Cross reactivity between IA-2 and phogrin/IA-2β in binding of autoantibodies in IDDM

Summary Patients with insulin-dependent diabetes mellitus (IDDM) possess antibodies to the cytoplasmic domains of two closely related tyrosine phosphatase-like proteins, IA-2 and phogrin, previously detected as 40 kDa and 37 kDa tryptic fragments, respectively. A higher proportion of IDDM patients possess antibodies to IA-2 than to phogrin, and autoimmunity to phogrin might arise through cross-reactivity with the highly homologous IA-2. In this study, we have investigated the major regions of IA-2 recognized by antibodies in IDDM patients and examined the ability of phogrin to block antibody binding to these regions as a measure of cross-reactivity. Analysis of antibody binding to in vitro transcribed and translated polypeptides representing different regions of the cytoplasmic domain of IA-2 identified five different patterns of reactivity with antibodies in IDDM. Protein footprinting analysis, whereby polypeptide fragments generated on protease treatment of immune complexes are studied, indicated considerable heterogeneity in antibody recognition of IA-2, even between sera with similar reactivity to deletion mutants. Blocking studies with recombinant phogrin indicated that IA-2 antibodies recognize epitopes that are both unique to IA-2 and shared with phogrin. The amino-terminal 150 amino acids of the cytoplasmic domain of IA-2 encompass epitopes that are not represented on phogrin, whereas shared epitopes are localized within the carboxy-terminal 220 amino acids. The results demonstrate considerable heterogeneity between IDDM patients in autoantibody recognition of IA-2 in IDDM, whereas antibody recognition of phogrin is restricted in most patients to epitopes also present on IA-2. [Diabetologia (1997) 40: 1327–1333] Received: 4 April 1997 and in revised form: 2 July 1997     

Mycoplasma pneumoniae Induces Chronic Respiratory Infection, Airway Hyperreactivity, and Pulmonary Inflammation: a Murine Model of Infection-Associated Chronic Reactive Airway Disease

Because chronic Mycoplasma pneumoniae respiratory infection is hypothesized to play a role in asthma, the potential of M. pneumoniae to establish chronic respiratory infection with associated pulmonary disease was investigated in a murine model. BALB/c mice were intranasally inoculated once with M. pneumoniae and examined at 109, 150, 245, 368, and 530 days postinoculation. M. pneumoniae was detected in bronchoalveolar lavage fluid by culture or PCR in 70 and 22% of mice at 109 and 530 days postinoculation, respectively. Lung histopathology was normal up to 368 days postinoculation. At 530 days, however, 78% of the mice inoculated with M. pneumoniae demonstrated abnormal histopathology characterized by peribronchial and perivascular mononuclear infiltrates. A mean histopathologic score (HPS) at 530 days of 5.1 was significantly greater (P < 0.01) than that for controls (HPS score of 0). Serum anti-M. pneumoniae immunoglobulin G was detectable in all of the mice inoculated with M. pneumoniae and was inversely correlated with HPS (r = -0.95, P = 0.01) at 530 days postinoculation. Unrestrained whole-body plethysmography measurement of enhanced pause revealed significantly elevated airway methacholine reactivity in M. pneumoniae-inoculated mice compared with that in controls at 245 days (P = 0.03) and increased airway obstruction at 530 days (P = 0.01). Murine M. pneumoniae respiratory infection can lead to chronic pulmonary disease characterized by airway hyperreactivity, airway obstruction, and histologic inflammation.     

Characterization of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli serotypes isolated from sheep.

The isolation and characterization of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) strains from sheep are described. One flock was investigated for E. coli O157:H7 over a 16-month period that spanned two summer and two autumn seasons. Variation in the occurrence of E. coli O157:H7-positive sheep was observed, with animals being culture positive only in the summer months but not in the spring, autumn, or winter. E. coli O157:H7 isolates were distinguished by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA and toxin gene restriction fragment length polymorphism (RFLP) analysis. Ten PFGE patterns and five RFLP patterns, identified among the isolates, showed that multiple E. coli O157:H7 strains were isolated from one flock, that a single animal simultaneously shed multiple E. coli O157:H7 strains, and that the strains shed by individuals changed over time. E. coli O157:H7 was isolated only by selective enrichment culture off 10 g of ovine feces. In contrast, strains of eight STEC serotypes other than O157:H7 were cultured from feces of sheep from a separate flock without enrichment. The predominant non-O157 STEC serotype found was O91:NM (NM indicates nonmotile), and others included O128:NM, O88:NM, O6:H49, and O5:NM. Irrespective of serotype, 98% of the ovine STEC isolates possessed various combinations of the virulence-associated genes for Shiga toxin(s) and the attaching-and-effacing lesion (stx1, stx2, and eae), suggesting their potential for human pathogenicity. The most common toxin-eae genotype was positive for stx1, stx2, and eae. A Vero cell cytotoxicity assay demonstrated that 90% of the representative STEC isolates tested expressed the toxin gene. The report demonstrates that sheep transiently shed a variety of STEC strains, including E. coli O157:H7, that have potential as human pathogens.     

Locating sequence on FPC maps and selecting a minimal tiling path

This study discusses three software tools, the first two aid in integrating sequence with an FPC physical map and the third automatically selects a minimal tiling path given genomic draft sequence and BAC end sequences. The first tool, FSD (FPC Simulated Digest), takes a sequenced clone and adds it back to the map based on a fingerprint generated by an in silico digest of the clone. This allows verification of sequenced clone positions and the integration of sequenced clones that were not originally part of the FPC map. The second tool, BSS (Blast Some Sequence), takes a query sequence and positions it on the map based on sequence associated with the clones in the map. BSS has multiple uses as follows: (1) When the query is a file of marker sequences, they can be added as electronic markers. (2) When the query is draft sequence, the results of BSS can be used to close gaps in a sequenced clone or the physical map. (3) When the query is a sequenced clone and the target is BAC end sequences, one may select the next clone for sequencing using both sequence comparison results and map location. (4) When the query is whole-genome draft sequence and the target is BAC end sequences, the results can be used to select many clones for a minimal tiling path at once. The third tool, pickMTP, automates the majority of this last usage of BSS. Results are presented using the rice FPC map, BAC end sequences, and whole-genome shotgun from Syngenta.     

The relationship of age and cardiovascular fitness to cognitive and motor processes

Older and younger aerobically trained and sedentary adults participated in an S1-S2-S3 paradigm designed to elicit event-related potential (ERP) and behavioral responses to determine the influence of cardiovascular fitness on cognitive and motor processes. The paradigm provided warning (S1) as to the difficulty level of an upcoming decision task (S2). Participants had to decide the taller of two bars on presentation of S2 but hold their response until S3, to which they indicated their choice motorically. Results revealed age-related differences for ERP measures as older participants showed increased amplitude of the stimulus preceding negativity (SPN) prior to S2, and longer latencies and equipotentiality of P3 in response to S2. Fitness effects were also observed for the contingent negative variation (CNV) with decreased amplitude for fit relative to sedentary individuals. Age interacted with fitness for P3 latency to S2 as older sedentary individuals showed the longest latency followed by older fit and both younger groups. No significant group differences were observed for reaction time (RT) to S3. Therefore, physical fitness is associated with attenuation of cognitive decline in older individuals and greater economy of motor preparation for both young and older participants.     

An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT

A new tetrazolium salt XTT, sodium 3'-[1-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6- nitro)benzene-sulfonic acid hydrate, was evaluated for use in a colorimetric assay for cell viability and proliferation by normal activated T cells and several cytokine dependent cell lines. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells yields a highly colored formazan product which is water soluble. This feature obviates the need for formazan crystal solubilization prior to absorbance measurements, as required when using other tetrazolium salts such as MTT. Bioreduction of XTT by all the murine cells examined was not particularly efficient, but could be potentiated by addition of electron coupling agents such as phenazine methosulfate (PMS) or menadione (MEN). Optimal concentrations of PMS or MEN were determined for the metabolism of XTT by the T cell lines HT-2 and 11.6, NFS-60 a myeloid leukemia, MC/9 a mast cell line and mitogen activated splenic T cells. When used in combination with PMS, each of these cells generated higher formazan absorbance values with XTT than were observed with MTT. Thus the use of XTT in colorimetric proliferation assays offer significant advantages over MTT, resulting from reduced assay time and sample handling, while offering equivalent sensitivity.     

Plant Antiviral Agents; VI.1 3-Methoxyflavones as Potent Inhibitors of Viral-Induced Block of Cell Synthesis

An investigation of the constituents responsible for the pronounced antiviral activity observed for extracts of EUPHORBIA GRANTII Oliv. stems has afforded four related 3-methoxyflavones exhibiting remarkable activities against picornaviruses and vesicular stomatitis virus. All compounds were found to be derivatives of 3-methylquercetin. The concentration of 3-methylquercetin (3-MQ) and 3,3'-dimethyl-quercetin (3,3'-DMQ) inhibiting 90 % of polio type 1 and coxsackie B4 viruses in tissue culture was about 0.01 microg/ml, whereas the 50 % cytotoxic concentration was 40 microg/ml. When administered intraperitoneally, 3-MQ protected mice from viremia and lethal infections from coxsac kie B4 virus at a daily dose of 20 mg/kg for a period of nine days. Biochemical studies on the mechanism of action of 3-MQ or 3,3'-DMQ on poliovirus replication suggested that these 3-methoxyflavones are able to protect the host cells from a viral induced shutdown of the cellular protein synthesis. Preliminary structure activity relationship studies have shown the 3-methoxyfunction of the flavones to be essential for the observed antiviral effects.