Mutation of hMSH3 and hMSH6 mismatch repair genes in genetically unstable human colorectal and gastric carcinomas

Mutations within microsatellite sequences, consisting of additions or deletions of repeat units, are known as the replication/repair error positive (RER+) phenotype or micorsatellite instability (MI). Microsatellite instability has been demonstrated in hereditary and sporadic colorectal carcinomas and is usually observed in noncoding regions of genomic DNA. However, relatively few coding region targets of MI have been identified thus far. Using PCR, we amplified regions encompassing (A)8 and (C)8 microsatellite tracts within hMSH3 and hMSH6 from 31 RER+ sporadic colorectal tumors, 8 hereditary colon cancers, 23 RER+ gastric carcinomas, and 32 RER- gastric tumors. Mutations were found in 11 (36%) of 31 sporadic colon carcinomas, 4 (50%) of 8 hereditary colorectal cancers, and 5 (22%) of 23 RER+ gastric carcinomas, but in only 2 (6%) of 32 RER- gastric carcinomas. These frameshift mutations cause premature stop codons downstream that are predicted to abolish normal protein function. Our results and those of others suggest that DNA mismatch repair genes, such as hMSH3 and hMSH6, are targets for the mutagenic activity of upstream mismatch repair gene mutations and that this enhanced genomic instability may accelerate the accumulation of mutations in RER+ tumors.     

High affinity recognition of serotonin transporter antagonists defined by species-scanning mutagenesis. An aromatic residue in transmembrane domain I dictates species-selective recognition of citalopram and mazindol

Human and Drosophila melanogaster serotonin (5-HT) transporters (SERTs) exhibit similar 5-HT transport kinetics and can be distinguished pharmacologically by many, but not all, biogenic amine transporter antagonists. By using human and Drosophila SERT chimeras, major determinants of potencies of two transporter antagonists, mazindol and citalopram, were tracked to the amino-terminal domains encompassing transmembrane domains I and II. Species-scanning mutagenesis, whereby amino acid substitutions are made switching residues from one species to another, was employed on the eight amino acids that differ between human and Drosophila SERTs in this region, and antagonist potencies were reassessed in 5-HT uptake assays. A single mutation in transmembrane domain I of human SERT, Y95F, shifted both citalopram and mazindol to Drosophila SERT-like potencies. Strikingly, these potency changes were in opposite directions suggesting Tyr95 contributes both positive and negative determinants of antagonist potency. To gain insight into how the Y95F mutant might influence mazindol potency, we determined how structural variants of mazindol responded to the mutation. Our studies demonstrate the importance of the hydroxyl group on the heterocyclic nucleus of mazindol for maintaining species-selective recognition of mazindol and suggest that transmembrane domain I participates in the formation of antagonist-binding sites for amine transporters.     

Positional firing properties of perirhinal cortex neurons

Neuronal activity in the perirhinal cortex was recorded while rats performed a spatial task on a four arm radial maze. The maze was defined by proximal multisensory cues on the arm surfaces and distal complex visual cues at the surround. During each recording session, rats were run in three conditions: baseline, a condition in which proximal and distal cues were manipulated, and a second baseline. Compared with the activity of hippocampal neurons in the same paradigm, a much smaller proportion of perirhinal neurons exhibited spatial selectivity and perirhinal place fields were larger than hippocampal place fields. Although perirhinal place fields exhibited a high degree of spatial tuning and reliability within a condition, they were not stable across conditions.     

An objective approach to antivenom therapy and assessment of first-aid measures in snake bite

Treatment of systemic envenoming in snake-bite victims has, in the past, depended almost entirely on the individual clinician's experience in assessing the severity of envenoming. The efficacy of treatment is obviously related to the neutralizing potency of the antivenom used, the route by which it is administered and the dose. The development of enzyme immunoassays has permitted a more scientific appraisal, allowing estimation of circulating specific venom and antivenom concentrations at any time after the bite in the patient's blood. It is therefore possible to measure accurately the efficacy of antivenom in the neutralization and clearance of venom antigen. In Brazil, it appears that clinicians treat patients with excessive amounts of highly efficient antivenoms and this results in an unacceptably high incidence of reactions. In Sri Lanka, the use of imported, Indian antivenom is relatively ineffective in neutralizing the venoms of Sri Lankan snakes, demonstrating the real problem of venom variability within individual species. In West Africa, the improved clearance of venom following treatment of Echis victims with a monospecific as opposed to a polyspecific antivenom has been demonstrated, and new, smaller fragment, Fab antivenoms have been developed and are now under clinical assessment. Such clinically-based immunological studies should result in more efficient and controlled use of expensive antivenoms for treatment of systemic envenoming and the accurate assessment of newly designed products. Such studies also emphasise the importance of individual countries producing their own antivenoms for treatment of systemic envenoming. Likewise, the use of such objective systems now enables the use of first-aid measures such as tourniquets to be properly assessed.     

Electrolyte quintet: Calcium

Abnormalities in serum calcium concentration may have profound effects on neurological, gastrointestinal, and renal function. Maintenance of the normal serum calcium is a result of tightly regulated ion transport by the kidney, intestinal tract, and bone, mediated by calcaemic hormones especially parathyroid hormone and 1,25-dihydroxyvitamin D3. Abnormalities in calcium transport that result in uncompensated influx into, or efflux from, the extracellular fluid, will result in hypercalcaemia or hypocalcaemia, respectively. When possible the biologically important ionised calcium concentration should be measured. A variety of common disorders are responsible for abnormalities in the serum calcium. Treatment of both hypercalcaemia and hypocalcaemia is dependent on the underlying disorder, the magnitude of the deviation of the serum calcium, and the severity of symptoms. Fortunately, in the case of hypercalcaemia, there is a broad selection of effective medications, especially the bisphosphonates. Treatment of hypocalcaemia relies on the provision of calcium and often vitamin D. In this article we review the mechanisms responsible for abnormalities in calcium homoeostasis, the differential diagnosis of hypercalcaemia and hypocalcaemia, and appropriate therapy.     

Antimicrobial therapies for Helicobacter pylori infection in gnotobiotic piglets

Gnotobiotic piglets infected with Helicobacter pylori were treated with various antimicrobials as monotherapy and dual therapy, and the results were compared to those for piglets treated with a triple-therapy regimen (bismuth subsalicyclate at 5.7 mg/kg of body weight, metronidazole at 4.4 mg/kg, and amoxicillin at 6.8 mg/kg four times a day [QID]). Clearance of infection was assessed after 7 days of treatment, and eradication was assessed following 7 days of treatment and a 14-day posttreatment observation interval. Monotherapy with amoxicillin, clarithromycin, and ciprofloxacin cleared and eradicated the organism from porcine stomachs; monotherapy with metronidazole cleared the infection and eradicated it from some piglets. Metronidazole-resistant microbes were recovered from treated piglets which cleared but did not eradicate the infection. Monotherapy with bismuth subsalicylate, erythromycin, nitrofurantoin, and tetracycline in the dosage range of 5.0 to 7.1 mg/kg QID was less than 100% effective in clearance and eradication, in that these drugs cleared and/or eradicated the infection from some of the piglets but did not eradicate the infection from all of the piglets. Monotherapy with an H-2 receptor antagonist (ranitidine) or a proton pump inhibitor (omeprazole) was ineffective at either clearance or eradication. In vivo dose titrations with several effective monotherapies were performed to determine the lowest effective in vivo dose of drug. In piglets, eradication was associated with a statistically significant decline in serum H. pylori-specific immunoglobulin M (IgM) antibodies; the titers of both IgA and IgG also declined, but the values were not statistically significant. For many antimicrobials, piglets are more sensitive indicators of clearance and eradication than humans. These data establish the H. pylori-infected gnotobiotic piglet as a useful model for the identification of novel antimicrobials for the treatment of this disease and for drug assessment during preclinical evaluations.     

Age-related alteration in processing of temporal sound features in the auditory midbrain of the CBA mouse

The perception of complex sounds, such as speech and animal vocalizations, requires the central auditory system to analyze rapid, ongoing fluctuations in sound frequency and intensity. A decline in temporal acuity has been identified as one component of age-related hearing loss. The detection of short, silent gaps is thought to reflect an important fundamental dimension of temporal resolution. In this study we compared the neural response elicited by silent gaps imbedded in noise of single neurons in the inferior colliculus (IC) of young and old CBA mice. IC neurons were classified by their temporal discharge patterns. Phasic units, which accounted for the majority of response types encountered, tended to have the shortest minimal gap thresholds (MGTs), regardless of age. We report three age-related changes in neural processing of silent gaps. First, although the shortest MGTs (1-2 msec) were observed in phasic units from both young and old animals, the number of neurons exhibiting the shortest MGTs was much lower in old mice, regardless of the presentation level. Second, in the majority of phasic units, recovery of response to the stimulus after the silent gap was of a lower magnitude and much slower in units from old mice. Finally, the neuronal map representing response latency versus best frequency was found to be altered in the old IC. These results demonstrate a central auditory system correlate for age-related decline in temporal processing at the level of the auditory midbrain.