Importance of the N-distal AP-2 binding element in Nef for simian immunodeficiency virus replication and pathogenicity in rhesus macaques

Point mutations in SIVmac239 Nef disrupting CD4 downmodulation and enhancement of virion infectivity attenuate viral replication in acutely infected rhesus macaques, but changes selected later in infection fully restore Nef function (A. J. Iafrate et al., J. Virol. 74:9836-9844, 2000). To further evaluate the relevance of these Nef functions for viral persistence and disease progression, we analyzed an SIVmac239 Nef mutant containing a deletion of amino acids Q64 to N67 (delta64-67Nef). This mutation inactivates the N-distal AP-2 clathrin adaptor binding element and disrupts the abilities of Nef to downregulate CD4, CD28 and CXCR4 and to stimulate viral replication in vitro. However, it does not impair the downmodulation of CD3 and class I major histocompatibility complex (MHC-I) or MHC-II and the upregulation of the MHC-II-associated invariant chain, and it has only a moderate effect on the enhancement of virion infectivity. Replication of the delta64-67Nef variant in acutely infected macaques was intermediate between grossly nef-deleted and wild-type SIVmac239. Subsequently, three of six macaques developed moderate to high viral loads and developed disease, whereas the remaining animals efficiently controlled SIV replication and showed a more attenuated clinical course of infection. Sequence analysis revealed that the deletion in nef was not repaired in any of these animals. However, some changes that slightly enhanced the ability of Nef to downmodulate CD4 and moderately increased Nef-mediated enhancement of viral replication and infectivity in vitro were observed in macaques developing high viral loads. Our results imply that both the Nef functions that were disrupted by the delta64-67 mutation and the activities that remained intact contribute to viral pathogenicity.     

Compensation of large motion sensor displacements during long recordings of limb movements

In motion capture applications using electromagnetic tracking systems the process of anatomical calibration associates the technical frames of sensors attached to the skin with the human anatomy. Joint centers and axes are determined relative to these frames. A change of orientation of the sensor relative to the skin renders this calibration faulty. This sensitivity regarding sensor displacement can turn out to be a serious problem with movement recordings of several minutes duration. We propose the “dislocation distance” as a novel method to quantify sensor displacement and to detect gradual and sudden changes of sensor orientation. Furthermore a method to define a so called fixed technical frame is proposed as a robust reference frame which can adapt to a new sensor orientation on the skin. The proposed methods are applied to quantify the effects of sensor displacement of 120 upper and lower limb movement recordings of newborns revealing the need for a method to compensate for sensor displacement. The reliability of the fixed technical frame is quantified and it is shown that trend and dispersion of the dislocation distance can be significantly reduced. A working example illustrates the consequences of sensor displacement on derived angle time series and how they are avoided using the fixed technical frame.     

Antibiosis against the green peach aphid requires the Arabidopsis thaliana MYZUS PERSICAE-INDUCED LIPASE1 gene

The green peach aphid (GPA) (Myzus persicae Sülzer) is an important sap-sucking pest of a large variety of plants, including Arabidopsis thaliana. Arabidopsis utilizes a combination of defenses that deter insects from settling on the plant, limit insect feeding and curtail insect reproduction. We demonstrate that the previously uncharacterized Arabidopsis MPL1 (MYZUS PERSICAE-INDUCED LIPASE1) gene has an important role in defense against the GPA. MPL1 expression was rapidly induced to high level in GPA-infested plants. Furthermore, the GPA population was larger on the mpl1 mutant than the wild-type (WT) plant. In contrast, constitutive over-expression of MPL1 from the Cauliflower mosaic virus 35S gene promoter curtailed the size of the GPA population. Insect settling and feeding behavior were unaffected on the mpl1 mutant. However, compared with the phloem-sap enriched petiole exudate from the WT plant, mpl1 petiole exudate was deficient in an activity that restricts insect reproduction on a synthetic diet. Furthermore, MPL1 was required for the heightened accumulation of an antibiotic activity in petiole exudate of the Arabidopsis ssi2 mutant, which exhibits enhanced resistance to GPA. These results indicate that MPL1 has an essential function in antibiosis against GPA. The MPL1 protein exhibits homology to lipases and recombinant MPL1 has lipase activity, thus suggesting that a MPL1-dependent lipid, or a product thereof, has an important role in antibiosis against GPA.     

The landscape of mitochondrial DNA variation in human colorectal cancer on the background of phylogenetic knowledge

Recently, an increasing number of studies indicate that mutations in mitochondrial genome may contribute to cancer development or metastasis. Hence, it is important to determine whether the mitochondrial DNA might be a good, clinically applicable marker of cancer. This review describes hereditary as well as somatic mutations reported in mitochondrial DNA of colorectal cancer cells. We showed here that the entire mitochondrial genome mutational spectra are different in colorectal cancer and non-tumor cells. We also placed the described mutations on the phylogenetic context, which highlighted the recurrent problem of data quality. Therefore, the most important rules for adequately assessing the quality of mitochondrial DNA sequence analysis in cancer have been summarized. As follows from this review, neither the reliable spectrum of mtDNA somatic mutations nor the association between hereditary mutations and colorectal cancer risk have been resolved. This indicates that only high resolution studies on mtDNA variability, followed by a proper data interpretation employing phylogenetic knowledge may finally verify the utility of mtDNA sequence (if any) in clinical practice.     

Structure of the O-polysaccharide of Proteus serogroup O34 containing 2-acetamido-2-deoxy-alpha-D-galactosyl phosphate

On mild acid degradation of the lipopolysaccharide of Proteus vulgaris O34, strain CCUG 4669, the O-polysaccharide was cleaved at a glycosyl-phosphate linkage that is present in the main chain. The resultant phosphorylated oligosaccharides and an alkali-treated lipopolysaccharide were studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, and the following structure of the branched tetrasaccharide phosphate repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text]The O-polysaccharide of Proteus mirabilis strain TG 276 was found to have the same structure and, based on the structural and serological data, this strain was proposed to be classified into the same Proteus serogroup O34.     

Aminoimidazole Carboxamide Ribonucleotide (AICAR) Inhibits the Growth of Retinoblastoma In Vivo by Decreasing Angiogenesis and Inducing Apoptosis

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an analog of AMP is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. Recently, we showed that AICAR-induced AMPK activation inhibits the growth of retinoblastoma cells in vitro by decreasing cyclins and by inducing apoptosis and S-phase arrest. In this study, we investigated the effects of AMPK activator AICAR on the growth of retinoblastoma in vivo. Intraperitoneal injection of AICAR resulted in 48% growth inhibition of Y79 retinoblastoma cell tumors in mice. Tumors isolated from mice treated with AICAR had decreased expression of Ki67 and increased apoptotic cells (TUNEL positive) compared with the control. In addition, AICAR treatment suppressed significantly tumor vessel density and macrophage infiltration. We also showed that AICAR administration resulted in AMPK activation and mTOR pathway inhibition. Paradoxically observed down-regulation of p21, which indicates that p21 may have a novel function of an oncogene in retinoblastoma tumor. Our results indicate that AICAR treatment inhibited the growth of retinoblastoma tumor in vivo via AMPK/mTORC1 pathway and by apoptogenic, anti-proliferative, anti-angiogenesis mechanism. AICAR is a promising novel non-chemotherapeutic drug that may be effective as an adjuvant in treating Retinoblastoma.     

Deprotection of centromeric cohesin at meiosis II requires APC/C activity but not kinetochore tension

Genome haploidization involves sequential loss of cohesin from chromosome arms and centromeres during two meiotic divisions. At centromeres, cohesin''s Rec8 subunit is protected from separase cleavage at meiosis I and then deprotected to allow its cleavage at meiosis II. Protection of centromeric cohesin by shugoshin‐PP2A seems evolutionarily conserved. However, deprotection has been proposed to rely on spindle forces separating the Rec8 protector from cohesin at metaphase II in mammalian oocytes and on APC/C‐dependent destruction of the protector at anaphase II in yeast. Here, we have activated APC/C in the absence of sister kinetochore biorientation at meiosis II in yeast and mouse oocytes, and find that bipolar spindle forces are dispensable for sister centromere separation in both systems. Furthermore, we show that at least in yeast, protection of Rec8 by shugoshin and inhibition of separase by securin are both required for the stability of centromeric cohesin at metaphase II. Our data imply that related mechanisms preserve the integrity of dyad chromosomes during the short metaphase II of yeast and the prolonged metaphase II arrest of mammalian oocytes.     

Effects of melatonin on luteinizing hormone secretion in anestrous ewes following dopamine and opiate receptor blockade

In the present investigation we have examined the ability of melatonin to modify the pulsatile LH secretion induced by treatment with a DA antagonist (sulpiride, SULP) or opioid antagonist (naloxone, NAL) in intact mid-anestrous ewes. The experimental design comprised the following treatments-in experiment 1: (1) intracerebroventricular (i.c.v.) infusion of vehicle (control I); (2) pretreatment with SULP (0.6 mg/kg subcutaneously) and then i.c.v. infusion of vehicle (SULP + veh); (3) pretreatment with SULP and then i.c.v. infusion of melatonin (SULP + MLT, 100 microg per 100 microl/h, total 400 microg). In experiment 2: (4) i.c.v. infusion of vehicle (control II); (5) i.c.v. infusion of NAL (NAL-alone, 100 microg per 100 microl/h, total 300 microg); (6) i.c.v. infusion of NAL in combination with MLT (NAL + MLT, 100 microg + 100 microg per 100 microl/h). All infusions were performed during the afternoon hours. Pretreatment with SULP induced a significant (P < 0.01) increase in LH pulse frequency, but not in mean LH concentration, compared with control I. In SULP + MLT-treated animals, the LH concentration was significantly (P < 0.01) higher during MLT infusion, but due to highly increased LH secretion in only one ewe. The significant changes in the SULP + MLT group occurred in LH pulse frequency. A few LH pulses were noted after melatonin administration compared with the number during the infusion (P < 0.05) and after vehicle infusion in the SULP + MLT group (P < 0.05). The i.c.v. infusion of NAL evoked a significant increase in the mean LH concentration (P < 0.001) and amplitude of LH pulses (P < 0.01) compared with these before the infusion. The enhanced secretion of LH was also maintained after i.c.v. infusion of NAL (P < 0.01) with a concomitant decrease in LH pulse frequency (P < 0.05). In NAL + MLT-treated ewes, mean plasma LH concentrations increased significantly during and after the infusion compared with that noted before ( P < 0.001). No difference in the amplitude of LH pulses was found in the NAL + MLT group, but this parameter was significantly higher in ewes during infusion of both drugs than during infusion of the vehicle (P < 0.01). The LH pulse frequency differed significantly (p < 0.05), increasing slightly during NAL + MLT administration and decreasing after the infusion. In conclusion, these results demonstrate that: (1) in mid-anestrous ewes EOPs, besides DA, are involved in the inhibition of the GnRH/LH axis; (2) brief administration of melatonin in long-photoperiod-inhibited ewes suppresses LH pulse frequency after the elimination of the inhibitory DA input, but seems to not affect LH release following opiate receptor blockade.