Impact of the CCR5 gene polymorphism on the survival of metastatic melanoma patients receiving immunotherapy

Purpose Chemokines influence both tumor progression and anti-tumor immune response. A 32-bp-deletion polymorphism in the chemokine receptor 5 gene (CCR5Δ32) has been shown to result in a non-functional protein. This study was aimed at evaluating the potential impact of this gene polymorphism on disease progression and treatment outcome in patients with melanoma. Patients and methods CCR5 genotyping was performed by PCR on DNA extracted from serum samples of 782 cutaneous melanoma patients with known disease history and long-term clinical follow-up. Genotypes were correlated with patient survival and types of treatment. Results Of 782 melanoma patients, 90 (11.5%) were heterozygous and 12 (1.5%) were homozygous for CCR5Δ32. Analyzing the complete cohort, the disease-specific survival from date of primary diagnosis was not influenced by CCR5 status. Similarly, no significant impact could be detected on the treatment outcome of stage III patients. In 139 stage IV patients receiving immunotherapy, CCR5Δ32 was associated with a decreased survival compared to patients not carrying the deletion (median 12.5 vs. 20.3 months, P = 0.029). Multivariate analysis revealed the CCR5 genotype as an independent factor impacting disease-specific survival in this patient population (P = 0.002), followed by gender (P = 0.019) and pathological classification of the primary (pT; P = 0.022). Conclusion The presence of the CCR5Δ32 polymorphism in patients with stage IV melanoma results in a decreased survival following immunotherapy and may help to select patients less likely to benefit from this type of treatment. Selma Ugurel and David Schrama have contributed equally to this work.     

Estradiol increases urethral tone through the local inhibition of neuronal nitric oxide synthase expression

Estrogens are known to modulate lower urinary tract (LUT) trophicity and neuronal nitric oxide synthase (nNOS) expression in several organs. The aim of this study was to explore the effects of endogenous and supraestrus levels of 17beta-estradiol (E2) on LUT and urethral nNOS expression and function. LUT function and histology and urethral nNOS expression were studied in adult female mice subjected either to sham surgery, surgical castration, or castration plus chronic E2 supplementation (80 microg.kg(-1).day(-1), i.e., pregnancy level). The micturition pattern was profoundly altered by long-term supraestrus levels of E2 with decreased frequency paralleled by increased residual volumes higher than those of ovariectomized mice. Urethral resistance was increased twofold in E2-treated mice, with no structural changes in urethra, supporting a pure tonic mechanism. Acute nNOS inhibition by 7-nitroindazole decreased frequency and increased residual volumes in ovariectomized mice but had no additive effect on the micturition pattern of long-term supraestrus mice, showing that long-term supraestrus E2 levels and acute inhibition of nNOS activity had similar functional effects. Finally, E2 decreased urethral nNOS expression in ovariectomized mice. Long-term supraestrus levels of E2 increased urethral tone through inhibition of nNOS expression, whereas physiological levels of E2 had no effect.     

Hypoxia effects on cell volume and ion uptake of cerebral microvascular endothelial cells

Increased transport of Na across an intact blood-brain barrier (BBB) contributes to cerebral edema formation in ischemic stroke. Our previous studies have shown that ischemic factors stimulate activity of a luminal BBB Na-K-Cl cotransporter, and we have hypothesized that during ischemia, the cotransporter together with the abluminal Na/K pump mediates increased transport of Na from blood into the brain. However, it is possible that elevated Na-K-Cl cotransporter activity could also cause cell swelling if it outpaces ion efflux pathways. The present study was conducted to evaluate the effects of hypoxia on intracellular volume of BBB cells. Cerebral microvascular endothelial cell (CMEC) monolayers were exposed to varying levels of hypoxia for 1 to 5 h in an O(2)-controlled glove box, and cell volume was assessed using 3-O-methyl-D-[(3)H]glucose and [(14)C]sucrose as markers of total and extracellular water space, respectively. Cells exposed to either 7.5%, 3%, or 1% O(2) showed gradual increases in volume (compared with 19% O(2) normoxic controls) that became significant after 3 or more hours. By ion chromatography methods, we also found that a 30-min exposure to 7.5% O(2) caused an increase in bumetanide-sensitive net Na uptake by the cells without increasing cell Na content. CMEC Na content was significantly increased, however, following 3 or more hours of exposure to 7.5% O(2). These findings are consistent with the hypothesis that during cerebral ischemia, the BBB Na-K-Cl cotransporter is stimulated to mediate transendothelial uptake of Na into the brain and that increased cotransporter activity also contributes to gradual swelling of the cells.     

Eukaryotic HMGB proteins as replacements for HU in E. coli repression loop formation

DNA looping is important for gene repression and activation in Escherichia coli and is necessary for some kinds of gene regulation and recombination in eukaryotes. We are interested in sequence-nonspecific architectural DNA-binding proteins that alter the apparent flexibility of DNA by producing transient bends or kinks in DNA. The bacterial heat unstable (HU) and eukaryotic high-mobility group B (HMGB) proteins fall into this category. We have exploited a sensitive genetic assay of DNA looping in living E. coli cells to explore the extent to which HMGB proteins and derivatives can complement a DNA looping defect in E. coli lacking HU protein. Here, we show that derivatives of the yeast HMGB protein Nhp6A rescue DNA looping in E. coli lacking HU, in some cases facilitating looping to a greater extent than is observed in E. coli expressing normal levels of HU protein. Nhp6A-induced changes in the DNA length-dependence of repression efficiency suggest that Nhp6A alters DNA twist in vivo. In contrast, human HMGB2-box A derivatives did not rescue looping.     

Structure-function analysis of the RNA helicase maleless

Loss of function of the RNA helicase maleless (MLE) in Drosophila melanogaster leads to male-specific lethality due to a failure of X chromosome dosage compensation. MLE is presumably involved in incorporating the non-coding roX RNA into the dosage compensation complex (DCC), which is an essential but poorly understood requirement for faithful targeting of the complex to the X chromosome. Sequence comparison predicts several RNA-binding domains in MLE but their properties have not been experimentally verified. We evaluated the RNA-binding characteristics of these conserved motifs and their contributions to RNA-stimulated ATPase activity, to helicase activity, as well as to the targeting of MLE to the nucleus and to the X chromosome territory. We find that RB2 is the dominant, conditional RNA-binding module, which is indispensable for ATPase and helicase activity whereas the N-terminal RB1 motif does not bind RNA, but is involved in targeting MLE to the X chromosome. The C-terminal domain containing a glycine-rich heptad repeat adds potential dimerization and RNA-binding surfaces which are not required for helicase activity.     

Baroreflex control of lumbar and renal sympathetic nerve activity in conscious rats

This study compared the baroreflex control of lumbar and renal sympathetic nerve activity (SNA) in conscious rats. Arterial pressure (AP) and lumbar and renal SNA were simultaneously recorded in six freely behaving rats. Pharmacological estimates of lumbar and renal sympathetic baroreflex sensitivity (BRS) were obtained by means of the sequential intravenous administration of sodium nitroprusside and phenylephrine. Sympathetic BRS was significantly (P < 0.05) lower for lumbar [3.0 +/- 0.4 normalized units (NU)/mmHg] than for renal (7.6 +/- 0.6 NU/mmHg) SNA. During a 219-min baseline period, spontaneous lumbar and renal BRS were continuously assessed by computing the gain of the transfer function relating AP and SNA at heart rate frequency over consecutive 61.4-s periods. The transfer gain was considered only when coherence between AP and SNA significantly differed from zero, which was verified in 99 +/- 1 and 96 +/- 3% of cases for lumbar and renal SNA, respectively. When averaged over the entire baseline period, spontaneous BRS was significantly (P < 0.05) lower for lumbar (1.3 +/- 0.2 NU/mmHg) than for renal (2.3 +/- 0.3 NU/mmHg) SNA. For both SNAs, spontaneous BRS showed marked fluctuations (variation coefficients were 26 +/- 2 and 28 +/- 2% for lumbar and renal SNA, respectively). These fluctuations were positively correlated in five of six rats (R = 0.44 +/- 0.06; n = 204 +/- 8; P < 0.0001). We conclude that in conscious rats, the baroreflex control of lumbar and renal SNA shows quantitative differences but is modulated in a mostly coordinated way.     

Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses.

The putative two-pore Ca(2+) channel TPC1 has been suggested to be involved in responses to abiotic and biotic stresses. We show that AtTPC1 co-localizes with the K(+)-selective channel AtTPK1 in the vacuolar membrane. Loss of AtTPC1 abolished Ca(2+)-activated slow vacuolar (SV) currents, which were increased in AtTPC1-over-expressing Arabidopsis compared to the wild-type. A Ca(2+)-insensitive vacuolar cation channel, as yet uncharacterized, could be resolved in tpc1-2 knockout plants. The kinetics of ABA- and CO(2)-induced stomatal closure were similar in wild-type and tpc1-2 knockout plants, excluding a role of SV channels in guard-cell signalling in response to these physiological stimuli. ABA-, K(+)-, and Ca(2+)-dependent root growth phenotypes were not changed in tpc1-2 compared to wild-type plants. Given the permeability of SV channels to mono- and divalent cations, the question arises as to whether TPC1 in vivo represents a pathway for Ca(2+) entry into the cytosol. Ca(2+) responses as measured in aequorin-expressing wild-type, tpc1-2 knockout and TPC1-over-expressing plants disprove a contribution of TPC1 to any of the stimulus-induced Ca(2+) signals tested, including abiotic stresses (cold, hyperosmotic, salt and oxidative), elevation in extracellular Ca(2+) concentration and biotic factors (elf18, flg22). In good agreement, stimulus- and Ca(2+)-dependent gene activation was not affected by alterations in TPC1 expression. Together with our finding that the loss of TPC1 did not change the activity of hyperpolarization-activated Ca(2+)-permeable channels in the plasma membrane, we conclude that TPC1, under physiological conditions, functions as a vacuolar cation channel without a major impact on cytosolic Ca(2+) homeostasis.     

Gymnosperm Orthologues of Class B Floral Homeotic Genes and Their Impact on Understanding Flower Origin

Class B floral homeotic genes play a key role in specifying the identity of male reproductive organs (stamens) and petals during the development of flowers. Recently, close relatives (orthologues) of these genes have been found in diverse gymnosperms, the sister group of the flowering plants (angiosperms). The fact that such genes have not been found so far, despite considerable efforts, in mosses, ferns or algae, has been taken as evidence to suggest that B genes originated 300–400 million years ago in a lineage that led to extant seed plants. Gymnosperms do not develop petals, and their male reproductive organs deviate considerably from angiosperm stamens. So what is the function of gymnosperm B genes? Recent experiments revealed that B genes from diverse extant gymnosperms are exclusively expressed in male reproductive organs (microsporophylls). At least for some of these genes it has been shown that they can partially substitute for the Arabidopsis B genes AP3 and PI in ectopic expression experiments, or even partially substitute these genes in different class B floral organ identity gene mutants. This functional complementation, however, is restricted to male organ development. These findings strongly suggest that gymnosperm and angiosperm B genes have highly related interaction partners and equivalent functions in the male organs of their different host species. It seems likely that in extant gymnosperms B genes have a function in specifying male reproductive organs. This function was probably established already in the most recent common ancestor of extant gymnosperms and angiosperms (seed plants) 300 million years ago and thus represents the ancestral function of seed plant B genes, from which other functions (e.g., in specifying petal identity) might have been derived. This suggests that the B gene function is part of an ancestral sex determination system in which B gene expression specifies male reproductive organ development, while the absence of B gene expression leads to the formation of female reproductive organs. Such a simple switch mechanism suggests that B genes might have played a central role during the origin of flowers. In the out-of-male and out-of-female hypotheses changes in B gene expression led to the origin of hermaphroditic flower precursors out of male or female gymnosperm reproductive cones, respectively. We compare these hypotheses with other recent molecular hypotheses on the origin of flowers, in which C/D and FLORICAULA/LEAFY-like genes is given a more prominent role, and we suggest how these hypotheses might be tested in the future.     

Properties of group I allergens from grass pollen and their relation to cathepsin B, a member of the C1 family of cysteine proteinases.

Expansins are a family of proteins that catalyze pH-dependent long-term extension of isolated plant cell walls. They are divided into two groups, alpha and beta, the latter consisting of the grass group I pollen allergens and their vegetative homologs. Expansins are suggested to mediate plant cell growth by interfering with either structural proteins or the polysaccharide network in the cell wall. Our group reported papain-like properties of beta-expansin of Timothy grass (Phleum pratense) pollen, Phl p 1, and suggested that cleavage of cell wall structural proteins may be the underlying mechanism of expansin-mediated wall extension. Here, we report additional data showing that beta-expansins resemble ancient and modern cathepsin B, which is a member of the papain (C1) family of cysteine proteinases. Using the Pichia pastoris expression system, we show that cleavage of inhibitory prosequences from the recombinant allergen is facilitated by its N-glycosylation and that the truncated, activated allergen shows proteolytic activity, resulting in very low stability of the protein. We also show that deglycosylated, full-length allergen is not activated efficiently and therefore is relatively stable. Motif and homology search tools detected significant similarity between beta-expansins and cathepsins of modern animals as well as the archezoa Giardia lamblia, confirming the presence of inhibitory prosequences, active site and other functional amino-acid residues, as well as a conserved location of these features within these molecules. Lastly, we demonstrate by site-directed mutagenesis that the conserved His104 residue is involved in the catalytic activity of beta-expansins. These results indicate a common origin of cathepsin B and beta-expansins, especially if taken together with their previously known biochemical properties.