The evolution of human chromosome 21: evidence from in situ hybridization in marsupials and a monotreme.

We have mapped five human chromosome 21 (HSA 21) markers in marsupials and a monotreme, two major groups of mammals that diverged from eutherians 130-150 and 150-170 million years before present (MYrBP), respectively. We have found that these genes map to two distinct autosomal sites, one containing SOD1/CBR/BCEI and the other containing ETS2/INFAR, in the marsupials Macropus eugenii and Sminthopsis macroura (which belong to orders that diverged 40-80 MYrBP), as well as in the monotreme Ornithorhynchus anatinus (the platypus). Since marsupials and monotremes diverged independently from eutherians, these data suggest that HSA 21 genes were originally located in two separate autosomal blocks. In another Sminthopsis species, SOD1 is linked to TRF (a marker on HSA 3q), suggesting that the ancestral SOD1/CBR/BCEI region also included HSA 3 markers. We suggest that these blocks became fused early in the eutherian evolution to form a HSA 3/21 chromosome, which has remained intact in artiodactyls, but has been independently disrupted in both the primate and rodent lineages.     

Electron microscopy of the fate of exogenous ferritin in the feline visual cortex

Summary The ultrastructural changes occurring in the feline visual cortex 3 hours after the injection of 0.02 mls of ferritin in 1% trypan blue in artificial cerebrospinal fluid have been studied.Near the site of injection, disrupted cells contained free and membrane-bound ferritin. In less damaged areas, some signs of oedema were present in the cells, especially in astrocytes. Membrane-bound ferritin occurred occasionally in neurones and more frequently in astrocytes and oligodendrocytes. Considerable amounts of ferritin were also accumulated in phagocytic cells of unknown origin. In blood vessels, ferritin collected in the basement membrane and around collagen and, in membrane-bound form, in pale cells at the periphery of the vessels. Ferritin occurred in all parts of the intercellular space except in interglial junctions and tight junctions between vascular endothelial cells.This work was supported by a research grant from the National Health and Medical Research Council of Australia to Professor M. J. Blunt, of the School of Anatomy, University of New South Wales. The author wishes to thank Professor Blunt for his constant encouragement and support. The assistance of Mrs. Ruth Mather is gratefully acknowledged.     

Sampling properties of DNA sequence data in phylogenetic analysis

We inferred phylogenetic trees from individual genes and random samples of nucleotides from the mitochondrial genomes of 10 vertebrates and compared the results to those obtained by analyzing the whole genomes. Individual genes are poor samples in that they infrequently lead to the whole-genome tree. A large number of nucleotide sites is needed to exactly determine the whole-genome tree. A relatively small number of sites, however, often results in a tree close to the whole-genome tree. We found that blocks of contiguous sites were less likely to lead to the whole-genome tree than samples composed of sites drawn individually from throughout the genome. Samples of contiguous sites are not representative of the entire genome, a condition that violates a basic assumption of the bootstrap method as it is applied in phylogenetic studies.      

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

Endocannabinoids and some phytocannabinoids bind to CB₁ and CB₂ cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 ^tm1Zim) CB₂ cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB₂ receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB₂ (Cnr2 ^Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 ^tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB₂ receptor deletion was lost. Likewise CB₁ receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB₁ receptor rather than a CB₂ receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.     

Diverse stability and catalytic properties of human tryptase alpha and beta isoforms are mediated by residue differences at the S1 pocket

Recombinant human tryptases (rHTs) corresponding to alpha and beta isoforms were characterized. rHTbeta was similar to tryptase isolated from skin (HST); it was a tetramer, hydrolyzed model substrates efficiently, and was functionally unstable when incubated under physiological conditions. Activity was lost rapidly (t(1/2) approximately 1 min) by a reversible process similar to that observed for the spontaneous inactivation of HST. Circular dichroism (CD) and intrinsic fluorescence emission (IFE) spectra of active rHTbeta corresponded to those of active HST and upon spontaneous inactivation IFE decreased in parallel to activity loss. rHTalpha differed from HST in catalytic ability and stability. rHTalpha did not react with model substrates, an active site titrant, or a competitive inhibitor of HST/rHTbeta. IFE and CD spectra were similar to those of the active and not the spontaneously inactivated form of HST. Under physiological conditions, rHTalpha IFE decreased at a rate 900-fold slower than that observed for HST, and rHTalpha remained tetrameric when examined by size exclusion chromatography at physiological salt concentration. Thus, rHTalpha is a stable "inactive" form of HT. Three active site variants of rHTalpha, K192Q, D216G, and K192Q-D216G were characterized. Residues 192 and 216 (chymotrypsinogen numbers for residues 191 and 215 of rHTalpha) lie at the entrance to the primary specificity (S1) pocket, and the mutations converted them to the residues of HTbeta. While K192Q displayed the same properties as rHTalpha, the catalytic and stability characteristics of D216G and K192Q-D216G progressively approached those of HST. Thus, the contrasting stability/activity properties of rHTalpha and rHTbeta are largely related to differences at the S1 pocket. On the basis of the properties of the variants, we suggest that the side chain of Asp216 is blocking and stabilizing the S1 pocket and that this stabilization is sufficient to prevent spontaneous inactivation.     

Cardiac gene expression profile and lipid accumulation in response to starvation

Starvation induces many biochemical and histological changes in the heart; however, the molecular events underlying these changes have not been fully elucidated. To explore the molecular response of the heart to starvation, microarray analysis was performed together with biochemical and histological investigations. Serum free fatty acids increased twofold in both 16- and 48-h-fasted mice, and cardiac triglyceride content increased threefold and sixfold in 16- and 48-h-fasted mice, respectively. Electron microscopy showed numerous lipid droplets in hearts of 48-h-fasted mice, whereas fewer numbers of droplets were seen in hearts from 16-h-fasted mice. Expression of 11,000 cardiac genes was screened by microarrays. More than 50 and 150 known genes were detected by differential expression analysis after 16- and 48-h-fasts, respectively. Genes for fatty acid oxidation and gluconeogenesis were increased, and genes for glycolysis were decreased. Many other genes for metabolism, signaling/cell cycle, cytoskeleton, and tissue antigens were affected by fasting. These data provide a broad perspective of the molecular events occurring physiologically in the heart in response to starvation.     

Potent bivalent inhibition of human tryptase-beta by a synthetic inhibitor

Human tryptase-beta (HTbeta) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HTbeta has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HTbeta interaction were defined in this study. Tight-binding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7x10(7) M(-1) s(-1) and a relatively slow dissociation rate constant of 0.04 s(-1). Bivalent inhibition was demonstrated by displacement of p-aminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HTbeta]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HTbeta, 24% or 53% inhibited by pre-incubation with an irreversible inhibitor. Two interactions were observed consistent with mono- and bi-valent binding; the Ki value for bivalent inhibition was at least 10(4)-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HTbeta finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HTbeta. The potency of CRA-2059 is thus attributable to effective bivalent binding.     

Some observations on the effect of Daflon (micronized purified flavonoid fraction of Rutaceae aurantiae) in bancroftian filarial lymphoedema

BACKGROUND: Morbidity management is a core component of the global programme for the elimination of lymphatic filariasis. In a double-blind clinical trial, the tolerability and efficacy of Daflon (500 mg) + DEC (25 mg) or DEC (25 mg) alone, twice daily for 90 days, was studied in 26 patients with bancroftian filarial lymphoedema. RESULTS: None of the patients in either drug group reported any adverse reaction throughout the treatment period (90 days). Haematological and biochemical parameters were within normal limits and there was no significant difference between the pre-treatment (day 0) and post-treatment (day 90) values. The group receiving Daflon showed significant reduction in oedema volume from day 90 (140.6 PlusMinus; 18.8 ml) to day 360 (71.8 PlusMinus; 20.7 ml) compared to the pre-treatment (day 0, 198.4 PlusMinus; 16.5 ml) value. This accounted for a 63.8% reduction in oedema volume by day 360 (considering the pre-treatment (day 0) as 100%). In the DEC group, the changes in oedema volume (between day 1 and day 360) were not significant when compared to the pre-treatment (day 0) value. The percentage reduction at day 360 was only 9%, which was not significant (P > 0.05). CONCLUSION: This study has shown that Daflon (500 mg, twice a day for 90 days) is both safe and efficacious in reducing oedema volume in bancroftian filarial lymphoedema. Further clinical trials are essential for strengthening the evidence base on the role of this drug in the morbidity management of lymphatic filariasis.