Seven New Mutations in hMSH2, an HNPCC Gene, Identified by Denaturing Gradient-Gel Electrophoresis

Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of hMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup.     

The interaction of gadolinium complexes with isolated rat hepatocytes

The lanthanide metal, gadolinium, is currently used in contrast agents for magnetic resonance imaging. We have performed a study of the interaction between isolated rat hepatocytes and ¹⁵³Gd complexed to diethylene-triamine pentaacetic acid (DTPA) or to DTPA-albumin conjugates. The study shows that isolated hepatocytes are able to take up both types of ¹⁵³Gd complexes. The ¹⁵³Gd-DTPA-albumin complexes are apparently taken up by pinocytosis, and possibly receptor-mediated endocytosis and/or adsorptive endocytosis, whereas the uptake mechanism of ¹⁵³Gd-DTPA is unknown. The ¹⁵³Gd-DTPA-albumin complexes, but not the ¹⁵³Gd-DTPA complex, are degraded by the cell. The degradation is inhibited by ammonium chloride. Gadolinium is slowly released back to the medium after loading of the cells with both complex types. In the experiments reported here no evidence of any adverse effects on the hepatocyte resulting from exposure to the ¹⁵³Gd-complexes were observed.     

Transferrin as a donor of iron to mitochondria Effect of pyrophosphate and relationship to mitochondrial metabolism and heme synthesis

Rat liver mitochondria accumulate iron mobilized from transferrin by pyrophosphate. The uptake has a very low energy dependence, but it is highly dependent on a functioning respiratory chain. Reduction of the ferric-iron-pyrophosphate complex is not linked to any specific respiratory complex. Half of the amount of iron accumulated is passed into heme. Iron once accumulated is very little accessible to chelation by added ferric or ferrous iron chelators. Iron uptake and heme synthesis are maximal if a suitable porphyrin substrate is added simultaneously with iron. The results represent further evidence that pyrophosphate is a possible candidate for intracellular iron transport. Also, the results suggest that iron uptake is coupled to simultaneous porphyrin uptake and heme synthesis.     

Substructure of inner mitochondrial membranes of myocardial cells as shown by cryo-ultramicrotomy

Summary The substructure of the inner mitochondrial membranes has been studied by cryo-ultramicrotomy under conditions during which denaturation of proteins by treatment with chemical solutes has been totally avoided. In such preparations, the inner membrane has a substructure consisting of globular subunits. These subunits have an average diameter of ca. 20Å–ca. 62Å and are fairly regularly spaced. Intracristal space is absent in the unstained, freeze-dried preparations, whereas a space of ca 40Å is seen in preparations lightly treated by OsO₄-vapour. It is concluded that the subunits of the inner mitochondrial membranes probably consist either of single protein molecules or of complexes of protein molecules.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice with single-sex schistosome infections

Primary Schistosoma mansoni single-sex infections in mice, i.e., either male only or female only, did not stimulate any detectable level of heterologous resistance to challenge with Fasciola hepatica after 22 to 76 days, while statistically significant resistance to a challenge with F. hepatica was demonstrated in the presence of patent mixed-sex S. mansoni infections. Simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction in the number of schisto-some worms established, i.e., the burden being reduced by 40.1 and 43.9%, respectively. There was no reduction of the F. hepatica worm burden. Similar features could be observed with a time interval of 48 hr between the S. mansoni infection and the F. hepatica challenge, i.e., the schistosome burden being reduced by 34.2 and 45.6%, respectively. Furthermore, simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction of the egg production capacity per paired female schistosome worm as compared with that of the S. mansoni control group. Tissue egg counts of the various intestinal sections were reduced by 92.8–99.6%.     

Purification and reaction mechanism of the primary inhibitor of plasmin from human plasma

The primary inhibitor of plasmin in human plasma was purified by a four-step procedure involving fractional (NH(4))(2)SO(4) precipitation, ion-exchange chromatography on a column of DEAE-Sepharose CL-6B and affinity chromatography on both a plasminogen-CH-Sepharose 4B column and a column of 6-aminohexanoic acid covalently coupled through the carboxylate function to AH-Sepharose 4B. No impurities in the final preparation could be detected when tested by immunoelectrophoresis against a range of specific antisera or against rabbit anti-human serum. On polyacrylamide-gel electrophoresis the inhibitor preparation showed a single band. The dissociation constant for the inhibitor-plasminogen complex was determined to be approx. 3mum at pH7.8. The reactions of the inhibitor with human plasmin and with bovine trypsin were studied. Comparison of the results obtained confirms the hypothesis previously presented, namely that the reaction of the inhibitor with plasmin involves at least two steps, the initial rapid formation of an enzyme-inhibitor complex followed by a slow irreversible transition to another complex. The results also indicate that the reaction of the inhibitor with trypsin involves just a single, irreversible step, so that this reaction seems to be less complicated than that of the inhibitor with plasmin. The ways in which 6-aminohexanoic acid influences the reactions were studied. The same value for the dissociation constant (approx. 26mum) for 6-aminohexanoic acid is obtained for both its effect on the reaction of the inhibitor with trypsin and for competitive inhibition of trypsin. The inhibitory effect of 6-aminohexanoic acid thus seems to be due to its blocking of the active site of trypsin. In contrast with this, the inhibitory effects of l-lysine and 6-aminohexanoic acid on the inhibitor-plasmin reaction occur at concentrations much too low to affect the active site of plasmin. The possible dependence of the reaction of the inhibitor with plasmin on a second site(s) on plasmin is discussed.     

Population structure,growth and fecundity of perch (Perca fluviatilis L.) in an acidified river system in Southern Norway

Population structure, density and reproductive potential of perch (Perca fluviatilis L.) from 4 acidified lakes in a river system in Southern Norway were investigated. The upper 3 lakes were most affected by acidification and the number of perch caught per unit effort indicate three-fold increase in density from the upper Gjerstadvann to the lower Brøbørvann. Low density of perch in the upper lakes is explained by; 1) Abnormal mortality occurring in episodes due to acidification, probably caused by aluminium toxicity at pH = 5.2–5.4. 2). Acid water (pH ≤ 5.0) during spawning and development of eggs and larvae, resulting in recruitment failure. Juvenile mortality of perch may depend on the density of the parental stock. In the 3 upper lakes, the density of adult perch probably was too low to produce significant juvenile mortality, and in these lakes the recruitment probably depended more on the May water quality. Improved water quality increases egg hatching and survival and also benefits zooplankton production, the main food for perch during the first summer. Low population density has reduced competition for food and thus improved growth of perch in the 3 upper lakes compared to the lower lake. The perch in Brøbørvann mature at higher age and have lower individual fecundity than perch in Gjerstadvann, an effect of both slower growth and lower length specific fecundity in Brøbørvann. In Gjerstadvann, the perch therefore have a higher reproductive potential relative to the stock density, and may, when the water quality is good enough, give rise to strong year-classes and more frequent year-class fluctuations than perch in the less acid Brøbørvann.     

Biometeorology in austria: past,present and future

Biometeorology in Austria has been shaped by concepts, personalities, and technology. In early times, the branches of biometeorology that are usual today were already evident: agricultural and forest meteorology, phenology, medical biometeorology and balneology, aerial biometeorology, urban housing and stabling meteorology all started to emerge several centuries ago. From the 1920 up to 1936, Wilhelm Schmidt at the Agricultural University of Austria laid the foundations of modern biometeorology. He was followed by Franz Sauberer, who headed a Department of Biometeorology at the National Weather Service and devoted his active life totally to biometeorology. Several years after his untimely death, the Department was dissolved. Not until 1981 was biometeorology taken up again at the Agricultural University, where the tradition of Schmidt and Sauberer now lives on in several courses within the area of applied biometeorology: Micro-and Topoclimatology, Agricultural and Forest Meteorology and Atmospheric Radiation. Biometeorology, being an experimental science, has also been influenced by new technological developments. The early period was exclusively observational. During the late nineteenth and early twentieth centuries mechanical and simple electric instruments were used with strip-chart recorders. These time consuming methods have now been replaced by electronic devices, including data loggers and portable computers along with many new electronic sensors, which provide additional insight into biometeorological problems. Since computers also make it possible to solve some of the complicated equations of biometeorology, the future of this science seems to be bright, not only in Austria but throughout the world.     

Dissolved organic carbon in a humic lake: effects on bacterial production and respiration

Allochthonous matter was the main source of carbon for pelagic bacteria in a humic lake, accounting for almost 90% of the carbon required to support observed bacterial growth. The estimated contribution from zooplankton excretion was of the same magnitude as direct phytoplankton release, both accounting for 5–7% of bacterial demands for dissolved carbon. Bacteria were an important source of carbon both for heterotrophic phytoplankton and for filter feeding zooplankton species, further stressing the role of humus DOC in overall lake productivity. The high contribution of allochthonous DOC implies a stoichiometry of dissolved nutrients with a surplus of C relative to P. The high P cell quota of bacteria suggest that under such conditions they are P-limited and act like net consumers of P. Excess C will be disposed of, and bacterial respiration rate will increase following a transition from carbon-limited bacterial growth towards mineral-nutrient-limited growth. Thus the high community respiration and frequent CO₂-supersaturation in humic lakes may be caused not only by the absolute supply of organic C, but also by the stoichiometry of the dissolved nutrient pool.