Detection of novel sequence heterogeneity and haplotypic diversity of HLA class II genes

Nucleic acid sequences of the second exons of HLA-DRB1, –DRB3/4/5, –DQB1, and –DQA1 genes were determined from 43 homozygous cell lines, representing each of the known class II haplotypes, and from 30 unrelated Caucasian subjects, comprising 60 haplotypes. This systematic sequence analysis was undertaken in order to a) determine the existence of sequence microheterogeneity among cell lines which type as identical by methods other than sequencing; b) determine whether direct sequencing of class II genes will identify the presence of more extensive sequence polymorphism at the population level than that identified with other typing methods; c) accurately determine the molecular composition of the known class II haplotypes; and d) study their evolutionary relatedness by maximum parsimony analysis. The identification of seven previously unidentified haplotypes carrying five new allelic amino acid sequences suggests that sequence microheterogeneity at the population level may be more frequent than previously thought. Maximum parsimony analysis of these haplotypes allowed their evolutionary classification and indicates that the higher mutation rate at DRB1 compared to DQB1 loci in most haplotypic groups is inversed in specific haplotype lineages. Furthermore, the extent and localization of gene conversions and point mutations at class II loci in the evolution of these haplotypes is significantly different at each locus. Identification of additional HLA class II molecular microheterogeneity suggests that direct sequence analysis of class II HLA genes can uncover new allelic sequences in the population and may represent a useful alternative to current typing methodologies to study the effects of sequence allelism in organ transplantation.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M35890 through M35953.     

Uterine electromyographic activity in horse mares as measured by radiotelemetry

The objective of this study was to determine if radiotelemetry could be used to measure myometrial electromyographic (EMG) activity. A radio transmitter with one pair of biopotential leads was implanted in the flank ipsilateral to the pregnant uterine horn at least five weeks prior to the expected date of parturition in two mares. The biopotential leads were implanted in the base of the pregnant uterine horn. Telemetered data were received by a pair of antennae placed at right angles in a 3.3 by 6.6-m stall. Data were recorded on VHS format videocassette tapes continuously for the 24h prior to and following parturition. Simultaneous physiograph recordings were made as a hard copy reference. In addition, 10 mg of prostaglandin F(2alpha) was administered to two mares in the luteal phase of the estrous cycle. Myometrial EMG during parturition was increased similarly to that of previously published reports that used myometrial electrodes wired directly to a physiograph. Prostaglandin F(2alpha) also caused an increase in myometrial EMG activity within 8 min of administration. This study demonstrated that radiotelemetry can be used for measuring myometrial EMG activity.     

A comparative assessment of purification techniques for mesophyll protoplasts of Brassica napus L.

The effects of three different general purification protocols have been assessed quantitatively using mesophyll protoplasts of Brassica napus. Within the initial sample two distinct sub-populations were determined. The methods used influenced the ratio of the vacuolated to chloroplastic type protoplast sub-populations. Overall recovery rates of the initial sample varied according to the method used from 38% to 27%, but the relative recovery of the sub-populations varied considerably with a purified ratio of between 1.0:0.78 to 1.0:7.0. Size distribution profiles of the initial and purified populations are also presented.     

A method for determining overall protein fold from NMR distance restraints

Summary We describe a simple method for determining the overall fold of a polypeptide chain from NOE-derived distance restraints. The method uses a reduced representation consisting of two particles per residue, and a force field containing pseudo-bond and pseudo-angle terms, an electrostatic term, but no van der Waals or hard shell repulsive terms. The method is fast and robust, requiring relatively few distance restraints to approximate the correct fold, and the correct mirror image is readily determined. The method is easily implemented using commercially available molecular modeling software.     

Structure of equine corticotropin releasing factor

A 41 amino acid peptide, probably identical in structure to human corticotropin releasing factor, was isolated from 70 equine hypothalami by methanol extraction, immunoaffinity chromatography and single step of reverse phase HPLC. The amino acid sequence was determined by gas phase sequence analysis. Probable carboxyl terminal amidation was demonstrated by similar retention times for equine and human corticotropin releasing factor on reverse phase HPLC at pH 8. The likely structure of equine corticotropin releasing factor is: Ser-Glu-Glu-Pro-Pro- Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn- Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂. The purified peptide is equipotent with human corticotropin releasing factor in an in vitro bioassay and in a human plasma binding protein assay.     

Calretinin and calbindin in the retina of the developing chick

Summary Calretinin and calbindin-D28k are two calcium-binding proteins that are present in largely different sets of nerve cells in the central nervous system. Their appearance during development of the chick retina was studied by immunohistochemistry and Western blots. The patterns are mature one day before hatching. Each cell type acquires its characteristic calcium-binding protein several days after its differentiation has started, but in most cases before morphological maturation is complete. There is also an early phase of calbindin immunoreactivity in many immature amacrine cells, and of calretinin immunoreactivity in the presumptive photoreceptor layer, suggesting that these proteins may have distinct functions in differentiating cells.Abbreviations CR+ Immunoreactive for calretinin only - CB+ immunoreactive for calbindin only - CR+CB+ immunoreactive for both antisera - IPL inner plexiform layer - OPL outer plexiform layer     

Statistical analysis of the migration component of genetic drift

Statistical methods are introduced for analysis of the migration component of genetic drift, i.e., of the stochastic changes that affect allele frequencies during migration between local groups. Attention focuses on alpha M, a parameter that measures the extent to which this component of drift departs from the ideal of independent random sampling, and which can be interpreted as a measure of the extent to which migration is kin-structured. It is shown that alpha M can be estimated from genetic data, even in the absence of information about the genealogical relationships of migrants, and Monte-Carlo simulations are used to approximate the sampling distribution of the estimator under the null hypothesis of independent random sampling. Application of these methods to data from the Aland Islands, Finland, shows that the migration pattern there is consistent with the hypothesis of independent random sampling.     

Factors influencing nitrate depletion in a rural stream

A mass balance procedure was used to analyze rates of nitrate depletion in three adjacent reaches of West Duffin Creek, Ontario, Canada. Daily nitrate losses in individual reaches were highly variable (0.5–24 kg N) during low and moderate stream flows in May–October, 1982–1985. Nitrate removal efficiency (nitrate loss as a % of nitrate input) showed a rapid exponential decline with increased nitrate inputs to each reach. Nitrate losses and nitrate removal efficiency also had a significant negative correlation with stream discharge. The association of large nitrate loads with high stream discharge reduced the nitrate removal capacity of the stream because of shorter residence times and a higher ratio of water volume to stream bed area. Water temperature exhibited a significant positive correlation with nitrate loss which may reflect increased denitrification at higher temperatures.Variations in nitrate losses and nitrate removal efficiency between the three reaches were highly influenced by differences in water residence time. Standarized nitrate losses with respect to water residence time revealed a longitudinal decline in nitrate depletion between the reaches which was associated with a downstream decrease in stream nitrate concentration and in the organic carbon content of fine textured sediments from pool habitats.     

Nitrate depletion in the riparian zone of a small woodland stream

Field enrichments with nitrate in two spring-fed drainage lines within the riparian zone of a small woodland stream near Toronto, Ontario showed an absence of nitrate depletion. Laboratory experiments with riparian substrates overlain with nitrate enriched solutions revealed a loss of only 5–8% of the nitrate during 48 h incubation at 12°C. However, 22–24% of the initial nitrate was depleted between 24 and 48 h when a second set of substrate cores was incubated at 20°C. Short-term (3 h) incubations of fresh substrates amended with acetylene were used to estimate in situ denitrification potentials which varied from 0.05–3.19 g N g^–1 d^–1 for organic and sandy sediments. Denitrification potentials were highly correlated with initial nitrate content of substrate samples implying that low nitrate levels in ground water and riparian substrates may be an important factor in controlling denitrification rates. The efficiency of nitrate removal in spring-fed drainage lines is also limited by short water residence times of < 1 h within the riparian zone. These data suggest that routes of ground water movement and substrate characteristics are important in determining nitrate depletion within stream riparian areas.