4-1BB signaling synergizes with programmed death ligand 1 blockade to augment CD8 T cell responses during chronic viral infection

Previous studies have identified the inhibitory role that the programmed death 1 (PD-1) pathway plays during chronic infection. Blockade of this pathway results in rescue of viral-specific CD8 T cells, as well as reduction of viral loads in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). We tested the effect of combining PD ligand 1 (PD-L1) blockade with an agonistic regimen that induces 4-1BB costimulation during chronic LCMV infection. There is a boosting effect in the rescue of LCMV-specific CD8 T cell responses after dual treatment with PD-L1 blockade and 4-1BB agonistic Abs when the amount and timing of 4-1BB costimulation are carefully controlled. When PD-L1-blocking Abs are given together with a single low dose of anti-4-1BB agonistic Abs, there is an enhanced and stable expansion of viral-specific CD8 T cells. Conversely, when blocking Abs to PD-L1 are given with a repetitive high dose of anti-4-1BB, there is an initial synergistic expansion of viral-specific CD8 T cells by day 7, followed by dramatic apoptosis by day 14. Viral control paralleled CD8 T cell kinetics after dual treatment. By day 7 posttreatment, viral titers were lower in both of the combined regimens (compared with PD-L1 blockade alone). However, whereas the high dose of anti-4-1BB plus PD-L1 blockade resulted in rebound of viral titers to original levels, the low dose of anti-4-1BB plus PD-L1 blockade resulted in a stable reduction of viral loads. These findings demonstrate the importance of carefully manipulating the balance between activating and inhibitory signals to enhance T cell responses during chronic infection.     

IL-17 in obesity and adipogenesis

The pro-inflammatory cytokine interleukin (IL)-17 (also known as IL-17) has been associated with induction of tissue inflammation. Obese individuals exhibit many symptoms of chronic low-grade inflammation, suggesting that IL-17 may impact adipose tissue. However, the role of IL-17 in obesity is largely unexplored. Emerging studies indicate that obesity selectively promotes expansion of the Th17 T-cell lineage, exacerbating disease in murine models of autoimmunity such as EAE and colitis. Human studies support this concept, as new clinical studies suggest that IL-17 is expressed at elevated levels in obese individuals. Conversely, however, an anti-adipogenic role for IL-17 is becoming evident, and therefore the interconnections between IL-17 and fat metabolism may be quite complex. Here, we consolidate the potential implications of IL-17 in relation to obesity and describe the emerging data regarding the role of IL-17 in adipose tissue.     

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

In mouse intestine, caveolae and caveolin‐1 (Cav‐1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L‐type Ca²⁺ channels, Na⁺‐Ca²⁺ exchanger type 1 (NCX1), plasma membrane Ca²⁺ pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo‐sarcoplasmic reticulum (ER‐SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L‐type Ca ⁺ channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav‐1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium‐free media with 100 mM ethylene glycol tetra‐acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium‐free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L‐type Ca²⁺ channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca²⁺‐ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav‐1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav‐1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L‐type Ca²⁺ channels or an increase in the distance between caveolae and SR affected calcium handling.     

A region of maize chromosome 2 affects response to downy mildew pathogens

Quantitative trait loci (QTLs) for downy mildew resistance in maize were identified based on co-segregation with linked restriction fragment length polymorphisms or simple sequence repeats in 220 F2 progeny from a cross between susceptible and resistant parents. Disease response was assessed on F3 families in nurseries in Egypt, Thailand, and South Texas and after inoculation in a controlled greenhouse test. Heritability of the disease reaction was high (around 93% in Thailand). One hundred and thirty polymorphic markers were assigned to the ten chromosomes of maize with LOD scores exceeding 4.9 and covering about 1,265 cM with an average interval length between markers of 9.5 cM. About 90% of the genome is located within 10 cM of the nearest marker. Three putative QTLs were detected in association with resistance to downy mildew in different environments using composite interval mapping. Despite environmental and symptom differences, one locus on chromosome 2 had a major effect and explained up to 70% of the phenotypic variation in Thailand where disease pressure was the highest. The other two QTLs on chromosome 3 and chromosome 9 had minor effects; each explained no more than 4% of the phenotypic variation. The three QTLs appeared to have additive effects on resistance, identifying one major gene and two minor genes that contribute to downy mildew resistance.     

Optimization of Insecticidal Triterpene Derivatives by Biomimetic Oxidations with Hydrogen Peroxide and Iodosobenzene Catalyzed by MnIII and FeIII Porphyrin Complexes

Semisynthetic functionalized triterpenes (4α,14‐dimethyl‐5α,8α‐8,9‐epoxycholestan‐3β‐yl acetate; 4α,14‐dimethyl‐5α‐cholest‐8‐ene‐3,7,11‐trione; 4α,14‐dimethyl‐5α‐cholesta‐7,9(11)‐dien‐3‐one and 4α,14‐dimethyl‐5α‐cholest‐8‐en‐3β‐yl acetate), previously prepared from 31‐norlanostenol, a natural insecticide isolated from the latex of Euphorbia officinarum, have been subjected to oxidation with hydrogen peroxide (H₂O₂) and iodosobenzene (PhIO) catalyzed by porphyrin complexes (cytochrome P‐450 models) in order to obtain optimized derivatives with high regioselectivity. The main transformations were epoxidation of the double bonds and hydroxylations of non‐activated C–H groups and the reaction products were 25‐hydroxy‐4α,14‐dimethyl‐5α‐cholesta‐7,9(11)‐dien‐3β‐yl acetate (59 %), 25‐hydroxy‐4α,14‐dimethyl‐5α‐cholest‐8‐ene‐3,7,11‐trione (60 %), 4α,14‐dimethyl‐5α,7β‐7,8‐epoxycholest‐9(11)‐en‐3‐one (22 %), 8‐hydroxy‐4α,14‐dimethyl‐5α‐cholest‐9(11)‐ene‐3,7‐dione (16 %), 12α‐hydroxy‐4α,14‐dimethyl‐5α,7β‐7,8‐epoxycholest‐9(11)‐en‐3‐one (16 %), and 4α,14‐dimethyl‐5α,8α‐8,9‐epoxycholestan‐3β‐yl acetate (26 %), respectively. We also investigated the insect (Myzus persicae, Rhopalosiphum padi and Spodoptera littoralis) antifeedant and postingestive effects of these terpenoid derivatives. None of the compounds tested had significant antifeedant effects, however, all were more effective postingestive toxicants on S. littoralis larvae than the natural compound 31‐norlanostenol, with 4α,14‐dimethyl‐5α,8α‐8,9‐epoxycholestan‐3β‐yl acetate being the most active. The study of their structure–activity relationships points out at the importance of C3 and C7 substituents.     

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.     

Anti-hypercholesterolemic impacts of barley and date palm fruits on the ovary of Wistar albino rats and their offspring

A high cholesterol diet is related to ovarian dysfunction and infertility which has been increased among young ages consuming processed food products. The present study was conducted to evaluate the role of a high cholesterol diet on the ovaries of young female rats via assessments of histopathology, immunohistochemistry, oxidative stress and apoptic markers. Also, mating of hypercholesterolemic female rats was carried out to measure the fertility and numbers of their offspring. At the same time, phytotherapy was carried out through supplementing the diet with barley and/ or date palm fruits (10%) during the experiment to assess the phyto-therapeutic impacts in attenuation of drastic hypercholesterolemic effects.Hypercholesterolemic diet-fed rats exhibited damage of the ovarian follicles and increased follicular atresia. Furthermore, expression of cleaved caspase-3 was upregulated, while PCNA was downregulated in granulosa, theca and stroma cells. Hypercholesterolemic female rats showed marked depletion of antioxidative enzymes, increased lipid peroxidation and apoptotic markers. Alterations to the female serum hormones were detected. Offspring maternally fed on hypercholesterolemic diet showed a significant decrease of body weight and altered sex ratio. However, concomitant supplementation of barley and or date fruits to hypercholesterolemic groups revealed marked improvement of ovarian structure and function.On the basis of these evidences, it is believed that the enhanced synergistic effects of barley and/or date palm fruits in the amelioration of ovarian structure and functions were elicited by the potential antioxidant activity of their phytomicronutrients, polyphenols, β-glucan and trace elements. These materials scavenge free radicals from inflamed cells that can be used to establish an effective and novel therapeutic strategy for activating ovarian cell regeneration.     

Enzymatic and structural analysis of inhibitors designed against Mycobacterium tuberculosis thymidylate kinase. New insights into the phosphoryl transfer mechanism

The chemical synthesis of new compounds designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogues modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogues are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg(2+) ion. The crystallographic structure of the complex of one of the compounds, 5-CH(2)OH-dUMP, with TMPK has been determined at 2.0 A. It reveals a major conformation for the hydroxyl group in contact with a water molecule and a minor conformation pointing toward Ser(99). Looking for a role for Ser(99), we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu(6), Ser(99), Arg(95), and Asp(9)) that probably serves to protonate the transferred PO(3) group. The crystallographic structure of the commercially available bisubstrate analogue P(1)-(adenosine-5')-P(5)-(thymidine-5')-pentaphosphate bound to TMPK is also reported at 2.45 A and reveals an alternative binding pocket for the adenine moiety of the molecule compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors.     

Studies on chromatin-associated protein phosphokinase of submandibular gland from isoproterenol-treated rats

Water-soluble chromatin from rat submandibular gland nuclei was isolated, and had a DNA: RNA:protein ratio of 8:1:20. The spectral properties of this preparation were similar to those described for chromatins from other tissues. The rat submandibular gland chromatin possessed protein phosphokinase activity. It was able to incorporate ³²P from [γ-³²P]-ATP into chromatin proteins, and into dephospho-phosvitin. The chromatin-associated protein phosphokinase activity (measured with dephospho-phosvitin as substrate) required Mg²⁺, Na⁺ or K⁺ and dithiothreitol for optimal activity. A single injection of isoproterenol influenced the activity of this enzyme system, so that it was decreased at 2 h, showed a transient increase at 4 h, and a large increase at 10–16 h after the injection. This event appears to precede the increase in ribosomal RNA induced by Ipr [13]. By 48 h the chromatin-associated protein kinase returned to the normal control levels. These changes appeared to be commensurate with the corresponding alterations in the non-histone acidic protein complement of these chromatins. Actinomycin D or cycloheximide, when administered 30 min prior to isoproterenol, blocked the increase in chromatin-associated protein kinase at 4 as well as 10 h after the injection of isoproterenol. Injection of pilocarpine did not influence the chromatin-associated protein phosphokinase activity. Dichloroisoproterenol appeared to be antagonistic to the influence of isoproterenol in mediating changes in chromatin-associated protein kinase. The results suggest that the isoproterenol-induced increase in chromatin-bound protein phosphokinase which precedes the increase in RNA synthesis is related to the eventual onset of DNA synthesis in rat submandibular gland stimulated by isoproterenol. The chromatin-bound protein phosphokinase activity (or activities) may have a regulatory role on gene action, mediated through the control of phosphorylation of nuclear non-histone acidic proteins [26].     

dam methylation and the initiation of DNA replication on oriC plasmids

Summary Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oric region.