Changes in cellular structure of heat-treated Salmonella in low-moisture environments.

AIMS: Salmonella cells desiccated in an environment with low-water activity (aw ) show longer survival times and enhanced resistance to heat. However, little is known about the cellular ultrastructure of Salmonella in low-aw environment in relation to the survival and persistence during desiccation. MATERIALS AND RESULTS: In this study, Salmonella Enteritidis strain PT30 was dehydrated by exposure to air or by mixing with wheat flour (aw 0·30 at room temperature) for 7 days followed by heat treatment at 80°C for 10, 20, 60 min respectively. Transmission electron microscopy (TEM) was employed to examine and compare the ultrastructure of heat-treated S. Enteritidis cells after desiccation with the cells suspended in trypticase soy broth (TSB). Cells suspended in TSB broth showed disrupted ribosomes, congregated proteins and denatured DNA. However, no significant alterations were observed in the ultrastructure of the desiccated cells after heat treatment. The number of desiccated S. Enteritidis cells decreased by <1·5 log CFU per gram after 80°C treatment for 60 min, however, cells suspended in TSB declined more than 5 log10 CFU per mL at 80°C within 5 min. CONCLUSIONS: A drastic difference in the number of survivors and cellular ultrastructure was observed between vegetative and air or food-dried S. Enteritidis cells after subjecting to heat treatment at 80°C. No significant ultrastructure changes were observed in desiccated cells after heat treatment except for roughening and corrugating surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a direct comparison to illustrate how desiccation influences the cell ultrastructure before/after heat treatment, which will aid in better understanding of the fundamental mechanism underlying the increased thermal resistance of Salmonella cells in low-aw environment.

Rapid differentiation of Mycobacterium bovis and Mycobacterium tuberculosis based on a 12.7-kb fragment by a single tube multiplex-PCR.

The aim of this work was the design and validation of a rapid and easy single tube multiplex-PCR (m-PCR) assay for the unequivocal differential detection of Mycobacterium bovis and Mycobacterium tuberculosis. Oligonucleotide primers were based on the uninterrupted 229-bp sequence in the M. bovis genome and a unique 12.7-kb insertion sequence from the M. tuberculosis genome, which is responsible for species-specific genomic polymorphism between these two closely related pathogens. The m-PCR assay was optimized and validated using 22 M. bovis and 36 M. tuberculosis clinical strains isolated from diverse host species and 9 other non-tuberculous mycobacterial (NTM) strains. The designed primers invariably amplified a unique 168-bp (M. bovis-specific) and 337-bp (M. tuberculosis-specific) amplicon from M. bovis and M. tuberculosis strains, respectively. The accuracy of the assay, in terms of specificity, was 100%, as none of the NTM strains tested revealed any amplification product. As little as 20 pg of genomic DNA could be detected, justifying the sensitivity of the method. The m-PCR assay is an extremely useful, simple, reliable and rapid method for routine differential identification of cultures of M. bovis and M. tuberculosis. This m-PCR may be a valuable diagnostic tool in areas of endemicity, where bovine and human tuberculosis coexist, and the distinction of M. bovis from M. tuberculosis is required for monitoring the spread of M. bovis to humans.

Alpha-adrenergic agents. 1. Direct-acting alpha 1 agonists related to methoxamine.

A series of phenylethylamines related to methoxamine has been prepared and evaluated for direct alpha 1-receptor agonist activity. It has been observed that for open-chain compounds such as methoxamine, in which the amine-containing portion is free to adopt numerous conformations, an hydroxyl group is necessary for direct alpha 1-adrenergic activity. When the hydroxyl is removed, however, the direct component of activity is greatly reduced unless the amine is incorporated into a more sterically defined structure. From our studies we have concluded that in order for a phenylethylamine to be active as a direct alpha 1-receptor agonist it should have a beta nitrogen in a fully extended conformation relative to a substituted phenyl ring. For optimum potency, the nitrogen should be exocyclic to a saturated six-membered ring. It may be further incorporated exocyclic or endocyclic into an additional ring as long as the amine occupies a well-defined region of space relative to the aromatic portion of a molecule. The ED50 values of some of the more potent compounds as alpha 1-receptor agonists are on the order of 1 X 10(-7) M.

alpha-Adrenergic agents. 2. Synthesis and alpha 1-agonist activity of 2-aminotetralins.

Substituted 2-aminotetralins are potent, selective, direct-acting agonists at postjunctional alpha 1 receptors. Within this series, substituent alterations on the ring, as well as on the nitrogen, change the potency of compounds by over three orders of magnitude (EC50 = 12 to greater than 10 000 nM). It has been demonstrated experimentally that substitution at both the 5 and 8 positions of the aromatic ring produces optimum agonist potency. Removal of either substituent results in a loss of potency and efficacy relative to norepinephrine. Substitution at positions 6 and/or 7 is generally detrimental to activity. Methyl, ethyl, or dimethyl substitution on nitrogen is compatible with high agonist potency, while substitution with larger groups is not. The most potent agonist in this series is 5-(thiomethyl)-8-methoxy-2-aminotetralin, which has an EC50 of 12 nM.

Conformationally constrained peptides and semipeptides derived from RGD as potent inhibitors of the platelet fibrinogen receptor and platelet aggregation.

Structure-activity studies have been pursued on cyclo-S,S-[Ac-Cys-(N alpha-Me)Arg-Gly-Asp-Pen]-NH2, 2 (SK&F 106760), a potent inhibitor of platelet aggregation, in an effort to improve potency and affinity for the GPIIb/IIIa receptor. Modifications on the N- and C-termini of 2 produced a series of peptides which indicate that the C-terminal carboxylate group may be a secondary receptor-binding element. Further modification by replacing the disulfide tether N alpha-acetylcysteine/penicillamineamide with the novel, inexpensive, achiral, constrained, and more lipophilic tether 2-mercaptobenzoyl/2-mercaptoaniline (Mba/Man) afforded the semipeptide cyclo-S,S-[Mba-(N alpha-Me)Arg-Gly-Asp-Man], 18 (SK&F 107260), which exhibited significant enhancement in both affinity and potency. To further investigate the effect of the phenyl ring at the C-terminus, peptides bearing the novel (2R,3S)- and (2R,3R)-beta-phenylcysteines were synthesized, which culminated in the cyclo-S,S-[Ac-Cys-(N alpha-Me)Arg-Gly-Asp-(2R,3S)-beta-phenylCys]-OH peptide, 22, which displayed substantial affinity and potency. We describe, herein, the development of both 18 and 22 and the additional structural modifications within the constrained cyclic disulfide ring to probe the stereochemical and steric requirements for receptor interaction.

Pyranenamines: a new series of antiallergic compounds.

Condensation of 3,5-diacylpyrantriones with various aromatic amines gave a new class of potent, orally active, antiallergic compounds, the 3-[(arylamino)ethylidene]-5-acylpyrantriones, hereafter referred to as pyranenamines, as evaluated not only in the traditional rat passive cutaneous anaphylaxis (PCA) assay but also in the in vitro fragmented rat and primate lung assay. Potencies in the PCA system, when measured intravenously, reached a maximum ID50 of 0.9 mu/kg (1000 times more potent than disodium chromoglycate) with 5-acetyl-4-hydroxy-3-[1-[(3,5-bis-glyceramoylphenyl)amino]ethylidene]-2H-pyran-2,6(3H)-dione (100), as predicted by structure-activity relationship (SAR) analysis. Potencies in the iv PCA system correlated well with potencies in the in vitro rat lung system but not with potencies in the oral PCA system or the in vitro primate lung system. Several compounds had good oral potency, and one analogue, 3-acetyl-4-hydroxy-3-[1-[3-amino-4-hydroxyphenyl)amino]ethylidene]-2H-pyran-2,6(3H)-dione hydrochloride (78), reached an oral ID50 of less than 1 mg/kg and was better than 10 times more effective than disodium chromoglycate at inhibiting the release of histamine and slow-reacting substance of anaphylaxis in the fragmented primate lung assay.

Development of an affinity ligand for purification of alpha 2-adrenoceptors from human platelet membranes.

Human platelets contain alpha 2-adrenoceptors which are negatively coupled to the enzyme adenylate cyclase. In order to better understand the interaction of this subtype of alpha receptor with this key enzyme, we have initiated a program to isolate and characterize the alpha 2-adrenoceptor. This report describes the synthesis and biological characterization of a series of molecules that were prepared as affinity ligands for this purpose. The best of these is 9-(allyloxy)-6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F 101253). This compound is an alpha 2-adrenoceptor antagonist, which was obtained by synthetic modification of 6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F 86466), a novel antagonist with high affinity for the alpha 2-receptor.

Pulmonary metastases in children and adolescents with well-differentiated thyroid cancer.

UNLABELLED: In this study, 27 patients less than 18 yr old with pulmonary metastases from well-differentiated thyroid carcinoma were evaluated to determine their response to (131)I therapy. METHODS: Of 121 children and adolescents treated with (131)I between 1963 and 1996, 27 patients had pulmonary metastases associated with nodal disease. Treatment response from (131)I was measured by three parameters: chest radiograph, scintigraphic images and serum thyroglobulin levels. Total activity of (131)I administered ranged from 4.6 GBq (125 mCi) to 38.7 GBq (1.05 Ci). Four patients were given one treatment, 8 were given two treatments, 4 were given three treatments and 11 were given more than three treatments. Radiation doses to the lungs were estimated in 14 patients using the MIRD methodology. The minimum duration of follow-up was 6 mo. RESULTS: At the time of initial presentation, diagnostic (131)I studies revealed bilateral radioiodine uptake in the lungs in 19 (70.4%) patients, whereas 12 (44.4%) patients had abnormal chest radiographs. One patient was lost to follow-up and was excluded from the study. Of the 26 patients studied, complete ablation of pulmonary metastases was observed in 8 (30.8%), partial ablation in 17 (65.4%) and there was no response to treatment in 1 (3.8%). Dosimetric parameters such as radioiodine uptake as a percentage of therapeutic activity, effective half-life and radiation dose delivered to the lungs were evaluated with each therapy. There was a progressive decline in each of these parameters with successive therapies. No correlation was observed between the radiation dose delivered and the response of pulmonary metastases to therapy. The number of therapies and amount of radioiodine administered had no influence on the ablation response. Of the 26 patients, 13 had a follow-up duration of less than 5 yr, 7 had 5-10 yr and 6 had more than 10 yr. One patient developed new metastases after 7 yr of diagnosis and treatment. One patient died of the disease after 4 yr. All surviving patients were asymptomatic and leading normal lives. CONCLUSION: Complete response of pulmonary metastases after (131)I therapy is difficult to achieve. A partial response with reduction of metastatic disease is possible and, in general, the patients had a good quality of life with no further disease progression and a low mortality rate.

Modifications in biphasic liquid-scintillation vial system for radiometry.

Several modifications of the biphasic liquid-scintillation vial system for radiometry have been tried in order to improve the counting efficiency. The biphasic system consisted of an inner sterile vial containing medium and substrate, and an outer liquid-scintillation vial lined on the inside with filter paper impregnated with scintillation fluors and alkali. The system gave an overall counting efficiency of 14.6%. Substitution of methanolic NaOH for impregnation of the paper raised the counting efficiency to 29.1%. This could be further enhanced to 33.8% by lining only half of the outer vial with filter paper, thereby allowing improved optical transmission of scintillation light. Increasing the amount of fluor did not change the efficiency significantly. A complete interchange in the system, whereby half of the inner vial was lined with filter paper and was otherwise empty, while the outer vial contained the medium and substrate, gave the highest efficiency (36.9%). This also allowed the use of larger amounts of medium and the inoculum.

Radiorespirometric testing of antibiotic sensitivity in urinary tract infections: concise communication.

A radiometric method, based on inhibition of 14CO2 release from bacterial metabolism of C-14-labeled glucose, was applied to test the susceptibility of urinary organisms to antibiotics. The testing was also carried out by the routine disc diffusion method after isolation of the organisms. Results of susceptibility to antibiotics could be obtained within 2 to 4 hr by the radiometric technique, compared with the 48 hr required for the disc method.

A multiplex-PCR for the differentiation of Mycobacterium bovis and Mycobacterium tuberculosis.

A multiplex-polymerase chain reaction (PCR) assay based on one-step amplification and detection of two different mycobacterial genomic fragments was designed for differentiation of Mycobacterium bovis and Mycobacterium tuberculosis. The oligonucleotide primers were chosen from a 500-bp genomic fragment which is well conserved in M. bovis and the pncA gene (based on M. tuberculosis-specific nucleotide polymorphism, a cytosine residue at position 169), specific for M. tuberculosis. The multiplex-PCR allowed detection of a single product of 500 bp in M. bovis isolates while M. tuberculosis isolates generated a single product of 185 bp, with or without an additional product of 500 bp. None of the atypical mycobacterial isolates revealed any amplification products. The method was found to be highly specific and could detect as little as 20 pg of pure DNA. This multiplex-PCR assay, based on the 500-bp fragment and the pncA gene, may be very useful for the rapid and specific differentiation of these two closely related mycobacteria and easy to use in medical and veterinary microbiological laboratories.

Malic enzyme activity in the developing rat brain in relation to thyroid status.

Malic enzyme activity in the soluble fraction of neonate brains from mothers fed with propylthiouracil (0.015% w/v) in drinking water from day 12 of the gestation period was significantly lowered (P less than 0.01) as compared to the offspring of normal mothers. Supplementation of triiodothyronine to the neonates from experimental mothers restored the malic enzyme activity to normal levels. However, administration of triiodothyronine to adult control rats did not influence malic enzyme activity in the brains of these animals. Our data suggest that during the initial critical period of brain maturation, malic enzyme is under the control of thyroid hormones. The response of malic enzyme towards thyroid hormones is lost once the brain has matured.

Metastasis to the liver in well-differentiated carcinoma of the thyroid.

Eleven patients (6 women; 5 men) of a series of 1,916 developed liver metastasis from differentiated thyroid cancer within 3 months to 202 months after the initial diagnosis. Liver metastasis generally appeared after the onset of metastases at other sites. The metastasis to the liver was clinically suspected in 8 patients whereas it was accidentally diagnosed on ultrasound examination of abdomen for a complaint of back pain in 1 patient, on a whole body diagnostic radioiodine scan in 1 case, and on radionuclide liver scan in 1 patient. Three patients had noniodide concentrating hepatic metastasis which were treated with chemotherapy but with poor response. The remaining 8 patients were treated with radioiodine. The survival rate was poor but could not be attributed to liver metastasis per se because of the extensive metastatic disease at other sites.

Significance of antithyroglobulin autoantibodies in differentiated thyroid carcinoma.

The incidence of antithyroglobulin autoantibodies (ATA) was 17.7% in 963 patients (who attended the clinic from 1981 to 1990) with differentiated thyroid carcinoma (DTC). Another 12 patients developed ATA for a transient period after the treatment with radioiodine. The prevalence of ATA in females (21.5%, 123/572) was significantly higher (p < 0.001) than that seen in males (12.0%, 47/391). Age-dependent occurrence of ATA was not seen for the various age decades. The ATA was more prevalent (p < 0.01) with the papillary type of tumor (118/564) as compared to the follicular variety (51/398). ATA did not influence the metastatic spread of the tumor at the initial presentation (105/170 for the ATA-positive group and 445/793 for the ATA-negative group). However, within the group with metastases, 82.9% (87/105) of patients had local spread into the neck in the presence of ATA, which was significantly higher (p < 0.01) than that seen for patients without ATA (63.8%, 284/445). For assessment of the influence of ATA on the outcome of the disease, the data from 222 patients (46 positive and 176 negative for ATA), with a minimum follow-up of 5 years (mean follow-up of 7.4 years), was considered suitable for analysis. The outcome of the disease was comparable in the presence and the absence of ATA (38/46 and 137/176 patients became disease-free in ATA-positive and -negative groups, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Malic enzyme response to triiodothyronine in the brain of neonatal rats.

Malic enzyme activity in the soluble fraction of the neonatal brain of hypothyroid rats was observed to be lowered as compared to that of the control animals (p less than 0.01). Administration of triiodothyronine to the neonates of control animals resulted in significant enhancement (p less than 0.001) in the activity of the Malic enzyme. Our studies show that brain malic enzyme which is involved in lipogenesis and hence in myelination responds to triiodothyronine in the early stage of life.

A simple radioassay for detection of antithyroglobulin autoantibodies.

Radioassay technique for detection of anti-thyroglobulin autoantibodies (ATA) in serum samples using protein-A rich Staphylococcus aureus as an immune complex separating agent was comparable (r = 0.99; n = 42; P < 0.001) to that evaluated with anti-human gammaglobulin (AHGG). The inter- and intra-assay coefficient of variations were lower (7.4 and 2.2% respectively) using Staph. aureus as compared to that observed with AHGG (10.4 and 4.7% respectively). A highly significant correlation was observed (r = 0.53; n = 71; P < 0.001) between per cent bound radiolabel thyroglobulin in radioassay and log reciprocal titre of ATA by haemagglutination method. Radioassay scored more number of positive sera as compared to haemagglutination method for normal controls (6 vs 1; n = 46) and patients of thyroid diseases (151 vs 70; n = 238). Due to the relatively poor stability of tracer on storage larger inter-assay coefficient of variations were observed with the radiolabelled preparations older than 10-12 days. The radioassay method evaluated is sensitive, reproducible and useful in detecting presence of anti-thyroglobulin autoantibodies.

An immunoradiometric assay for measurement of serum thyroglobulin.

A two-site immunoradiometric assay (IRMA) for estimation of serum thyroglobulin (tg) is optimized using partially purified rabbit anti-tg antibodies (ammonium sulphate precipitated) as a solid phase antibody and affinity purified 125I labeled anti-tg antibodies as a tracer. The IRMA correlated well (r = 0.93, n = 200, P < 0.001) with conventional radioimmunoassay (RIA). The inter- and intra-assay coefficient of variation were comparable for both methods. The IRMA method gave a superior sensitivity and working range (2 ng/ml, 3-1000 ng/ml) as compared to that of RIA method (6 ng/ml, 9-500 ng/ml). Using IRMA the total incubation time was curtailed to 4 h as compared to 90 h required for conventional RIA. The shelf-life of tracer antibody used in IRMA was much longer (3 months) as compared to tracer tg (3-4 wk) used in RIA. The optimized IRMA for quantitation of serum tg has superior assay characteristics as compared to RIA.

Filter paper blood spot test for detection of anti-ND-BSA antibodies in school children.

For identifying individuals at 'high risk' for developing leprosy, a simplified technique of collection of blood samples on filter paper for detection of anti -ND-BSA antibodies, was optimised. Anti-ND-BSA antibody reactivity on the filter paper was lost on storage at room temperature, but was stable at least for a period of 8 wk at lower temperature. Among the 1495 children screened, 166 (11.1%) were lepromin negative and 122 (8.2%) positive for anti-ND-BSA antibody. In the first phase, 7 of 871 children followed up for 2 yr developed leprosy, while in the second phase 2 of 624 children followed-up for 9 yr developed leprosy. The positivity and negativity of anti-ND-BSA antibodies and lepromin in these I children indicate that the positive status for anti-ND-BSA antibodies has a better predictive value than negative lepromin reactivity. Measurement of anti-ND-BSA antibodies was also of value for monitoring the efficacy of therapy and course of the disease. Extrapolation of the two tests to the total population of children evaluated showed that an individual with negative lepromin reactivity along with presence of anti-ND-BSA antibodies is at a higher risk for developing leprosy than those who have both the tests normal. Lepromin reactivity alone was not of much value for the prediction for development of the disease. However, the presence of anti-ND-BSA antibody was a better indicator for the development of the disease. The probability of developing leprosy in a child with any one of the tests abnormal was higher as compared to a child having all the tests normal.

Clinical utility of serum thyroglobulin in metastatic disease.

We examined the usefulness of serum thyroglobulin (Tg) levels in 196 patients with metastatic disease. Of these, 51 patients had a thyroidal primary (40 differentiated, 7 medullary and 4 undifferentiated), 35 patients had a nonthyroidal primary and in 110 patients the primary site was not known. Serum Tg was raised in 74.5 per cent (38 of 51) patients with carcinoma of the thyroid and in 92.5 per cent (37 of 40) patients with differentiated thyroid carcinoma (DTC). Twelve (34.3%) patients with proved nonthyroidal malignancies and 34 (30.9%) patients with unknown primary origin had elevated serum Tg levels. Of the 110 patients with unknown primary site thyroidal primary in 10 patients (all with elevated serum Tg levels) and nonthyroidal primary in 38 patients (7 had raised serum Tg levels) could be established. The sensitivity (for DTC) and the specificity (for nonthyroidal primary) of serum Tg estimation were 94.0 per cent (47 of 50) and 74.0 per cent (54 of 73) respectively and for patients with distant metastases were 100 (29 of 29) and 85.1 per cent (40 of 47) respectively. The positive and the negative predictive values of serum Tg were 71.2 (47 of 66) and 94.7 per cent (54 of 57) respectively and for patients with distant metastases were 80.6 (29 of 36) and 100 per cent (40 of 40) respectively. Our findings suggest that for patients presenting with metastases (particularly distant metastases) of an unknown primary site, serum Tg estimation is of great value to identify or rule out the involvement of the thyroid as the primary organ.

TSH receptors in human thyroid tumours.

To evaluate the binding of human TSH (h-TSH) to various human thyroid tumours using radio receptor assay technique, 26 thyroid tumour specimens were examined. Five specimens did not show displacement by stable h-TSH. A wide variation was observed in B0, non specific binding, affinity and capacity of TSH in all the tumours examined. The Scatchard analysis of the binding of h-TSH to thyroid membranes suggested the presence of the receptors in 57.7 per cent (15 of 26, Ka much greater than 10(9)) and more than one component in 46 per cent (12 of 26) of the tumours studied. There was no consistent pattern of the binding of TSH for thyroid tissue with respect to its pathology. However, with 35 pairs of observations log affinity appeared to be linearly related to log capacity with a slope -0.95, intercept 9.96 and r value -0.93.