Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5′-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4–6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.     

Screening for point mutations in exon 10 of the low density lipoprotein receptor gene by analysis of single-strand conformation polymorphisms: detection of a nonsense mutation — FH469→Stop

DNA from 40 unrelated familial hypercholesterolemia (FH) heterozygotes were subjected to analyses of single-strand conformation polymorphisms (SSCPs) of exon 10 of the low density lipoprotein receptor (LDLR) gene. Four different SSCP patterns were observed. The underlying mutations were characterized by DNA sequencing. Three of the patterns represented the three genotypes of a recently described sense mutation in codon 450. A method based upon the polymerase chain reaction (PCR) was developed to analyze this mutation. The frequencies of the wild-type (G at nucleotide 1413) and mutant (A at nucleotide 1413) alleles were 0.56 and 0.44, respectively. The fourth pattern was found in only one FH heterozygote and was caused by heterozygosity at nucleotide 1469 (G/A). Nucleotide 1469 is the second base of codon 469_Trp(TGG). The GA mutation changes this codon into the amber stop codon, and is referred to as FH_469Stop. The mutant receptor consists of the amino terminal 468 amino acids. Because the truncated receptor has lost the membrane-spanning domain, it will not be anchored in the cell membrane. FH_469Stop destroys an AvaII restriction site, and this characteristic was used to develop a PCR method to establish its frequency in Norwegian FH subjects. Two out of 204 (1%) unrelated FH heterozygotes possessed the mutation.     

Retrospective molecular detection of transhyretin Met 111 mutation in a Danish kindred with familial amyloid cardiomyopathy,using DNA from formalin-fixed and paraffin-embedded tissues

Severe familial amyloid cardiomyopathy (FAC) in a Danish kindred is associated with a specific mutation (Met for Leu 111) in the transthyretin (TTR) gene. The mutation causes the loss of a DdeI restriction site in the gene, allowing molecular diagnostic studies. We studied formalin-fixed, paraffin-embedded tissues, up to 39 years old, from 29 family members of this kindred. DNA was partially purified from deparaffinized tissue sections and a DNA sequence of the TTR gene flanking the mutation site was amplified by the polymerase chain reaction (PCR), followed by restriction enzyme analysis. Amplified DNA was obtained from tissues representing 23 of the 29 persons. Ten out of the 23 family members were found to carry the TTR Met 111 mutation, whereas 13 were not affected. The results were consistent with known clinical data and with corresponding serum TTR examinations. This retrospective study shows that archival tissues can be used to confirm the diagnosis and disease pattern in members of families affected by hereditary diseases.     

Bayesian estimation of two-part joint models for a longitudinal semicontinuous biomarker and a terminal event with INLA: Interests for cancer clinical trial evaluation

Two-part joint models for a longitudinal semicontinuous biomarker and a terminal event have been recently introduced based on frequentist estimation. The biomarker distribution is decomposed into a probability of positive value and the expected value among positive values. Shared random effects can represent the association structure between the biomarker and the terminal event. The computational burden increases compared to standard joint models with a single regression model for the biomarker. In this context, the frequentist estimation implemented in the R package frailtypack can be challenging for complex models (i.e., a large number of parameters and dimension of the random effects). As an alternative, we propose a Bayesian estimation of two-part joint models based on the Integrated Nested Laplace Approximation (INLA) algorithm to alleviate the computational burden and fit more complex models. Our simulation studies confirm that INLA provides accurate approximation of posterior estimates and to reduced computation time and variability of estimates compared to frailtypack in the situations considered. We contrast the Bayesian and frequentist approaches in the analysis of two randomized cancer clinical trials (GERCOR and PRIME studies), where INLA has a reduced variability for the association between the biomarker and the risk of event. Moreover, the Bayesian approach was able to characterize subgroups of patients associated with different responses to treatment in the PRIME study. Our study suggests that the Bayesian approach using the INLA algorithm enables to fit complex joint models that might be of interest in a wide range of clinical applications.     

Energy Metabolism During Late Gestation and Lactation in Muciparous Sows in Relation to Backfat Thickness and the Interval from Weaning to First Oestrus

Ten crossbred, fourth or fifth parity sows were divided into 2 groups - high (H) and low (L) - according to their backfat thickness 9 days before parturition. Body weight, backfat thickness and litter weight were recorded repeatedly during a 5 week lactation period. The length of the interval from weaning to first oestrus was also noted. All sows were fed a commercial diet (11.9 MJ/kg, 14.5% crude protein). During gestation, daily food intake was 2.2 kg/sow, while during lactation it was 3.0 kg/sow plus 0.4 kg/piglet. Blood samples were drawn on day 9 before parturition and on days 2,7,14 and 21 of lactation. The samples were analysed to determine concentrations of glucose, urea nitrogen, creatinine, triglycerides, free fatty acids and beta-hydroxybutyric acid. In both groups, concentrations of free fatty acids and urea nitrogen were low on day 9 before parturition while those of triglycerides were high, indicating anabolism regardless of backfat thickness. During the first week of lactation, concentrations of free fatty acids increased in the H-group but not in the L-group, and concentrations of urea nitrogen were higher in the H-group. These differences, together with the greater loss of weight observed in the H-group, indicate that catabolism of maternal fat and protein depots was more pronounced in the Η-group than in the L-group during this time. On day 14 of lactation, both groups showed equally low concentrations of free fatty acids, decreasing creatinine concentrations and stable triglyceride and urea nitrogen concentrations. Furthermore, weight loss during the second and third weeks of lactation was low in both groups. These facts, taken together, indicate that the catabolic rate was decreasing in both groups during this period. No differences in return to oestrus interval were noted between the groups. The present study indicates that under a restricted feeding regime the catabolic rate during the first week of lactation is higher in sows with higher backfat thickness in late gestation. As lactation progresses, a more balanced metabolism is achieved regardless of backfat thickness, which may tend to reduce differences in return to oestrous interval.     

Variability of plant species diversity in Swedish natural forest and its relation to atmospheric deposition

Within a subprogram of Integrated Monitoring (IM), understorey vegetation in Swedish natural forests was observed at fifteen reference sites over the country for twelve seasons, 1982–1993. The main task of the subprogram was to assess the impact of atmospheric deposition, mainly sulphur and nitrogen, on natural vegetation through time. The present study is focused on the variability of plant species diversity at community level and the possible impact of sulphur and nitrogen deposition. Species richness, evenness and diversity varied greatly among the sites, and between years within each site. Regarding only coniferous forests the species richness was higher in the north than in the south. But the effects of site condition and atmospheric deposition were not clarified. Changes in species diversity through time differed from site to site. No overall temporal trend was found. The atmospheric deposition of sulphur and nitrogen demonstrated a clear geographical pattern being low in the north-west and high in the south-west. Sulphur deposition declined significantly in Southern Sweden during the period. We concluded that the species diversity of understorey vegetation at the Swedish IM sites was not significantly influenced by atmospheric deposition. The changes observed are explained as natural processes.     

Cataract Development in Atlantic Salmon (Salmo salar L) in Fresh Water

Irreversible bilateral cataracts were diagnosed by slit-lamp biomicroscopy in 178 of 200 farm-raised Atlantic salmon (Salmo salar L) fed a standard diet over a five-month period. Initial changes were anterior polar opacities, progressing to involve both the anterior and posterior cortex before changes in the lens nucleus were seen. The lens changes were recorded and given scores according to the severity of the cataracts. At each of 3 samplings, after 2, 4 and 5 months, 200 fish were measured, weighed and examined by slit-lamp biomicroscopy. At all 3 samplings, there was a significant correlation between body length and both cataract incidence and cataract severity. There was also a significant correlation between body weight and cataract incidence and severity for the 2 last samplings. There was a significant correlation between K-factor as a measure of the shape of the fish, and both cataract incidence and severity, at all 3 samplings. Evaluation of specific growth rate in the periods between the examinations showed that the rapidly-growing fish were most susceptible to cataract formation. After cataract developed, however, the growth rate slowed. Follow-up examination of severely affected fish 3 months after transfer to sea water showed a normal cortical zone in the periphery of the lens in 24 out of 28 fish.     

Geographical and latitudinal variation in growth patterns and adult body size of Swedish moose (Alces alces)

We examined the geographical pattern in growth and adult body size among 14 populations of Swedish moose (Alces alces) using data from 4,294 moose (1.5 years old) killed during the hunting season in 1989–1992. In both sexes, adult body mass was significantly positively correlated with latitude. Moose in northern populations had a 15–20% larger adult body mass than moose in the south. Juvenile body mass was correlated with neither latitude nor adult body mass. Thus, variation in time (years) and rate of body growth after the juvenile stage were responsible for most of the variation in adult body mass among populations. Moose in northern populations grew for approximately 2 more years of life than southern moose. In contrast to adult body mass, skeletal size (measured as jawbone length) was not correlated with latitude, suggesting that variation in adult body mass was primarily due to differences in fat reserves. Discrimination between population characteristics, such as moose density, climate, and the amount of browse available to moose, showed climatic harshness to be the most important variable explaining geographical variation in body mass among populations. The results support the notion that in mammals body size increases with latitude in accordance with Bergmann's rule. We conclude that (1) variation in patterns of growth after the juvenile stage is the main cause of the latitudinal trend in adult body size in moose, and (2) climatic conditions are a more important factor than population density and availability of food in explaining geographical variation in growth patterns and adult body mass between populations of Swedish moose.     

Urinary excretion of mercury, copper and zinc in subjects exposed to mercury vapour

The excretion of mercury, copper and zinc in urine, and mercury in whole blood andplasma, was determined in 40 chloralkali workers exposed to mercury vapour and 40age-matched referents. The Hg concentrations in whole blood, plasma and urine werehigher in the exposed group (35 nmol l, 30 nmol l,and 11.5 nmol mmol creatinine, respectively) in comparison with thereference group (15 nmol l, 6.3 nmol l, and 1.8nmol mmol creatinine, respectively). The urinary copper excretionwas similar in the two groups, while U-Zn excretion was significantly higher (P = 0.04)in the exposed group, median 0.83 mmol mmol creatinine versus 0.76mnmol mmol creatinine in the reference group. In a subgroup of exposedworkers with current U-Hg above 11.5 nmol lmmolcreatinine (20 mg g creatinine) the medianU-Zn was 1.1 mmol mmol creatinine. In both groups smokers had highU-Zn levels than non smokers. When both U-Hg and smoking were taken into account in alinear regression model, there was a significant association between U-Hg and U-Zn inthe combined group of exposed and referents (P = 0.002). This study indicates thatmercury exposure in humans, as in animals, causes increased urinary excretion of zinc.The mechanisms may be induced synthesis of metallothionein in the kidneys, displacementof Zn from preexisting metallothionein by Hg, or a decreased reabsorption of zinc in thekidneys owing to a slight tubular dysfunction.     

The ecology of ruffe,Gymnocephalus cernuus (Pisces: Percidae) introduced to Mildevatn,western Norway

Synopsis Diet, habitat use, diel and seasonal activity and a number of population parameters were studied on ruffe,Gymnocephalus cernuus, introduced to Mildevatn, western Norway. This lake is sited outside the natural range of the ruffe and has a lower fish diversity and a different fish species composition than within its native range. From June through September the ruffe was planktivorous and mainly caught at 4 to 6 m depth in the benthic zone. At other times of year ruffe was feeding on zoobenthos and caught deeper in the benthic zone. Ruffe was mainly day active. Zooplankton feeding during summer is the clearest difference compared to ruffe populations living within its natural range. Presence of large zooplankton organisms available for ruffe is suggested as the main reason for the difference found in food choice. The availability of large zooplankton is probably due to community structure caused by a predator and lack of interspecific competition for zooplankton in the deeper parts of the lake. Piscivorous brown trout.Salmo trutta, restrict the habitat of threespined stickleback,Gasterosteus aculeatus, to the zone of littoral vegetation, allowing high densities of larger zooplankton species likeBythotrephes longimanus to be present in the lake. Brown trout is present only in the upper light and well oxygenated parts of the lake, leaving a refuge for the ruffe, where they can feed on the rich zooplankton community.