Hypoglossal motoneurons in newborn mice receive respiratory drive from both sides of the medulla.

Respiratory motor output in bilateral cranial nerves is synchronized, but the underlying synchronizing mechanisms are not clear. We used an in vitro slice preparation from newborn mice to investigate the effect of systematic transsections on respiratory activity in bilateral XII nerves. Complete transsection at the midline resulted in desynchronized rhythm with reduced XII burst amplitude and duration. Transsections in the ventral or dorsal 1/3 of the midline did not desynchronize rhythm. However, transsections in the ventral 2/3 of the midline desynchronized rhythm with characteristic amplitude correlations, where large-amplitude XII-bursts on one side was synchronized with small-amplitude XII-burst on the contralateral side. These characteristic amplitude correlations suggest that hypoglossal motoneurons receive respiratory drive from bilateral sources. Retrograde labeling confirmed that commissural fibers from the pre-Bötzinger complex cross in the mid-1/3 of the midline, and that dendrites of hypoglossal motoneurons project into the contralateral XII nucleus. In conclusion, commissural fibers crossing in the mid-1/3 of the midline are required for synchronization of respiratory activity in bilateral XII nerves. Hypoglossal motoneurons receive respiratory drive from both sides of the medulla, possibly mediated by contralaterally projecting dendrites.

Magnitude of indoor NO2 from biomass fuels in rural settings of Ethiopia.

UNLABELLED: Half of the world's population and about 80% of households in Sub-Saharan Africa depend on biomass fuels. Indoor air pollution due to biomass fuel combustion may constitute a major public health threat affecting children and women. The purpose of this study was to measure levels of indoor NO(2) concentration in homes with under-five children in rural Ethiopia. The study was undertaken in the Butajira area in Ethiopia from March 2000 to April 2002. 24-h samples were taken regularly at about three month intervals in approximately 3300 homes. Indoor air sampling was done using a modified Willems badge. For each sample taken, an interview with the mother of the child was performed. A Saltzman colorimetric method using a spectrometer calibrated at 540 nm was employed to analyze the mass of NO(2) in field samples. Wood, crop residues and animal dung were the main household fuels. The mean (s.d.) 24-h concentration of NO(2) was 97 microg/m(3) (91.4). This is more than double the currently proposed annual mean of WHO air quality guideline. Highland households had significantly higher indoor NO(2) concentration. This study demonstrates high levels of indoor NO(2) in rural homes of Ethiopia. PRACTICAL IMPLICATIONS: Respiratory infection is a major cause of morbidity and mortality, globally. Acute respiratory symptoms are also related to high levels of air pollution. Interventions aimed at reducing exposure to indoor air pollution should focus on cooking and heating practices in developing countries. This study is not undermining the role of other biomass smoke constituents in determining respiratory infections.

Microsatellite instability as a predictor of a mutation in a DNA mismatch repair gene in familial colorectal cancer.

Germline alterations in human DNA mismatch repair genes are associated with hereditary nonpolyposis colorectal cancer (HNPCC). Mutation analysis of the genes reveals carriers with a high risk of colorectal cancer, who will benefit from surveillance. We wanted to find the best predictive parameter of a germline mutation in those genes among patients with familial colorectal cancer. Affected members from a total of 83 unrelated colorectal cancer families previously analyzed for mutations in MSH2 and MLH1 were used to evaluate different parameters' ability to predict a germline mutation. We studied various clinical criteria such as family structure, age of onset, and prevalence of endometrial cancer, as well as microsatellite instability in the tumors from the families. In total, 124 tumors from 59 of the families were tested for microsatellite instability (MSI) using PCR-based mono- and dinucleotide markers to establish whether the families could be scored as MSI-positive or -negative. The finding of MSI-positive tumors in a family was the best predictor of a germline mutation, and was found in 73% of the MSI-positive, but in less than 3% of the MSI-negative families (P < 0.0001). In contrast, MSI in unselected colorectal cancer is not as useful, since most of these MSI-positive tumors are sporadic. The finding of microsatellite instability in colorectal tumors seems efficient enough even to select those with germline mutations among families fulfilling HNPCC Amsterdam criteria, once used in identification of the DNA mismatch repair genes. Genes Chromosomes Cancer 27:17-25, 2000.

Germ-line msh6 mutations in colorectal cancer families.

Hereditary nonpolyposis colorectal carcinoma (HNPCC) is due primarily to inherited mutations in two mismatch repair genes, MSH2 and MLH1, whereas germ-line mutations in other mismatch repair genes are rare. We examined the frequency of germ-line msh6 mutations in a population-based series of 140 colorectal cancer patients, including 45 sporadic cases, 91 familial non-HNPCC cases, and 4 HNPCC cases. Among the 91 population-based familial non-HNPCC cases, germ-line msh6 mutations were found in 6 patients (7.1% of probands analyzed; median age at diagnosis, 61 years). These mutations included a splice site mutation, a frameshift mutation, two missense mutations that were demonstrated to be loss of function mutations, and two missense mutations for which functional studies were not possible. In contrast, germ-line msh6 mutations were not found in any of the 45 sporadic cases and the 4 HNPCC cases in the population-based series or in the second series of 58 clinic-based, primarily HNPCC families. Our data suggest that germ-line msh6 mutations predispose individuals to primarily late-onset, familial colorectal carcinomas that do not fulfill classic criteria for HNPCC.

Various mutation screening techniques in the DNA mismatch repair genes hMSH2 and hMLH1.

Germline alterations in one of five human DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, hPMS2, and hMSH6) cause hereditary nonpolyposis colorectal cancer. Mutation analyses of these genes reveal gene carriers with a high risk for colorectal cancer, who benefit from surveillance to prevent disease. Equally important, presymptomatic testing allows nondisposed individuals to discontinue surveillance. We tested different mutation screening methods to optimize mutation detection in hMSH2 and hMLH1. Affected members from a total of 142 unrelated colorectal cancer families were analyzed. Denaturant gradient gel electrophoresis (DGGE), RT-PCR, and the protein truncation test (PTT) were used to screen for mutations on a DNA or RNA basis, respectively. In addition, a mutation-specific test on genomic DNA was used to find the Finnish mutation no. 1, a deletion of hMLH1 exon 16. DGGE identified most of the mutations in the mismatch repair genes hMLH1 and hMSH2. The RNA-based techniques were used to identify large deletions; however, these were rare in our materials. We describe our compiled results and experience from all our mutation screening studies, as well as unpublished data from our last DGGE screening of 58 patients and RT-PCR and PTT screening of 73 patients.

Microsatellite instability and mismatch repair gene inactivation in sporadic pancreatic and colon tumours.

Genomic instability has been proposed as a new mechanism of carcinogenesis involved in hereditary non-polyposis colorectal cancer (HNPCC) and in a large number of sporadic cancers like pancreatic and colon tumours. Mutations in human mismatch repair genes have been found in HNPCC patients, but their involvement in sporadic cancer has not been clarified yet. In this study we screened 21 pancreatic and 23 colorectal sporadic cancers for microsatellite instability by ten and six different microsatellite markers respectively. Microsatellite alterations were observed at one or more loci in 66.6% (14/21) of pancreatic cancers and in 26% (6/23) colon tumours, but all the pancreatic and half of the colon samples showed a low rate of microsatellite instability. All the unstable samples were further analysed for mutations in the hMLH1 and hMSH2 genes and for hypermethylation of the hMLH1 promoter region. Alterations in the hMLH1 gene were found only in colorectal tumours with a large presence of microsatellite instability. None of the pancreatic tumours showed any alteration in the two genes analysed. Our results demonstrate that microsatellite instability is unlikely to play a role in the tumorigenesis of sporadic pancreatic cancers and confirm the presence of mismatch repair gene alterations only in sporadic colon tumours with a highly unstable phenotype.

Mutation analyses of KRAS exon 1 comparing three different techniques: temporal temperature gradient electrophoresis, constant denaturant capillary electrophoresis and allele specific polymerase chain reaction.

Mutations in the KRAS gene is a key event in the carcinogenesis of many human cancers and may serve as a diagnostic marker and a target for therapeutic intervention. In this study we have applied three different techniques for mutation detection of KRAS exon 1 mutations: Allele specific polymerase chain reaction (AS-PCR), temporal temperature gradient electrophoresis (TTGE) and constant denaturant capillary electrophoresis (CDCE). Samples from 191 sporadic colon carcinomas were analyzed. AS-PCR were performed with oligonucleotides specific for know mutations in codon 12 and 13 of the KRAS gene. In TTGE analyses, linear ramping of the temperature were performed during electrophoresis in a constant denaturant gel. CDCE analyses were performed using fluorescin labeled PCR-products. Separation was achieved under constant denaturing conditions using high temperature in a gel-filled capillary followed by laser detection. A mutated KRAS gene was found in 42/191 (22.0%) of the samples using AS-PCR, in 62/191 (32.5%) using TTGE and in 66/191 (34.6%) of the samples using CDCE. In the TTGE and CDCE analyses the sequence of the mutant were determined by comparing the electrophoretic pattern to that of known mutations or by mixing the sample with known mutations prior to reanalysis. In a titration experiment mixing mutant and wild-type alleles prior to PCR, the sensitivity for mutation detection was shown to be 10(-2) for TTGE and under optimized conditions 10(-3) for CDCE.

Ki-ras mutations and prognosis in colorectal cancer.

A total of 191 colorectal adenocarcinomas, obtained from consecutive patients with a median follow-up of 6 years, were studied in order to evaluate the possible association of Ki-ras mutations with tumour stage, tumour differentiation and survival time. Resected full-cross tumour samples were screened for Ki-ras mutations in codons 12 and 13 using temporal temperature gradient gel electrophoresis (TTGE). Ki-ras mutations were detected in 62 (32%) of the samples. The most frequent mutation, observed in 21 samples, was from GGT to GAT changing glycine to aspartic acid in codon 12. The study did not show any association between Ki-ras mutations and Dukes' stage or tumour differentiation. Patients with Ki-ras mutations had a marginally shorter survival time (median 50 months) compared with patients without (median 59 months), but the difference was not statistically significant. The results indicate that Ki-ras gene mutations have no relevant prognostic importance in this cohort of colorectal cancer patients.

DGGE screening of mutations in mismatch repair genes (hMSH2 and hMLH1) in 34 Swedish families with colorectal cancer.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited syndrome which confers an increased risk for colorectal cancer and endometrial cancer as well as other tumors. It is caused by germline DNA mismatch repair (MMR) gene mutations in five MMR genes, hMSH2, hMLH1, hPMS1, hPMS2 and hMSH6. Finding mutations in these high risk families means that you can offer presymptomatic carrier diagnosis and thereby identify individuals with a very high risk for cancer. These persons benefit from counseling and should be offered surveillance. We have used DGGE to screen members from 34 families for mutations in hMLH1 and hMSH2. Six mutations in five families were found, five of these mutations are new. Besides, three new polymorphisms were identified. The mutations were found in two of seven Amsterdam criteria HNPCC families and in three of four families with at least one case of early onset of CRC (before 35), suggesting there are appropriate families to be chosen for mutation screening in MMR genes.

Measurements of indoor and outdoor nitrogen dioxide concentrations using a diffusive sampler.

The Willems badge, a short-term diffusion sampler, was used to measure nitrogen dioxide concentrations inside and outside the homes of participants in the European study "PEACE' (Pollution Effects on Asthmatic Children in Europe). The main aim of the study was to determine levels of nitrogen dioxide concentrations both outside and inside children's homes, and to estimate the indoor/outdoor ratios for nitrogen dioxide in an urban area, in comparison with a less urbanized control area. We conducted measurements in 23 homes in Umeå, a city of about 100,000 inhabitants in the northern part of Sweden, in addition to 20 homes in a less urbanized control area situated about 20 km from Umeå. Measurements were made on two different occasions in each home during the period January-March, 1994. The houses were not equipped with any gas appliances. The mean outdoor 24-h concentration in Umeå was 28 micrograms m-3 and the mean indoor concentration was 11 micrograms m-3. The mean indoor: outdoor ratio was 0.44 (s = 0.23). The highest outdoor value, measured in the city centre of Umeå, was 54 micrograms m-3. In the control area the mean ambient 24-h concentration was 12 micrograms m-3, approximately half as high as in the urban area, and the mean indoor concentration was 6 micrograms m-3. The mean indoor: outdoor ratio was 0.67 (s = 0.55). The correlation coefficient between indoor and outdoor concentrations was higher in the control area, r = 0.79 (p < 0.001), in comparison with the urban area, r = 0.43 (p < 0.01). It is concluded that the outdoor as well as the indoor concentrations of nitrogen dioxide were approximately twice as high in Umeå as in the control area. This could be explained by heavier traffic density in Umeå. The mean 24-h concentration outside homes in Umeå was, however, below the 24-h national standard level of 75 micrograms m-3. The higher correlation between indoor and outdoor concentrations, combined with higher indoor: outdoor ratio, in the control area is interpreted as a sign of a lower level of penetration of outdoor air into the houses in the urban area. This was not explained by differences in types of buildings between the two areas, but possibly by differences in air-exchange rates and in habits of ventilating rooms with open windows.