Monomeric G-protein, Rhes, is not an imidazoline-regulated protein in pancreatic beta-cells

The monomeric G-protein, Rhes, is a candidate imidazoline-regulated molecule involved in mediating the insulin secretory response to efaroxan [S.L. Chan, L.K. Monks, H. Gao, P. Deaville, N.G. Morgan, Identification of the monomeric G-protein, Rhes, as an efaroxan-regulated protein in the pancreatic beta-cell, Br. J. Pharmacol. 136 (1) (2002) 31-36]. This suggestion was based on observations regarding changes in Rhes mRNA expression in rat islets and pancreatic beta-cells after prolonged culture with efaroxan, leading to desensitization of the insulin response to the compound. To verify this report, we have evaluated the effects of the imidazoline compounds efaroxan and BL11282 on Rhes mRNA expression in isolated rat pancreatic islets maintained in conditions identical to those used by Chan et al. The results demonstrate that desensitization of the insulin response to efaroxan, or to another imidazoline, BL11282, does not change Rhes mRNA expression levels. Transfection of MIN6 cells with plasmids containing Rhes or Rhes-antisense also does not alter efaroxan- or BL11282-induced insulin secretion. Together, these data do not support the hypothesis that Rhes is an imidazoline-regulated protein.     

A CAPS marker set for mapping in linkage group III of pea (Pisum sativum L.)

A set of twelve CAPS markers was mapped for linkage group III of pea (Pisum sativum L.). New primers were designed to use a polymerase chain reaction to amplify fragments of sequenced pea genes containing at least one large intron. Amplification products were tested for polymorphism across three pea lines (Chi115, Flagman and WL1238) using eleven four-base restriction endonucleases. Nine STS markers for linkage group III from the literature were also tested for polymorphism, and five of these were used in this mapping study as anchor points. All polymorphic loci were located by genetic analysis of the F(2)population from the cross Chi115 x WL1238, and a map of linkage group III consisting of one morphological and twelve CAPS markers was created. The map covers the full length of the chromosome and is about 162 cM long. All the CAPS markers in a set were used to test for polymorphism among 10 additional pea DNA samples extracted from different marker lines and cultivars.     

A model of the ternary complex of interleukin-10 with its soluble receptors

Background  

Interleukin-10 (IL-10) is a cytokine whose main biological function is to suppress the immune response by induction of a signal(s) leading to inhibition of synthesis of a number of cytokines and their cellular receptors. Signal transduction is initiated upon formation of a ternary complex of IL-10 with two of its receptor chains, IL-10R1 and IL-10R2, expressed on the cell membrane. The affinity of IL-10R1 toward IL-10 is very high, which allowed determination of the crystal structure of IL-10 complexed with the extracellular/soluble domain of IL-10R1, while the affinity of IL-10R2 toward either IL-10 or IL-10/sIL-10R1 complex is quite low. This so far has prevented any attempts to obtain structural information about the ternary complex of IL-10 with its receptor chains.     

The C-type lectin fold as an evolutionary solution for massive sequence variation

Only few instances are known of protein folds that tolerate massive sequence variation for the sake of binding diversity. The most extensively characterized is the immunoglobulin fold. We now add to this the C-type lectin (CLec) fold, as found in the major tropism determinant (Mtd), a retroelement-encoded receptor-binding protein of Bordetella bacteriophage. Variation in Mtd, with its approximately 10(13) possible sequences, enables phage adaptation to Bordetella spp. Mtd is an intertwined, pyramid-shaped trimer, with variable residues organized by its CLec fold into discrete receptor-binding sites. The CLec fold provides a highly static scaffold for combinatorial display of variable residues, probably reflecting a different evolutionary solution for balancing diversity against stability from that in the immunoglobulin fold. Mtd variants are biased toward the receptor pertactin, and there is evidence that the CLec fold is used broadly for sequence variation by related retroelements.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

Cardiotonic steroids differentially affect intracellular Na+ and [Na+]i/[K+]i-independent signaling in C7-MDCK cells

Recently, we reported that ouabain kills renal epithelial and vascular endothelial cells independently of elevation of the [Na(+)](i)/[K(+)](i) ratio. These observations raised the possibility of finding cardiotonic steroids (CTS) that inhibit the Na(+),K(+) pump without attenuating cell survival and vice versa. To test this hypothesis, we compared CTS action on Na(+),K(+) pump, [Na(+)](i) content, and survival of Madin-Darby canine kidney cells. At a concentration of 1 microM, ouabain and other tested cardenolides, as well as bufadienolides such as bufalin, cinobufagin, cinobufotalin, and telobufotoxin, led to approximately 10-fold inhibition of the Na(+),K(+) pump, a 2-3-fold decrease in staining with dimethylthiazol-diphenyltetrazolium (MTT), and massive death indicated by detachment of approximately 80% of cells and caspase-3 activation. In contrast, Na(+),K(+) pump inhibition and elevation of [Na(+)](i) seen in the presence of 3 microM marinobufagenin (MBG) and marinobufotoxin did not affect MTT staining and cell survival. Inhibition of the Na(+),Rb(+) pump in K(+)-free medium was not accompanied by a decline of MTT staining and cell detachment but increased sensitivity to CTS. In K(+)-free medium, half-maximal inhibition of (86)Rb influx was observed in the presence of 0.04 microM ouabain and 0.1 microM MBG, whereas half-maximal detachment and decline of MTT staining were detected at 0.03 and 0.004 microM of ouabain versus 10 and 3 microM of MBG, respectively. Both ouabain binding and ouabain-induced [Na(+)](i),[K(+)](i)-independent signaling were suppressed in the presence of MBG. Thus, our results show that CTS exhibit distinctly different potency in Na(+),K(+) pump inhibition and triggering of [Na(+)](i)/[K(+)](i)-independent signaling, including cell death.     

The Ras/Raf-1/MEK1/ERK signaling pathway coupled to integrin expression mediates cholinergic regulation of keratinocyte directional migration

The physiologic mechanisms that determine directionality of lateral migration are a subject of intense research. Galvanotropism in a direct current (DC) electric field represents a natural model of cell re-orientation toward the direction of future migration. Keratinocyte migration is regulated through both the nicotinic and muscarinic classes of acetylcholine (ACh) receptors. We sought to identify the signaling pathway mediating the cholinergic regulation of chemotaxis and galvanotropism. The pharmacologic and molecular modifiers of the Ras/Raf-1/MEK1/ERK signaling pathway altered both chemotaxis toward choline and galvanotropism toward the cathode in a similar way, indicating that the same signaling steps were involved. The galvanotropism was abrogated due to inhibition of ACh production by hemicholinium-3 and restored by exogenously added carbachol. The concentration gradients of ACh and choline toward the cathode in a DC field were established by high-performance liquid chromatographic measurements. This suggested that keratinocyte galvanotaxis is, in effect, chemotaxis toward the concentration gradient of ACh, which it creates in a DC field due to its highly positive charge. A time-course immunofluorescence study of the membrane redistribution of ACh receptors in keratinocytes exposed to a DC field revealed rapid relocation to and clustering at the leading edge of alpha7 nicotinic and M(1) muscarinic receptors. Their inactivation with selective antagonists or small interfering RNAs inhibited galvanotropism, which could be prevented by transfecting the cells with constitutively active MEK1. The end-point effect of the cooperative signaling downstream from alpha7 and M(1) through the MEK1/ERK was an up-regulated expression of alpha(2) and alpha(3) integrins, as judged from the results of real-time PCR and quantitative immunoblotting. Thus, alpha7 works together with M(1) to orient a keratinocyte toward direction of its future migration. Both alpha7 and M(1) apparently engage the Ras/Raf/MEK/ERK pathway to up-regulate expression of the "sedentary" integrins required for stabilization of the lamellipodium at the keratinocyte leading edge.     

Structural consequences of the inhibitor-resistant Ser130Gly substitution in TEM beta-lactamase

Beta-lactamase confers resistance to penicillin-like antibiotics by hydrolyzing their beta-lactam bond. To combat these enzymes, inhibitors covalently cross-linking the hydrolytic Ser70 to Ser130 were introduced. In turn, mutant beta-lactamases have emerged with decreased susceptibility to these mechanism-based inhibitors. Substituting Ser130 with glycine in the inhibitor-resistant TEM (IRT) mutant TEM-76 (S130G) prevents the irreversible cross-linking step. Since the completely conserved Ser130 is thought to transfer a proton important for catalysis, its substitution might be hypothesized to result in a nonfunctional enzyme; this is clearly not the case. To investigate how TEM-76 remains active, its structure was determined by X-ray crystallography to 1.40 A resolution. A new water molecule (Wat1023) is observed in the active site, with two configurations located 1.1 and 1.3 A from the missing Ser130 Ogamma; this water molecule likely replaces the Ser130 side-chain hydroxyl in substrate hydrolysis. Intriguingly, this same water molecule is seen in the IRT TEM-32 (M69I/M182T), where Ser130 has moved significantly. TEM-76 shares other structural similarities with various IRTs; like TEM-30 (R244S) and TEM-84 (N276D), the water molecule activating clavulanate for cross-linking (Wat1614) is disordered (in TEM-30 it is actually absent). As expected, TEM-76 has decreased kinetic activity, likely due to the replacement of the Ser130 side-chain hydroxyl with a water molecule. In contrast to the recently determined structure of the S130G mutant in the related SHV-1 beta-lactamase, in TEM-76 the key hydrolytic water (Wat1561) is still present. The conservation of similar accommodations among IRT mutants suggests that resistance arises from common mechanisms, despite the disparate locations of the various substitutions.     

The Y deletion gr/gr and susceptibility to testicular germ cell tumor

Testicular germ cell tumor (TGCT) is the most common cancer in young men. Despite a considerable familial component to TGCT risk, no genetic change that confers increased risk has been substantiated to date. The human Y chromosome carries a number of genes specifically involved in male germ cell development, and deletion of the AZFc region at Yq11 is the most common known genetic cause of infertility. Recently, a 1.6-Mb deletion of the Y chromosome that removes part of the AZFc region—known as the “gr/gr” deletion—has been associated with infertility. In epidemiological studies, male infertility has shown an association with TGCT that is out of proportion with what can be explained by tumor effects. Thus, we hypothesized that the gr/gr deletion may be associated with TGCT. Using logistic modeling, we analyzed this deletion in a large series of TGCT cases with and without a family history of TGCT. The gr/gr deletion was present in 3.0% (13/431) of TGCT cases with a family history, 2% (28/1,376) of TGCT cases without a family history, and 1.3% (33/2,599) of unaffected males. Presence of the gr/gr deletion was associated with a twofold increased risk of TGCT (adjusted odds ratio [aOR] 2.1; 95% confidence interval [CI] 1.3–3.6; P = .005) and a threefold increased risk of TGCT among patients with a positive family history (aOR 3.2; 95% CI 1.5–6.7; P = .0027). The gr/gr deletion was more strongly associated with seminoma (aOR 3.0; 95% CI 1.6–5.4; P = .0004) than with nonseminoma TGCT (aOR 1.5; 95% CI 0.72–3.0; P = .29). These data indicate that the Y microdeletion gr/gr is a rare, low-penetrance allele that confers susceptibility to TGCT.