Lower azure B methylene blue ratios in Giemsa type blood and malaria stains

Starting from ancient reports that rare samples of methylene blue were apparently sufficiently contaminated with azures to give red plasmodial and red purple nuclear chromatin in Chenzinsky type methylene blue eosin stains, it was decided to determine how little azure B would suffice for such staining in methylene blue eosin stains. The traditional 1902 Giemsa had an azure : methylene blue : eosin ratio of about 6 : 3 : 6.3 : 10; Lillie's 1943 formula had a 5 : 7 : 10 ratio. In the current series of tests 5 : 7 : 10 (I), 4 : 8 : 10 (II), 3 : 9 : 10 (III), 2 : 10 : 10 (IV), 1 : 11 : 10 (V), and 0 : 12 : 10 (VI) were used. Malaria and blood stains were better than the standard 5 : 7 : 10 (I) in III, IV and II in that order. Normal and leukemic human blood, mouse blood with Plasmodium berghei, and monkey blood with the CDC strain of Pl. falciparum were used as test materials. The staining mixtures were made from highly purified samples of azure B and methylene blue. Staining mixtures contained 12 ml 0.1% thiazin dye, 10 ml 0.1% eosin, 2 ml each of glycerol, methanol and 0.1 M phosphate buffer pH 6.5, 3 ml acetone as accelerator, and distilled water to make 40 ml; staining times of 10--30 min were used.     

Mortality in over 350,000 Insured Swedish Dogs from 1995–2000: II. Breed-Specific Age and Survival Patterns and Relative Risk for Causes of Death

This study continues analysis from a companion paper on over 350,000 insured Swedish dogs up to 10 years of age contributing to more than one million dog-years at risk during 1995–2000. The age patterns for total and diagnostic mortality and for general causes of death (trauma, tumour, locomotor, heart and neurological) are presented for numerous breeds. Survival estimates at five, eight and 10 years of age are calculated. Survival to 10 years of age was 75% or more in Labrador and golden retrievers, miniature and toy poodles and miniature dachshunds and lowest in Irish wolfhounds (91% dead by 10 years). Multivariable analysis was used to estimate the relative risk for general and more specific causes of death between breeds accounting for gender and age effects, including two-way interactions. Older females had tumour as a designated cause of death more often than males in most breeds, but not in the Bernese mountain dog. Information presented in this and the companion paper inform our understanding of the population level burden of disease, and support decision-making at the population and individual level about health promotion efforts and treatment and prognosis of disease events.     

The influence of formaldehyde fixation time on the rate of nitrous acid deamination of erythrocytes and spinal cord proteins

Summary Alcohol fixed blood films and fresh blocks of spinal cord were immersed in phosphate buffered neutral 10% formol for graded intervals, the films for 10, 30 min, 1, 2, 4, 8, 24 hr; the blocks for 2, 4, 6, 24 hr at 3 and 24° C; 1, 3, 7, 14, 21, 28, 42, 56 da, 3 and 14 mo at 24–26°. Graded deaminations in 2 N NaNO₂/HAc at 3° C were applied: 1, 2, 5, 10, 20, 30 min; 1, 2, 4, 6, 8, 12, 18, 24, 36 hr. Blood films were stained at pH 6 and 6.5, tissue at pH 4.5 and 5.0, both in azure A eosin B. The point at which erythrocytes reached a slightly bluish green was taken as the end point, since no further color change occurred on further exposure and erythrocytes were the last of usually deamination susceptible tissue elements to lose their oxyphilia on deamination. Deamination of alcohol fixed blood films is completed in about 2 min, of sublimate fixed spinal cord in about 1 hr. Progressive formaldehyde exposure increased deamination time of blood films to 10–20 min in 1 hr, to 6–8 hr in 4 hr and to 12 hr in 24 hr. The tissue deamination showed similar progressive increase of deamination time, slower with 3° C fixation than with 24–26°, reaching 18–36 hr by about 3 days formol, and remaining about the same thereafter.Supported by National Cancer Institute Grant No. C-04816, National Institutes of Health.     

Sequence variation at the major histocompatibility complex locus DQ beta in beluga whales (Delphinapterus leucas) [published erratum appears in Mol Biol Evol 1995 Nov;12(6):1174]

Genetic variation at the Major Histocompatibility Complex locus DQ beta was analyzed in 233 beluga whales (Delphinapterus leucas) from seven populations: St. Lawrence Estuary, eastern Beaufort Sea, eastern Chukchi Sea, western Hudson Bay, eastern Hudson Bay, southeastern Baffin Island, and High Arctic and in 12 narwhals (Monodon monoceros) sympatric with the High Arctic beluga population. Variation was assessed by amplification of the exon coding for the peptide binding region via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Five alleles were found across the beluga populations and one in the narwhal. Pairwise comparisons of these alleles showed a 5:1 ratio of nonsynonymous to synonymous substitutions per site leading to eight amino acid differences, five of which were nonconservative substitutions, centered around positions previously shown to be important for peptide binding. Although the amount of allelic variation is low when compared with terrestrial mammals, the nature of the substitutions in the peptide binding sites indicates an important role for the DQ beta locus in the cellular immune response of beluga whales. Comparisons of allele frequencies among populations show the High Arctic population to be different (P < or = .005) from the other beluga populations surveyed. In these other populations an allele, Dele-DQ beta*0101-2, was found in 98% of the animals, while in the High Arctic it was found in only 52% of the animals. Two other alleles were found at high frequencies in the High Arctic population, one being very similar to the single allele found in narwhal.      

Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects

We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.      

Histochemical Identification of Basic Proteins with Biebrich Scarlet at Alkaline pH

Staining at graded alkaline pH levels with the sulfonated dye, Biebrich scarlet, shows basic proteins in various histologic sites, and differentiates the sites according to their relative basicity. Certain structures stain at pH 6.0 but not at 8.0 or above. Others stain maximally up to pH 93 and a few stain strongly as high as pH 103. The most strongly basic sites resist more than the others the destruction of acidophilia by nitrosation, acetylation or exposure to formaldehyde.