A high incidence of Shigella-induced arthritis in a primate species: major histocompatibility complex class I molecules associated with resistance and susceptiblity, and their relationship to HLA-B27

 The human major histocompatibility complex (MHC) class I gene, HLA-B27, is a strong risk factor for susceptibility to a group of disorders termed spondyloarthropathies. Rodents that express HLA-B27 develop spondyloarthropathies, implicating HLA-B27 in the etiology of these disorders. To determine whether an HLA-B27-like molecule was associated with spondyloarthropathies in nonhuman primates, we analyzed the MHC class I cDNAs expressed in a cohort of rhesus macaques that developed reactive arthritis after an outbreak of shigellosis. We identified several cDNAs with only limited sequence similarity to HLA-B27. Interestingly, one of these MHC molecules had a B pocket identical to that of HLA-B39. Pool sequencing of radiolabeled peptides bound by this molecule demonstrated that, like HLA-B27 and HLA-B39, it could bind peptides with arginine at the second position. However, extensive analysis of the MHC class I molecules in this cohort revealed no statistically significant association between any particular MHC class I allele and susceptibility to reactive arthritis. Furthermore, none of the rhesus MHC class I molecules bore a strong resemblance to HLA-B27, indicating that reactive arthritis can develop in this animal model in the absence of an HLA-B27-like molecule. Surprisingly, there was a statistically significant association between the rhesus macaque MHC A locus allele, Mamu-A*12, and the absence of reactive arthritis following Shigella infection. Received: 26 July 1999 / Revised: 28 December 1999     

The human SWI-SNF complex protein p270 is an ARID family member with non-sequence-specific DNA binding activity

p270 is an integral member of human SWI-SNF complexes, first identified through its shared antigenic specificity with p300 and CREB binding protein. The deduced amino acid sequence of p270 reported here indicates that it is a member of an evolutionarily conserved family of proteins distinguished by the presence of a DNA binding motif termed ARID (AT-rich interactive domain). The ARID consensus and other structural features are common to both p270 and yeast SWI1, suggesting that p270 is a human counterpart of SWI1. The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. Although about a dozen ARID proteins can be identified from database searches, to date, only Bright (a regulator of B-cell-specific gene expression), dead ringer (a Drosophila melanogaster gene product required for normal development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) have been analyzed directly in regard to their DNA binding properties. Each binds preferentially to AT-rich sites. In contrast, p270 shows no sequence preference in its DNA binding activity, thereby demonstrating that AT-rich binding is not an intrinsic property of ARID domains and that ARID family proteins may be involved in a wider range of DNA interactions.     

Why Helicobacter pylori has Lewis antigens

In mimicry with human gastric epithelial cells, the lipopolysaccharide of Helicobacter pylori expresses Lewis blood group antigens. Recent data suggest that molecular mimicry does not promote immune evasion, nor does it lead to induction of autoantibodies, but that H. pylori Lewis X mediates adhesion to gastric epithelial cells and is essential for colonization.     

Purification and characteristics of recombinant human folylpoly-gamma-glutamate synthetase expressed at high levels in insect cells

Folylpoly-gamma-glutamate synthetase activity is central to the operation of folate metabolism and is essential for the survival of mammalian stem cell populations but the very low levels of endogenous expression of this enzyme have greatly limited its study. We now report the expression of cytosolic folylpoly-gamma-glutamate synthetase (FPGS) cloned from human leukemic cells in baculovirus-infected insect cells at levels of 4-5% of the total soluble protein of the cells. As was the case with endogenously expressed mammalian FPGS, recombinant enzyme was quantitatively blocked at the amino terminus in spite of the large-scale production in insect cells. A three-step purification procedure resulted in an overall yield of 7-35 mg per liter of culture with a recovery of about 50% and purity approximately 95%; pure enzyme was stable to storage for extended periods. Pure protein had a specific activity of 25 micromol h(-1)mg(-1) with aminopterin as a substrate and used a broad spectrum of folates as substrates. The pure enzyme also carried out ATP hydrolysis in the absence of a folate substrate or glutamic acid; this partial reaction occurred at a k(cat) about 0.4% that of the full reaction. In vitro, this single protein added several (1-8) moles of glutamic acid per mole of folate analog, the same spectrum of folate polyglutamates as seen in vivo. The quantities of pure enzyme achievable in insect cells should allow functional and structural studies on this enzyme.     

D/H amide kinetic isotope effects reveal when hydrogen bonds form during protein folding

We have exploited a procedure to identify when hydrogen bonds (H-bonds) form under two-state folding conditions using equilibrium and kinetic deuterium/hydrogen amide isotope effects. Deuteration decreases the stability of equine cytochrome c and the dimeric and crosslinked versions of the GCN4-p1 coiled coil by approximately 0. 5 kcal mol-1. For all three systems, the decrease in equilibrium stability is reflected by a decrease in refolding rates and a near equivalent increase in unfolding rates. This apportionment indicates that approximately 50% of the native H-bonds are formed in the transition state of these helical proteins. In contrast, an alpha/beta protein, mammalian ubiquitin, exhibits a small isotope effect only on unfolding rates, suggesting its folding pathway may be different. These four proteins recapitulate the general trend that approximately 50% of the surface buried in the native state is buried in the transition state, leading to the hypothesis that H-bond formation in the transition state is cooperative, with alpha-helical proteins forming a number of H-bonds proportional to the amount of surface buried in the transition state.     

Blue light activates potassium-efflux channels in flexor cells from Samanea saman motor organs via two mechanisms

Light-induced leaflet movement of Samanea saman depends on the regulation of membrane transporters in motor cells. Blue light (BL) stimulates leaflet opening by inducing K(+) release from the flexor motor cells. To elucidate the mechanism of K(+)-efflux (K(D))-channel regulation by light, flexor motor cell protoplasts were patch-clamped in a cell-attached configuration during varying illumination. Depolarization elicited outward currents through single open K(D) channels. Changes in cell membrane potential (E(M)) were estimated by applying voltage ramps and tracking the change of the apparent reversal potential of K(D)-channel current. BL shifted E(M) in a positive direction (i.e. depolarized the cell) by about 10 mV. Subsequent red light pulse followed by darkness shifted E(M) oppositely (i.e. hyperpolarized the cell). The BL-induced shifts of E(M) were not observed in cells pretreated with a hydrogen-pump inhibitor, suggesting a contribution by hydrogen-pump to the shift. BL also increased K(D)-channel activity in a voltage-independent manner as reflected in the increase of the mean net steady-state patch conductance at a depolarization of 40 mV relative to the apparent reversal potential (G(@40)). G(@40) increased by approximately 12 pS without a change of the single-channel conductance, possibly by increasing the probability of channel opening. Subsequent red-light and darkness reversed the change in G(@40). Thus, K(+) efflux, a determining factor for the cell-volume decrease of flexor cells, is regulated by BL in a dual manner via membrane potential and by an independent signaling pathway.     

Stress down regulates milk yield in cows by plasmin induced beta-casein product that blocks K+ channels on the apical membranes

Stress and stress related hormones such as glucocorticoids inhibit lactation in cows. In the present study we propose a novel mechanism connecting stress with plasminogen-plasmin system (PPS) (an enzymatic mechanism in milk, which leads to the breakdown of the major milk protein casein). We show that stress activates the PPS leading to an increase in plasmin activity, and that a distinct plasmin-induced beta-casein breakdown product (fraction 1-28) is a potent blocker of potassium channels in mammary epithelia apical membranes. The reduction in milk production due to dehydration stress or glucocorticoid (dexamethsone) was correlated with the activities of plasmin and channel blocking activity in the milk of the tested cows. The notion that the axis Stress-PPS-beta-casein fraction 1-28 is responsible for the reduction in milk yield is supported by the results of experiments showing that injecting solution composed of casein digest enriched with beta-casein fraction 1-28 to the udder lumen leads to a transient reduction in milk production. Furthermore, injecting a pure beta-casein fraction 1-28 to the udder lumen of goat's lead also to a transient reduction in milk production with kinetics that was similar to the kinetics observed in cows.     

Role of endogenous CCK in the inhibition of gastric emptying by peptone and Intralipid in rats

To assess the role of endogenous cholecystokinin in the control of gastric emptying of peptone solutions and Intralipid suspensions, we examined the ability of a dose range of the CCK-A antagonist, devazepide to accelerate the gastric emptying of various caloric concentrations of peptone and Intralipid in rats. In the absence of devazepide, both peptone and Intralipid emptying slowed with increasing concentration. Devazepide's effect on peptone gastric emptying diminished with increasing peptone concentration. The threshold dose for accelerating the emptying of 0.2 kcal/ml peptone was lower than the threshold dose for affecting 0.5 kcal/ml peptone and devazepide had no effect on the gastric emptying of 1.0 kcal/ml peptone. In contrast, devazepide affected Intralipid gastric emptying at all three Intralipid concentrations and the threshold dose decreased with increasing Intralipid concentration. However, the magnitude of the effect of devazepide on peptone or Intralipid gastric emptying was partial and did not increase as a function of concentration. These data demonstrate a role for endogenous CCK in the emptying of peptone and Intralipid but suggest that endogenous CCK does not account for the increased slowing of gastric emptying evident with increased caloric concentration.