Phylogenetic screening of the human genome: identification of differentially hybridizing repetitive sequence families

The phi-screen, a method of phylogenetic screening, can be employed to detect repetitive sequence families that differentially hybridize between closely related species. Such differences may involve sequence divergence or variations in copy number, including total presence versus absence of a family of repeated DNA. We present the results of a phi-screen comparing the human genome to that of the prosimian, Galago crassicaudatus. Three human repetitive families that are divergent or not present in galago have been detected. One of these families is described in detail; it is similar among the anthropoids but is present in a lower copy number and/or divergent form in prosimians. The family is clearly related to the transposon-like human element (THE) described by Paulson et al. (1985). THEs have long terminal repeats reminiscent of retroviruses but are unique in that they have no sequence similarity to known mammalian retroviruses. The sequence of a solo long terminal repeat, found unassociated with THE internal sequence, is presented. This family member, THE p2, is bordered by a 5-bp target-site repeat and is interrupted by the insertion of an Alu element. A solo THE element sequenced by Wiginton et al. (1986) contains an insertion of Alu at precisely the same position as does THE p2.      

Immunity in human schistosomiasis mansoni: cross-reactive IgM and IgG2 anti-carbohydrate antibodies block the expression of immunity

We have previously reported that the slow development of immunity to reinfection after treatment of Schistosoma mansoni infections is partly attributable to the continued presence of 'blocking' antibodies in young, susceptible children. A further analysis of this phenomenon supports the hypothesis that such blocking antibodies can be of the IgG2 as well as the IgM isotype, and that they react with carbohydrate epitopes expressed both on egg polysaccharides and on schistosomulum surface antigens, of particular importance being those antigens that are shed from the schistosomulum surface during the early stages of maturation in vitro. Evidence is also presented that, in those patients lacking high levels of IgG2 blocking antibodies, resistance to reinfection after treatment is associated with the presence of other IgG isotypes against the same shed antigens.     

Activators of protein kinase C depolarize insulin-secreting cells by closing K+ channels.

Carbohydrate stimuli of insulin secretion depolarize the pancreatic B cell and the B-cell line RINm5F by inhibiting ATP-sensitive K+ channels. We examined the possibility that this effect is mediated by activation of protein kinase C. In RINm5F cells, the triose D-glyceraldehyde evoked a rapid increase of the mass of 1,2-diacylglycerol, the endogenous activator of protein kinase C. This effect is mainly due to de novo synthesis of the lipid from glycolytic intermediates, as glyceraldehyde carbon was incorporated into 1,2-diacylglycerol within 1 min of exposure to 14C-labelled glyceraldehyde. The effects of two exogenous activators of kinase C, 4-beta-12-phorbol-myristate 13-acetate (PMA) and 1,2-didecanoylglycerol (DC10) on single K+ channel currents were examined in RINm5F cell-attached membrane patches. Both PMA and DC10 depolarized the cells and decreased the open-state probability of the ATP-sensitive K+ channels. These actions were not due to changes in cellular ATP content, since PMA, like glyceraldehyde, failed to alter cellular ATP. As is the case for glyceraldehyde, PMA and DC10 raised cytosolic free Ca2+ [( Ca2+]i) and stimulated insulin secretion. Both of these effects are inhibited in the absence of external Ca2+. This, and the attenuation of the [Ca2+]i rise by verapamil, suggest that all three stimuli raise [Ca2+]i by promoting Ca2+ influx through voltage-gated channels in turn leading to insulin secretion. As the exogenous activators of protein kinase C mimic the effects of glyceraldehyde, it is proposed that the carbohydrate-mediated production of 1,2-diacylglycerol constitutes the link between metabolism and membrane depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA divergence in and around the alcohol dehydrogenase locus in five closely related species of Hawaiian Drosophila

The alcohol dehydrogenase (Adh) region from five planitibia subgroup species of Hawaiian picture-wing Drosophila has been cloned. A total of 15 kb of DNA in and around the Adh gene has been compared among the five species. Genetic distances were calculated to determine evolutionary relationships. These distances agree with previous distances determined by protein polymorphism and DNA hybridization techniques and can be interpreted in terms of specific island colonization and speciation (founder) events over the past 5 Myr. Examination of the restriction maps of the cloned Adh region from the five species shows many instances of small deletions, insertion of a transposable element in D. heteroneura, and the existence of a highly variable region on the 3' side of the Adh gene. Clustering relationships and rates of DNA change are calculated and compared with the relationship found for other species of Drosophila.      

Galanin activates nucleotide-dependent K+ channels in insulin-secreting cells via a pertussis toxin-sensitive G-protein.

The effects of galanin (7-70 nM) on ATP-sensitive K+ channels (KATP channels), membrane potential and the release of insulin have been studied in the insulinoma cell line, RINm5F. Single-channel currents have been recorded from excised outside-out membrane patches as well as intact insulin-secreting cells and it is shown that galanin, added to the outside of the membrane, specifically activates KATP channels. Studies carried out using the fluorescent probe bisoxonol demonstrate that galanin hyperpolarizes RINm5F cells. Galanin was also found to abolish glyceraldehyde-stimulated immunoreactive insulin release from the insulinoma cells. Both the galanin-evoked hyperpolarization and inhibition of insulin release were abolished in cells pre-exposed to pertussis toxin. The possibility that the gating of KATP channels could be mediated by a G-protein was studied in patch-clamp experiments by adding F- to the solution bathing the inside of the cell membranes (open-cell), in order to generate the alumino-fluoride complex AlF4-. F- (1-10 mM) evoked dose-dependent activation of KATP channels and this effect was fully reversible. F- was also able to activate K+ channels inhibited by ATP. That the fluoride activation of KATP channels is mediated by the complex AlF4- was indicated by experiments in which AlCl3 (10 microM) was found to enhance further the activation of K+ channels evoked by 1 mM F- and by results showing that F(-)-stimulation of KATP channels was (i) abolished in the continued presence of F- by the Al3+ chelator deferoxamine (0.5 mM) and (ii) could be mimicked by VO4(3-) which has a structure similar to that of the AlF4- complex.     

Frequency of HLA antigens in patients with psoriasis or psoriatic arthritis.

A study of 138 patients with psoriasis--74 with psoriasis alone and 64 with psoriatic arthritis--revealed a significantly increased frequency of the HLA antigens A1, A28, B13, DR7 and MT3 in those with psoriasis alone and of Bw39 in those with psoriatic arthritis. The frequency of B17 was higher in both patient groups than in a control group of healthy individuals. The frequency of DRw6 was slightly higher in the patients with psoriasis alone (17.8%) than in the controls (4.7%), and that of DR7 was higher in the patients with psoriatic arthritis (52.9%) than in the controls (32.6%). Elevated levels of serum IgG and IgA along with positive results of tests for antinuclear antibody or rheumatoid factor or both were present in less than a tenth of the patients with psoriatic rash alone and in up to a third of those with psoriatic arthritis. Psoriatic arthritis was found to be less likely to develop in patients with purely guttate psoriasis than in those with other types of psoriasis. Clinical subtypes of psoriatic rash or psoriatic arthritis were not associated with the presence of particular HLA antigens.     

Purification of cytochrome b-245 from human neutrophils.

The low potential cytochrome b (b-245) of the microbicidal oxidase of phagocytic cells has been purified from neutrophils from patients with chronic myeloid leukaemia. Cells were homogenized in the presence of proteinase inhibitors and centrifuged to remove the cytoplasm. The pellets containing membranes, granules and other organelles (15 mg/ml) were then washed with buffered sodium cholate (5 mg/ml). Residual pellets were subsequently solubilized with the non-ionic detergent Triton N 101 (10 mg/ml) which extracted about 60% of the cytochrome b. About 10% of the cytochrome b was of mitochondrial origin which was removed on a column of n-amino-octyl-Sepharose that did not adsorb cytochrome b-245. Cytochrome b-245 was chromatographed on a column of heparin-agarose and eluted with NaCl to give a peak specific content of 11-16 nmol of cytochrome b-245/mg of protein, representing a 140-200-fold purification with a recovery of 15%. This technique results in the purification of approx. 100-150 nmol of highly purified cytochrome b-245 from (3-5) X 10(11) cells within 4 days. The most purified material gave a broad band with an apparent Mr of between 68 000 and 78 000 on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, but gel filtration indicated an aggregated form of the protein in Triton N101 . Purified protein (14 nmol of haem/mg of protein) did not contain FAD or FMN and had no NADPH-dependent O2--generating activity.     

How does coarse gravel augmentation affect early‐stage Chinook salmon Oncorhynchus tshawytscha embryonic survivorship?

Early‐stage Chinook salmon Oncorhynchus tshawytscha embryos were incubated in artificial redds that mimicked hyporheic conditions in gravel‐augmented habitat to assess survivorship. Two complementary experiments were conducted where units varied along gradients of (1) increasing interstitial flow velocity (0·05–2·50 cm s^?1) in a uniformly coarse (particles ≥22 mm) sediment mixture and (2) increasing sediment porosity with interstitial flow velocity held constant. Embryonic survivorship increased moderately along a gradient of interstitial flow velocity, while survivorship among units with varying sediment porosities was consistent. No evidence for flow‐induced agitation and mortality was observed. Results suggest that high interstitial flow velocities may confer a moderate advantage for incubating salmonid embryos when conditions that typically reduce embryonic mortality (i.e. low concentrations of fine particles) are ideal.     

Ketamine Does Not Produce Relief of Neuropathic Pain in Mice Lacking the β-Common Receptor (CD131)

Neuropathic pain (NP) is a debilitating condition associated with traumatic, metabolic, autoimmune and neurological etiologies. Although the triggers for NP are diverse, there are common underlying pathways, including activation of immune cells in the spinal cord and up-regulation of the N-methyl-D-aspartate receptor (NMDAR). Ketamine, a well-known NDMAR antagonist, reduces neuropathic pain in a sustained manner. Recent study has shown that the novel 11-amino acid peptide erythropoietin derivative ARA290 produces a similar, long-lasting relief of NP. Here, we show that both drugs also have similar effects on the expression of mRNA of the NMDAR, as well as that of microglia, astrocytes and chemokine (C-C motif) ligand 2, all-important contributors to the development of NP. Although the effects of ketamine and ARA 290 on NP and its molecular mediators suggest a common mechanism of action, ARA 290 has no affinity for the NMDAR and acts specifically via the innate repair receptor (IRR) involved in tissue protection. We speculated therefore, that the IRR might be critically involved in the action of ketamine on neuropathic pain. To evaluate this, we studied the effects of ketamine and ARA 290 on acute pain, side effects, and allodynia following a spared nerve injury model in mice lacking the β-common receptor (βcR), a structural component of the IRR. Ketamine (50 mg/kg) and ARA 290 (30 µg/kg) produced divergent effects on acute pain: ketamine produced profound antinociception accompanied with psychomotor side effects, but ARA290 did not, in both normal and knock out mice. In contrast, while both drugs were antiallodynic in WT mice, they had no effect on NP in mice lacking the βcR. Together, these results show that an intact IRR is required for the effective treatment of NP with either ketamine or ARA 290, but is not involved in ketamine’s analgesic and side effects.