Curtis L. Atkin

The Mycoplasma arthritidis T-cell mitogen MAM: a model superantigen

The superantigens are receiving a great deal of attention as a new group of potent immunomodulatory molecules. They are produced by diverse microbial agents including staphylococci, streptococci and mycoplasmas and are also encoded by murine tumor viruses (the Mls antigens). Superantigens activate T cells by a unique pathway which can lead to modification of the T-cell repertoire and induction of autoimmunity. Here, Barry Cole and Curtis Atkin review their observations on the Mycoplasma arthritidis superantigen, MAM, and discuss how MAM might contribute to the acute and chronic inflammatory disease mediated by this organism.

Stable isotopes of calcium as tracers: methodology

While radioisotopic tracers have been used extensively, stable isotopes may, in principle, give the same information and offer several advantages for some investigations. Since there is no radiation involved, experiments using such high risk groups as children and pregnant women become possible. In addition, some studies may require using several different tracers. In the case of calcium, there are five stable isotopes ( 42Ca, 43Ca, 44Ca, &Ca and 48Ca) which may be used as tracers. If stable isotopic tracers are used, experiments may be done independent of isotope production schedules and the samples may be stored indefinitely. In spite of these advantages, stable isotopes have rarely been used as tracers because there has not been a satisfactory method of detection. Although isotopic abundances of calcium have been determined by both neutron activation analysis [1,2] and thermal ionization mass spectrometry [3,4], neither method has been used extensively for clinical investigation. This is presumably due to requirements for extensive sample preparation, and the necessary instrumentation. It has recently been shown [5] that fast atom bombardment mass spectrometry can be used to measure tracer levels of stable isotopes of calcium in plasma and urine. This technique offers good precision, high sensitivity, rapid analysis, and requires little or no sample preparation. This paper describes the first application of fast atom bombardment mass spectrometry and the double isotope technique to measure the fractional true absorption of calcium.

Intact form of myeloperoxidase from normal human neutrophils.

Abstract Myeloperoxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7) of human polymorphonuclear neutrophils was purified rapidly in the presence of the protease inhibitors phenylmethanesulfonyl fluoride and pepstatin A. The purified enzyme behaved as a single molecular species in several nondenaturing electrophoretic and chromatographic systems. Peroxidase activity in fresh extracts of neutrophils from 20 normal persons and from 5 patients with polycythemia was electrophoretically identical to purified enzyme. Treatment with trypsin converted myeloperoxidase to multiple electrophoretic forms of active enzyme. Size (Mr ca. 15,000 and ca. 55,000) and stoichiometry of the subunits of purified enzyme, and enzyme Mr ca. 140,000, were compatible with intact myeloperoxidase having an α2β2 structure. We found no evidence for electrophoretically detectable genetic polymorphism of myeloperoxidase. Proteolytic degradation of myeloperoxidase probably accounted for electrophoretic heterogeneity of enzyme and for some constituent peptides described previously.

Efficient detection of alport syndrome COL4a5 mutations with multiplex genomic PCR-SSCP

We have performed effective mutation screening of COL4A5 with a new method of direct, multiplex genomic amplification that employs a single buffer condition and PCR profile. Application of the method to a consecutive series of 46 United States patients with diverse indications of Alport syndrome resulted in detection of mutations in 31 cases and of five previously unreported polymorphisms. With a correction for the presence of cases that are not likely to be due to changes at the COL4A5 locus, the mutation detection sensitivity is greater than 79%. The test examines 52 segments, including the COL4A6/COL4A5 intergenic promoter region, all 51 of the previously recognized exons and two newly detected exons between exons 41 and 42 that encode an alternatively spliced mRNA segment. New genomic sequence information was generated and used to design primer pairs that span substantial intron sequences on each side of all 53 exons. For SSCP screening, 16 multiplex PCR combinations (15 4-plex and 1 3-plex) were used to provide complete, partially redundant coverage of the gene. The selected combinations allow clear resolution of products from each segment using various SSCP gel formulations. One of the 29 different mutations detected initially seemed to be a missense change in exon 32 but was found to cause exon skipping. Another missense variant may mark a novel functional site located in the collagenous domain.

High-density genetic and physical mapping of DNA markers near the X-linked Alport syndrome locus: definition and use of flanking polymorphic markers

SummaryTo refine the genetic and physical mapping of the locus for Alport syndrome (ATS), 22 X-chromosome restriction fragment length polymorphism (RFLP) markers that fall between Xq21.3 and Xq25 were tested for genetic linkage with the disease and also mapped with respect to a series of physical breakpoints in this region. The location of the COL4A5 gene, which has recently been shown to be mutated in at least some families with Alport syndrome, was determined with respect to the same physical breakpoints. Two large Utah kindreds were included in the genetic studies, kindreds P and C, with 125 and 63 potentially informative meioses, respectively. Both kindreds have essentially identical nephritis; however, kindred P has sensorineural hearing loss associated with the nephritis, while kindred C does not. A mutation in COL4A5 has been demonstrated for kindred P, but no change in this gene has yet been detected for kindred C. Twelve informative probes did not recombine with the disease locus in either kindred (θ= 0.0, with combined lod scores for the two kindreds ranging from 7.7 to 30.0). The closest markers that could be demonstrated to flank the disease locus were the same for each kindred and thus the locations of the mutations causing the two disease phenotypes are not distinguishable at the current level of genetic resolution. The flanking markers are also useful for the resolution of questionable diagnoses and allow accurate estimates for these families of the rate of sporadic hematuria in noncarrier females (7%) and the penetrance of hematuria for carrier females (93%).

Common ancestry of three Ashkenazi-American families with Alport syndrome and COL4A5 R1677Q

Abstract Mutations in the basement membrane collagen gene COL4A5 cause the progressive renal glomerular nephropathy and typical hearing loss that occur in X-linked Alport syndrome. Nearly all cases involve distinct mutations, as expected for an X-linked disease that significantly reduces the fitness of affected males. A few exceptional COL4A5 mutations appear to be associated with a reduced disease severity and may account for a significant proportion of late-onset Alport syndrome in populations where a founder effect has occurred. The novel mutation reported here, COL4A5 arg1677gln, has been detected in three independently ascertained Ashkenazi-American families, causes a relatively mild form of nephritis with typical onset in the fourth or fifth decade, and may be involved in the etiology of a large proportion of adult-onset hereditary nephritis in Ashkenazi Jews.

Expression of mRNA for type IV collagen α1, α5 and α6 chains by cultured dermal fibroblasts from patients with X-linked Alport syndrome

Abstract COL4A5 mutations causing X-linked Alport syndrome (XLAS) are frequently associated with absence of the α3, α4, α5 and α6 chains of type IV collagen from basement membranes and increased amounts of the α1(IV) and α2(IV) chains in glomerular basement membrane. Although many COL4A5 mutations have been described in XLAS, the mechanisms by which these mutations influence the basement membrane appearance of chains other than α5(IV) remain poorly understood. In this study, we used dermal fibroblasts from eight normal individuals and nine males with XLAS to test the hypotheses that COL4A5 mutations increase transcription of COL4A1 and suppress transcription of COL4A6. Ribonuclease protection assays revealed that α1(IV), α5(IV) and α6(IV) transcripts were expressed in cultures of dermal fibroblasts. The mRNA levels for α1(IV) in eight nine patients with XLAS were not increased compared to controls; one patient with a large COL4A5 deletion showed significant elevation of α1(IV) mRNA levels. No differences in steady-state mRNA levels for α6(IV) were found when XLAS fibroblasts were compared with controls, even though little or no α6(IV) protein was detectable at the dermal-epidermal junction by immunofluorescence study. This finding suggests that post-transcriptional events account for the absence of α6(IV) in the Alport dermal-epidermal junction.

Abnormal neutrophil myeloperoxidase from a patient with chronic myelocytic leukemia

Abstract Neutrophil myeloperoxidase from a patient with chronic myelocytic leukemia was isolated under conditions designed to minimize proteolysis. Those methods yielded an α 2 β 2 form of myeloperoxidase from normal individuals. Purified enzyme from the patient had electronic absorbances ( A 430 A 280 = 0.85 ), enzymatic activity, and electrophoretic and Chromatographic behavior indistinguishable from that of normal myeloperoxidase. Edman degradation and physical studies after reduction and denaturation, however, showed that as compared to normal enzyme, one 55,000-dalton α subunit of the patients myeloperoxidase was replaced by a 39,000-dalton peptide with a different amino-terminal sequence, a mixture of smaller peptides, and an heme derivative. Myeloperoxidase from the leukemic neutrophils appeared to have been partially degraded in vivo by lysosomal proteases.

F6 – IDENTIFICATION, CHARACTERIZATION, AND PURIFICATION OF MYCOPLASMAL SUPERANTIGENS

This chapter focuses on identification, characterization, and purification of mycoplasmal superantigens (SAg). SAg can be secreted products such as the staphylococcal and streptococcal exotoxins or can be a part of the cell wall such as the streptococcal M proteins, Yersinia enterocolitica, and others. SAgs are T-cell mitogens that are dependent on major histocompatibility complex (MHC) accessory molecules. However, unlike classic antigens, they do not require processing by accessory cells but bind directly to MHC molecules outside of the antigen-binding groove. The Mycoplasma arthritidis superantigen, MAM, was first recognized by its ability to induce cytotoxic lymphocytes and lymphocyte proliferation depending on the expression of certain MHC molecules. The current interest in superantigens relates to their ability to modulate the immune system in vivo in a V β -specific manner resulting in expansion or deletion of specific V β -bearing T cells as well as a V β -specific polyclonal B-cell activation with enhanced immunoglobulin production. It has been hypothesized that these pathways may lead to the development or enhancement of human autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, and multiple sclerosis.