change in the relative rate of transcription or mRNA turnover. Furthermore, the data suggest that the mutation rna2 has no additional effect on ribosomal protein mRNA metabolism in sporulating cells.

Gene Expression Regulation↗