Determination of the CCR5∆32 frequency in Emiratis and Tunisians and the screening of the CCR5 gene for novel alleles in Emiratis

Background

The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5?32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5?32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians).

Methodology

The prevalence of CCR5 gene variants including CCR5?32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5?32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing.

Results

Among Emiratis, the allele frequency of the CCR5?32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5?32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans.

Conclusion

We conclude that the allele frequency of the most critical CCR5 polymorphism (?32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.     

Comparison of the mutagenic activity of 5-hydroxymethyldeoxyuridine with 5-substituted 2′-deoxyuridine analogs in the Ames Salmonella/microsome test

4 antiviral drugs 5-hydroxymethyldeoxyuridine (HMUdR), 5-trifluorothymidine (F₃TdR), 5-methoxymethyldeoxyuridine (MMUdR) and 5-ethyldeoxyuridine (EtUdR) have been evaluated for mutagenic activity in the Ames Salmonella/microsome test. The antimetabolites F₃TdR and HMUdR were mutagenic in a dose-dependent manner in strain TA100. F₃TdR also was mutagenic in strain TA1535. Rat-liver post-mitochondrial supernatant (S9) was not required for mutagenicity.     

Kinesin-5 mitotic motors: Is loop5 the on/off switch?

The microtubule-based mitotic spindle orchestrates chromosome segregation, facilitated by many microtubule-associated proteins. Kinesin-5 proteins are important components of the cell division machinery, and are involved in generation of mitotic spindle bipolarity by cross-linking microtubules. Kinesin-5s are members of the ATP- and MT-dependent kinesin superfamily of molecular motors. Human kinesin-5 is also a target for small molecule inhibitors that specifically bind to the motor domain and are currently in cancer clinical trials. The regulatory mechanisms that control kinesin-5 activity during mitosis and the effects of regulation on the kinesin-5-microtubule interaction remain unknown. Recent work has focused on a kinesin-5 specific region within the motor domain, loop5, as a potential regulatory switch. Kinesin-5-specific small molecule inhibitors bind beneath loop5, loop5 is rearranged when kinesin-5 binds to microtubules and residues adjacent to loop5 are subject to cell cycle-dependent tyrosine phosphorylation which could affect its conformation. It will be essential to consider these studies, which shed light on potential kinesin-5 regulatory mechanisms, as part of efforts to develop clinically effective kinesin-5 inhibitors.     

Structural insight into the binding complex: β-arrestin/CCR5 complex

The chemokine receptor 5 (CCR5) belongs to the superfamily of serpentine G protein-coupled receptors (GPCRs). The DRY motif (Asp, Arg, Tyr) of the intracellular loop 2 (ICL2), which is highly conserved in the GPCRs has been shown to be essential for the stability of folding of CCR5 and the interaction with β-arrestin. But the molecular mechanism by which it recognizes and interacts with β-arrestin has not been elucidated. In the present study, we described the active state of the β-arrestin structure using normal mode analysis and characterized the binding cleft of CCR5-ICL2 with β-arrestin using SABRE© docking tool and molecular dynamics simulation. Based on our computational results, we proposed a mode of binding between the ICL2 loop of CCR5 and β-arrestin structure, and modeled the energetically stable β-arrestin/CCR5 complex. In view of CCR5’s importance as a therapeutic target for the treatment of HIV, this observation provides novel insight into the β-arrestin/CCR5 pathway. As a result, the current computational study of the detailed β-arrestin/CCR5 binding complex could provide the rationale for the development of next generation of HIV peptide inhibitors as therapeutic agents.     

Codisterol and other Δ5-sterols in the seeds of Cucurbita maxima

A substantial amount (ca 18%) of the sterol found in the seeds of Cucurbita maxima had a Δ-bond and consisted of seven components. They were identified as 25(27)-dehydroporiferasterol, clerosterol, isofucosterol, stigmasterol, sitosterol, campesterol and codisterol. The C-24 configuration of each of the sterols was unequivocally established by a ¹H NMR spectral comparison with authentic standards. This is the first time codisterol has been found in a higher plant and also the first time the structures and configurations of the Δ⁵-sterols from a Cucurbitaceae species have been clearly characterized.     

Synthesis and Functional Identification of Oligopeptides Derived from the α3/5-Conotoxins

Conotoxins are small peptides comprising of 12–50 residues. The α3/5-conotoxins have the primary sequence motif -CCX3CX5C- and are paralytic, presumably targeted to the muscle nicotinic acetylcholine receptors. Some oligopeptide motifs chosen from the two loops of the α3/5-conotoxins, were synthesized and evaluated for biological activities on the rat nerve-skeletal muscle contractility, muscle cell contraction, electrophysiology and cytotoxicity. Three peptides (HPA, NPA and WNPA) showed high inhibitory effects and low cytotoxicities. They had the similar bioactivities as the anti-wrinkle product SYN-AKE did. The results in the present study indicated that these oligopeptides may be potential ingredients for anti-wrinkle product. However, further studies using in vivo animal models and human clinical trials are necessary before consumer use.     

Localization of 5′-ectonucleotidase/phosphatase activity within the olfactory sensilla of the spiny lobster,Panulirus argus

Summary Previous electrophysiological studies have shown that the olfactory organ (antennule) of the spiny lobster, Panulirus argus, has external chemoreceptors, which are selectively stimulated by adenosine 5-monophosphate (AMP) when present in seawater. Subsequent biochemical investigations revealed that AMP can be rapidly dephosphorylated by 5-ectonucleotidase/phosphatase activity associated with the olfactory sensilla (aesthetascs). In this study the deposition of cerium phosphate was used to examine the ultrastructural distribution of 5-ectonucleotidase/phosphatase activity in aesthetascs. Utilizing AMP as substrate, we found dephosphorylating activity to be associated with the outer membranes of both dendrites and auxiliary cells. Moreover, this activity was specifically localized to a narrow band that approximately corresponds to the transitional zone where dendrites develop cilia and branch extensively to form the outer dendritic segments. A similar distribution of the cerium phosphate reaction product was found when -glycerol phosphate was substituted for AMP. The alkaline-phosphatase inhibitor, levamisole, had no apparent effect on the deposition of reaction product when either AMP or -glycerol phosphate was used as substrate. The ectoenzymatic activity in the transitional zone may be of importance in clearing exogenous chemoexcitatory nucleotides from this region.Abbreviations ADP adenosine 5-diphosphate - AMP adenosine 5-monophosphate - ATP adenosine 5-triphosphate - EM electron microscopy     

Novel cross-strand three-purine stack of the highly conserved 5′-GA/AAG-5′ internal loop at the 3′-end termini of Parvovirus Genomes

We have used two-dimensional nuclear magnetic resonance (2D-NMR), distance geometry (DG) and molecular dynamics / energy minimization (MD/EM) methods to study a 2×3 asymmetric internal loop structure of the highly conserved `5-(GA)/(AAG)-5 bubble' present at the 3-end hairpin of the single-stranded DNA genome of parvoviruses. This motif contains an unpaired adenosine stacked between two bracketed sheared GA pairs. However, the phenomenal cross-strand G-G and A-A stacking in the tandem sheared GA pairs has undergone considerable change. A novel three-purine stacking pattern is observed instead; the inserted A18 base is completely un-stacked from its neighboring G17 and A19 bases, but well stacked with the cross-strand A4 and G3 bases to form a novel A4/A18/G3 stack that is different from the double G/G, A/A or quadruple G/G/G/G stack present in the 5-(GA)/(AG)-5 or 5-(GGA)/(AGG)-5 motifs. Unlike the bulged purine residue that usually causes about 20 degree kink in the helical axis of the parent helix when bracketed by canonical GC or AT base pairs, no significant kink is observed in the present helix containing a bulged-adenine that is bracketed by sheared G A pairs. The phosphodiesters connecting G3-A4 and G17-A18 residues adopt unusual torsional angles close to the trans domain, yet that connecting A18-A19 residues resumes the normal (g <sup>–</sup>) value. The well structured `5-(GAA)/(AG)-5' internal loop in the parvovirus genomes explains its resistance to single-strand specific endonuclease susceptibility.     

Possible association of the allele status of the CS.7/HhaI polymorphism 5′ of the CFTR gene with postnatal female survival

Cystic fibrosis (CF) patients show a high degree of linkage disequilibrium between the CF transmembrane conductance regulator (CFTR) gene and polymorphisms 5′ of that gene. To determine whether the region 5′ of CFTR contains biologically important sequences, the allele frequencies of six CFTR-linked polymorphisms (metH/MspI, XV-2c/TaqI, CS.7/HhaI, KM19/PstI, MP6-d9/MspI, J44/XbaI) were determined in 417 randomly selected elderly individuals (over 75 years of age) from the Czech population. The elderly individuals were considered “escapees” of strong selective pressures that had operated during their lifetime, prior to the introduction of modern health care since 1950. The pooled allele frequencies of the analyzed marker polymorphisms in the elderly did not significantly differ from published data. However, when analyzed by sex, the allele frequencies of markers CS.7/HhaI and KM19/PstI differed significantly (P < 0.05) between elderly females and males. The allele frequencies of the six polymorphisms were then determined in 646 newborns and 345 young adults of reproductive age; these individuals were selected in a similar manner and drawn from the same population. In these control groups, the studied marker polymorphisms exhibited no statistically significant differences between sexes and/or between individuals of the same sex, only between different age groups. A gradual relative increase in the frequency of allele “2” of marker CS.7/HhaI was observed from newborn females to elderly women, the overall difference in allele frequencies of this marker polymorphism between newborn females and elderly women reaching statistical significance (P < 0.05). Interestingly, allele “2” is the major constituent of the extended “B-haplotype”, which is in strong linkage disequilibrium with common CF alleles. Taken together, our data suggest that the region spanning markers CS.7 and KM19 is associated with a genetic factor that influences postnatal female survival, providing a possible mechanism for increasing the frequency of particular mutations in the adjacent CFTR gene. Received: 30 January 1996 / Revised: 16 December 1996     

5′-Nucleotidase activity in the pineal organ of the pike

Summary To date, it is still unknown whether the metabolism of purine nucleotides and nucleosides plays an important role in the pineal organ of lower vertebrates. We have therefore investigated the sites of 5-nucleotidase activity in the pineal organ of the pike (Esox lucius L.). Various ultracytochemical procedures were used. An intense ecto-5-nucleotidase activity was characteristic of the entire plasma membrane of the phototransducers (cone-like and modified photoreceptor elements) and the interstitial cells, with exception of the portions facing the basal lamina of the pericapillary spaces. Additionally, intracellular sites of activity were also visualized in the inner segment and the pedicle of the phototransducers. Most of the intracellular deposits were apparently cytosolic and only few seemed to be associated with the membrane of the clear synaptic vesicles of the pedicle. Phagocytotic cells in the pineal lumen also showed a strong enzymatic activity on the outer surface of their plasmalemma (in ectoposition). This was apparently not the case for the cell types of the tissues surrounding the pineal vesicle. The present study emphasizes the importance of the occurrence and metabolism of purine nucleotides and nucleosides in a photoreceptive pineal organ.     

Wnt5b stimulates adipogenesis by activating PPARγ, and inhibiting the β-catenin dependent Wnt signaling pathway together with Wnt5a

Correct Wnt signaling is required for adipogenesis and alterations occur in Type 2 diabetes mellitus (T2DM). Gene expression studies showed that β-catenin independent Wnt5b was down-regulated in T2DM preadipocytes, while its paralog Wnt5a was unchanged. Our study aimed at defining the expression profile and function of Wnt5a and Wnt5b during adipogenesis by determining their effect on aP2 and PPARγ expression and assessing the level of β-catenin translocation in mouse 3T3-L1 preadipocytes. Additionally, we explored the effect on adipogenic capacity by Wnt5b overexpression in combination with stimulation of the β-catenin dependent or β-catenin independent Wnt signaling. Expression of Wnt5b was, like Wnt5a, down-regulated upon induction of differentiation and both inhibit β-catenin dependent Wnt signaling at the initiation of adipogenesis. Wnt5b additionally appears to be a potent enhancer of adipogenic capacity by stimulation of PPARγ and aP2. Down-regulation of Wnt5b could therefore contribute to decreased adipogenesis observed in T2DM diabetic subjects.     

Schistosoma japonicum: Cloning, expression and characterization of a gene encoding the α5-subunit of the proteasome

The development of an effective vaccine against the schistosome is thought to be the most desirable means to control schistosomiasis, even though there is an effective means of chemotherapy with praziquantel. A full-length cDNA encoding the Schistosoma japonicum proteasome subunit alpha type 5 protein (SjPSMA5) was first isolated from 18-day-schistosomulum cDNAs. The cDNA had an open reading frame (ORF) of 747 bp and encoded 248 amino acids. Real-time quantitative RT-PCR analysis revealed that SjPSMA5 is up-regulated in 18-day and 32-day schistosomes, and the level of expression in male is around fourfold higher than that in female worms at 42 days. The SjPSMA5 was subcloned into pET28a(+) and expressed as inclusion bodies in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjPSMA5 (rSjPSMA5) was immunogenic. After immunization of BALB/c mice with rSjPSMA5, reductions of 23.29% and 35.24% were obtained in the numbers of worms and eggs in the liver, respectively. The levels of specific IgG antibodies and cells were significantly higher (P < 0.01) in the group vaccinated with rSjPSMA5 combined with Seppic 206 adjuvant than in the other groups, as detected by enzyme linked immunosorbent assay (ELISA) and flow cytometry. The study suggested that rSjPSMA5 induced partial immunoprotection against S. japonicum in BALB/c mice, and it could be a potential vaccine candidate against schistosomiasis.     

Studies on the effect of 5α-androstane-3α, 17α-diol in the canine prostate in vivo

Adult beagle dogs, castrated one month previously, were treated with 5α-androstane-3α, 17α-diol (total dose 300 mg) over a period of one month. Examination of the prostate after treatment showed no significant change in size, weight or histological appearance from the castrate dog prostate. Subsequent administration of 5α-androstane-3α, 17β-diol (300 mg) over the same period of time resulted in restoration of the prostate size, weight and histological appearance to that of the normal intact dog prostate. It is concluded that exogenously administered 5α-androstane-3α, 17α-diol does not promote prostatic growth in the castrate beagle dog.     

新型重组AAV5/5载体及包装系统

本研究组建了一种可用于规模化生产的以重组单纯疱疹病毒为辅助病毒的AAV5/5载体包装系统。首先,将5型腺相关病毒 (AAV5) 的rep和cap基因插入I型单纯疱疹病毒 (HSV-1) 基因组非必需基因UL2中,获得重组病毒rHSV1-rep5cap5。其次,构建一种携带AAV5 ITR的通用型载体质粒pAAV5neo,将报告基因EGFP插入pAAV5neo中,得到pAAV5neo-EGFP质粒。将pAAV5neo-EGFP质粒导入BHK-21细胞,用G418选择培养,挑选出表达EGFP并在重组病毒rHSV1-rep5cap5感染下能高效产生rAAV5/5-EGFP的单克隆载体细胞株C020。用rHSV1-rep5cap5感染C020细胞制备rAAV5/5-EGFP,用“氯仿处理-聚乙二醇/氯化钠-氯仿抽提”方法粗纯化rAAV5/5-EGFP。用100 kDa分子量截流超滤方法进一步纯化和浓缩,获得高纯度的rAAV5-EGFP。SDS-PAGE电泳分析可见3条特征性外壳蛋白带。电镜分析显示病毒颗粒以实心颗粒为主。用rAAV5/5-EGFP病毒按1×105 vg/cell感染体外培养的HEK293细胞,可见30％细胞呈现绿色荧光。本研究提出了一种高效AAV5/5载体生产系统和纯化方法,为重组AAV5载体的进一步应用提供了基础。