A reinvestigation of residues 64–68 and 175 in papain. Evidence that residues 64 and 175 are asparagine

The tryptophan-containing peptides were isolated from the chymotryptic digest of S-carboxymethylated papain. Residue 175, which is strongly hydrogen-bonded to the active-site histidine residue in the tertiary structure of papain, is asparagine, confirming the work of Kimmel, Rogers & Smith (1965). Its function is probably to maintain the orientation and tautomeric state of the imidazole ring of histidine-159. The amino acid sequence predicted from the electron-density map of papain for residues 64-68 was confirmed, but residue 64 is asparagine, not aspartic acid. This residue, which is about 10 A from the thiol group of the active-site cysteine-25, cannot therefore be a site of electrostatic attraction for substrates of basic amino acids.     

Influence of Process and Formulation Parameters on Dissolution and Stability Characteristics of Kollidon? VA 64 Hot-Melt Extrudates

The objective of the present study was to investigate the effects of processing variables and formulation factors on the characteristics of hot-melt extrudates containing a copolymer (Kollidon® VA 64). Nifedipine was used as a model drug in all of the extrudates. Differential scanning calorimetry (DSC) was utilized on the physical mixtures and melts of varying drug–polymer concentrations to study their miscibility. The drug–polymer binary mixtures were studied for powder flow, drug release, and physical and chemical stabilities. The effects of moisture absorption on the content uniformity of the extrudates were also studied. Processing the materials at lower barrel temperatures (115–135°C) and higher screw speeds (50–100 rpm) exhibited higher post-processing drug content (~99–100%). DSC and X-ray diffraction studies confirmed that melt extrusion of drug–polymer mixtures led to the formation of solid dispersions. Interestingly, the extrusion process also enhanced the powder flow characteristics, which occurred irrespective of the drug load (up to 40% w/w). Moreover, the content uniformity of the extrudates, unlike the physical mixtures, was not sensitive to the amount of moisture absorbed. The extrusion conditions did not influence drug release from the extrudates; however, release was greatly affected by the drug loading. Additionally, the drug release from the physical mixture of nifedipine–Kollidon® VA 64 was significantly different when compared to the corresponding extrudates (f₂ = 36.70). The extrudates exhibited both physical and chemical stabilities throughout the period of study. Overall, hot-melt extrusion technology in combination with Kollidon® VA 64 produced extrudates capable of higher drug loading, with enhanced flow characteristics, and excellent stability.KEY WORDS: extrusion, Kollidon® VA 64, moisture absorption, nifedipine, solid dispersion     

Deubiquitinase USP47/UBP64E Regulates β-Catenin Ubiquitination and Degradation and Plays a Positive Role in Wnt Signaling

Wnt signaling plays important roles in development and tumorigenesis. A central question about the Wnt pathway is the regulation of β-catenin. Phosphorylation of β-catenin by CK1α and GSK3 promotes β-catenin binding to β-TrCP, leading to β-catenin degradation through the proteasome. The phosphorylation and ubiquitination of β-catenin have been well characterized; however, it is unknown whether and how a deubiquitinase is involved. In this study, by screening RNA interference (RNAi) libraries, we identified USP47 as a deubiquitinase that prevents β-catenin ubiquitination. Inactivation of USP47 by RNAi increased β-catenin ubiquitination, attenuated Wnt signaling, and repressed cancer cell growth. Furthermore, USP47 deubiquitinates itself, whereas β-TrCP promotes USP47 ubiquitination through interaction with an atypical motif in USP47. Finally, in vivo studies in the Drosophila wing suggest that UBP64E, the USP47 counterpart in Drosophila, is required for Armadillo stabilization and plays a positive role in regulating Wnt target gene expression.     

Prevalence and Incidence of Memory Complaints in Employed Compared to Non-Employed Aged 55–64 Years and the Role of Employment Characteristics

Objectives

To examine the association of employment status and characteristics with prevalent and incident memory complaints (MC) in 55–64-year-olds.

Methods

Subjects were participants of the Longitudinal Aging Study Amsterdam (LASA). Respondents with baseline data were selected to examine the association of employment status (n = 1525) and employment characteristics (n = 1071) with prevalent MC (i.e., MC at baseline). Respondents without MC at baseline were selected to examine the association of employment (n = 526) and employment characteristics (n = 379; working hours, job prestige, job level, psychological job demands, iso-strain) with incident MC (i.e., no MC at baseline and MC at three-year follow-up). Associations were adjusted for relevant covariates (demographics, memory performance, physical health, mental health, personality traits). Logistic regression was applied. Data were weighed according to gender and age of the Dutch population.

Results

At baseline 20.5% reported MC. At three-year follow-up, 15.4% had incident MC. No associations were found between employment status and MC. Adjusted analysis revealed that individuals with high occupational cognitive demands were more likely to have prevalent MC.

Conclusions

Middle-aged workers are equally as likely to experience MC as non-working age-peers. Among workers, those with cognitively demanding work were more likely to experience MC, independent of memory performance. Memory decline due to ageing may be noticed sooner in 55–64-year-olds performing cognitively demanding work.     

Skin and Soft Tissue Infections and Associated Complications among Commercially Insured Patients Aged 0–64 Years with and without Diabetes in the U.S.

Introduction

Skin and soft tissue infections (SSTIs) are common infections occurring in ambulatory and inpatient settings. The extent of complications associated with these infections by diabetes status is not well established.

Methods

Using a very large repository database, we examined medical and pharmacy claims of individuals aged 0–64 between 2005 and 2010 enrolled in U.S. health plans. Diabetes, SSTIs, and SSTI-associated complications were identified by ICD-9 codes. SSTIs were stratified by clinical category and setting of initial diagnosis.

Results

We identified 2,227,401 SSTI episodes, 10% of which occurred in diabetic individuals. Most SSTIs were initially diagnosed in ambulatory settings independent from diabetes status. Abscess/cellulitis was the more common SSTI group in diabetic and non-diabetic individuals (66% and 59%, respectively). There were differences in the frequencies of SSTI categories between diabetic and non-diabetic individuals (p<0.01). Among SSTIs diagnosed in ambulatory settings, the SSTI-associated complication rate was over five times higher in people with diabetes than in people without diabetes (4.9% vs. 0.8%, p<0.01) and SSTI-associated hospitalizations were 4.9% and 1.1% in patients with and without diabetes, respectively. Among SSTIs diagnosed in the inpatient setting, bacteremia/endocarditis/septicemia/sepsis was the most common associated complication occurring in 25% and 16% of SSTIs in patients with and without diabetes, respectively (p<0.01).

Conclusions

Among persons with SSTIs, we found SSTI-associated complications were five times higher and SSTI-associated hospitalizations were four times higher, in patients with diabetes compared to those without diabetes. SSTI prevention efforts in individuals with diabetes may have significant impact on morbidity and healthcare resource utilization.     

The β3-adrenergic receptor in the obesity and diabetes prone rhesus monkey is very similar to human and contains arginine at codon 64

The β₃-adrenergic receptor (ADRβ₃) is a seven-membrane spanning, G-protein linked receptor expressed in brown adipose tissue in rodents, and visceral adipose tissue in humans. Stimulation of the receptor by norepinephrine leads to lipolysis and thermogenesis. In rodent models of obesity and diabetes, administration of β₃-agonists results in weight loss and improved glucose tolerance. Studies indicate that the pharmacological properties of the ADRβ₃ differ markedly between rodents and humans, making generalizations of rodent studies to humans difficult. We hypothesized that the obesity and diabetes prone rhesus monkey (Macaca mulatta) would provide an excellent animal model to study the role of the ADRβ₃ in the development of obesity and diabetes as well as for assessment of the therapeutic efficacy of β₃-agonists. We sequenced the entire coding region of the rhesus ADRβ₃ gene. Like humans, the rhesus ADRβ₃ has two exons. There is 89% amino acid (aa) identity between human and rhesus compared to 82% aa identity between human and mouse. A single base deletion results in divergence of the intracellular carboxy terminus accounting for 26 of the 45 aa changes and 10 additional aa. Of the 15 rhesus monkeys studied, all were homozygous for Arg⁶⁴. In humans, Arg⁶⁴ (rather than Trp) is associated with increased body mass index, insulin resistance, and an earlier onset of type II diabetes mellitus. We conclude that the rhesus ADRβ₃ is more similar to the human ADRβ₃ than to the rodent ADRβ₃ suggesting that this primate model may be more appropriate for physiologic and therapeutic studies of the ADRβ₃ axis, and that Arg⁶⁴ may influence susceptibility in this species to obesity, insulin resistance, and type II diabetes.     

Bone “Awls” and Utilized Antler Tines From Arnold Research Cave, 23Cy64, Missouri

Abstract

Bone awls and utilized antler tines from Arnold Research Cave in central Missouri were microscopically examined to determine the type, location, and orientation of wear on them. It is assumed that similar wear patterns indicate similar use and, conversely, dissimilar wear patterns indicate divergent use. The awls were first divided into morphological types as proposed by Kidder (1932). The wear patterns on the awls were then compared both on members within a single morphological type and on members in different morphological types. It is concluded: (1) awls within a single morphological type may exhibit evidence of divergent use, and (2) members of different morphological types may exhibit evidence of similar use. Since the antler tines are morphologically similar, they are classified on the basis of wear patterns. Four different patterns of striations and the presence of abrasion on the distal end indicates there are five classes of antler tine tools in the sample     

Cervical Screening at Age 50–64 Years and the Risk of Cervical Cancer at Age 65 Years and Older: Population-Based Case Control Study

Background

There is little consensus, and minimal evidence, regarding the age at which to stop cervical screening. We studied the association between screening at age 50–64 y and cervical cancer at age 65–83 y.

Methods and Findings

Cases were women (n = 1,341) diagnosed with cervical cancer at age 65–83 y between 1 April 2007 and 31 March 2012 in England and Wales; age-matched controls (n = 2,646) were randomly selected from population registers. Screening details from 1988 onwards were extracted from national databases. We calculated the odds ratios (OR) for different screening histories and subsequent cervical cancer. Women with adequate negative screening at age 65 y (288 cases, 1,395 controls) were at lowest risk of cervical cancer (20-y risk: 8 cancers per 10,000 women) compared with those (532 cases, 429 controls) not screened at age 50–64 y (20-y risk: 49 cancers per 10,000 women, with OR = 0.16, 95% CI 0.13–0.19). ORs depended on the age mix of women because of the weakening association with time since last screen: OR = 0.11, 95% CI 0.08–0.14 at 2.5 to 7.5 y since last screen; OR = 0.27, 95% CI 0.20–0.36 at 12.5 to 17.5 y since last screen. Screening at least every 5.5 y between the ages 50 and 64 y was associated with a 75% lower risk of cervical cancer between the ages 65 and 79 y (OR = 0.25, 95% CI 0.21–0.30), and the attributable risk was such that in the absence of screening, cervical cancer rates in women aged 65+ would have been 2.4 (95% CI 2.1–2.7) times higher. In women aged 80–83 y the association was weaker (OR = 0.49, 95% CI 0.28–0.83) than in those aged 65–69 y (OR = 0.12, 95% CI 0.09–0.17). This study was limited by an absence of data on confounding factors; additionally, findings based on cytology may not generalise to human papillomavirus testing.

Conclusions

Women with adequate negative screening at age 50–64 y had one-sixth of the risk of cervical cancer at age 65–83 y compared with women who were not screened. Stopping screening between ages 60 and 69 y in women with adequate negative screening seems sensible, but further screening may be justifiable as life expectancy increases. Please see later in the article for the Editors'' Summary     

CstF-64 and 3′-UTR cis-element determine Star-PAP specificity for target mRNA selection by excluding PAPα

Almost all eukaryotic mRNAs have a poly (A) tail at the 3′-end. Canonical PAPs (PAPα/γ) polyadenylate nuclear pre-mRNAs. The recent identification of the non-canonical Star-PAP revealed specificity of nuclear PAPs for pre-mRNAs, yet the mechanism how Star-PAP selects mRNA targets is still elusive. Moreover, how Star-PAP target mRNAs having canonical AAUAAA signal are not regulated by PAPα is unclear. We investigate specificity mechanisms of Star-PAP that selects pre-mRNA targets for polyadenylation. Star-PAP assembles distinct 3′-end processing complex and controls pre-mRNAs independent of PAPα. We identified a Star-PAP recognition nucleotide motif and showed that suboptimal DSE on Star-PAP target pre-mRNA 3′-UTRs inhibit CstF-64 binding, thus preventing PAPα recruitment onto it. Altering 3′-UTR cis-elements on a Star-PAP target pre-mRNA can switch the regulatory PAP from Star-PAP to PAPα. Our results suggest a mechanism of poly (A) site selection that has potential implication on the regulation of alternative polyadenylation.     

64DP与人染色质的结合反应及细胞核内64DP的检测

现有结果表明64 DP(DNA结合蛋白)可能是一种急性期反应蛋白质。正常人每百毫升血清中含有约50mg的64 DP。目前,关于64 DP的生物学功能国内外尚无研究和报道。这方面的研究对阐明肿瘤及其它病理条件下血清64 DP含量增高的机制和意义有可能提供线索。 已知64 DP在体外具有和DNA结合的特性.它在体内的生理作用是否也和DNA有关呢?本实验首先分离纯化了人肝细胞染色质,用同位素标记的64 DP与染色质进行的结合实验及结合抑制实验表明64 DP能特异地与人染色质结合。进一步用~(125)I标记的兔抗64 DP抗体对人染色质作了固相放射免疫测定,结果定性地显示出染色质内可能含有64 DP。用兔抗64 DP抗体作为第一抗体,利用PAP免疫组织化学染色研究了16例正常肝组织切片,发现大多数切片(80％)中部分肝细胞核内有明显64DP显色颗粒。以上诸实验结果均提示细胞核内有64 DP存在,表明64 DP的生物学功能可能与它的DNA结合特性有关。     

中医中药救治蝮蛇咬伤64例

自１９９０年以来,笔者以中医中药救治蝮蛇咬伤６４例,皆获成功,现小结如下。１临床资料１．１一般资料６４例病人中,男性４２例,女性２２例,年龄最大者６４岁,最小者６岁,平均３７岁,以青壮年为多。１．２临床分型及疗效评定根据国家中医药管理局医政司分型标准...     

64例肺部感染病原菌调查

我们用经纤维支气管镜双防污毛刷从６４例肺部感染病灶中刷取标本,同时进行需、厌菌培养,结果５８例阳性,共培养出需氧菌３３株,厌氧菌６２株,厌氧菌的感染率明显高于需氧菌的感染率;对６２株厌氧菌用７种常用抗菌素做药敏试验,发现常用治疗厌氧菌的青霉和灭滴灵的敏感率仅为６９％和７２％。     

面癣64例临床分析

目的探讨面癣的临床特点,误诊分析,诊断治疗。方法选择有病史记录的面癣患者作一回顾性分析,所有患者作真菌镜检,部分患者加真菌培养;根据皮损的表现,给予单纯外用抗真菌药膏或外用加口服联合治疗。结果 64例中初次诊断面癣25例,外院或本院第1次误诊,复诊诊断面癣39例,64例中难辨认癣15例。结论面癣易误诊,对于疑似面癣的患者一定要作真菌镜检或培养以明确诊断,并给予规范化治疗。     

Structure, Mechanistic Action, and Essential Residues of a GH-64 Enzyme, Laminaripentaose-producing ��-1,3-Glucanase

Laminaripentaose-producing β-1,3-glucanase (LPHase), a member of glycoside hydrolase family 64, cleaves a long-chain polysaccharide β-1,3-glucan into specific pentasaccharide oligomers. The crystal structure of LPHase from Streptomyces matensis DIC-108 was solved to 1.62 Å resolution using multiple-wavelength anomalous dispersion methods. The LPHase structure reveals a novel crescent-like fold; it consists of a barrel domain and a mixed (α/β) domain, forming a wide-open groove between the two domains. The liganded crystal structure was also solved to 1.80 Å, showing limited conformational changes. Within the wide groove, a laminaritetraose molecule is found to sit in an electronegatively charged central region and is proximal to several conserved residues including two carboxylates (Glu¹⁵⁴ and Asp¹⁷⁰) and four other sugar-binding residues (Thr¹⁵⁶, Asn¹⁵⁸, Trp¹⁶³, and Thr¹⁶⁷). Molecular modeling using a laminarihexaose as a substrate suggests roles for Glu¹⁵⁴ and Asp¹⁷⁰ as acid and base catalysts, respectively, whereas the side chains of Thr¹⁵⁶, Asn¹⁵⁸, and Trp¹⁶³ demarcate subsite +5. Site-directed mutagenesis of Glu¹⁵⁴ and Asp¹⁷⁰ confirms that both carboxylates are essential for catalysis. Together, our results suggest that LPHase uses a direct displacement mechanism involving Glu¹⁵⁴ and Asp¹⁷⁰ to cleave a β-1,3-glucan into specific α-pentasaccharide oligomers.Glycoside hydrolases (GHs,³ EC 3.2.1.x) hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and non-carbohydrate moiety (1). These enzymes play diverse roles in nature; they breakdown cellulose into smaller carbohydrates (i.e. during biomass degradation by cellulases), they function during pathogenesis such as the activity of influenza virus neuraminidase (2), and they are engaged in normal cellular metabolic processes that involve the formation and breakage of glycosidic bonds along with glycosyl transferase (3). GHs can be classified as exo- or endo-type of glycoside hydrolases that catalyze the hydrolysis of the glycosidic bond from the end or at the middle, respectively, of a polysaccharide chain. GHs can also be classified as the inverting or the retaining enzymes with respect to their distinct stereochemical mechanisms during catalysis (3). Sequence-based classification of GHs into various families has been proposed by Henrissat et al. (4, 5). Additionally, numerous structures of GHs have revealed details of their catalytic mechanisms as well as the basis for their diverse substrate specificity (6). Based on sequence comparisons and structural analyses, the carbohydrate-active enzymes data base (CAZy) provides continuously updated information on the GH families (7).Two key residues among GHs, generally found as carboxylates, are involved in the hydrolysis of the glycosidic bond: a proton donor and a nucleophile/base (3). In either the retaining or the inverting enzymes, the position of the proton donor is found within hydrogen-bonding distance of the glycosidic oxygen. Active sites that consist of the key residues have been classified into three topologies by Davies and Henrissat (1): (i) a pocket or a crater that preferentially recognizes a saccharide molecule with a non-reducing end, presenting the exo-type hydrolysis, (ii) a cleft or groove that accommodates a large substrate for endo-type cleavage, and (iii) a tunnel that enables a polysaccharide chain to be threaded through for efficient endo-hydrolase processivity.Among the current 114 families of GHs, β-1,3-glucanases, namely exo-β-1,3-glucanases, (E.C. 3.2.1.58) and endo-β-1,3-glucanases (E.C 3.2.1.39) that degrade β-1,3-glucans into smaller biological response modifiers (8) are found in seven families. GH-3 and GH-5 are found to be exo-type; GH-16 and GH-17 are in the endo-type category. Both endo- and exo-type enzymes are found in GH-55. However, GH-64 and GH-81 remain unclear (7). Three-dimensional structures of members from GH-3, GH-5, GH-16, GH-17, and GH-55 have been solved, providing detailed structure-activity information (9–13). Members of the GH-5 and GH-17 families contain a (β/α)₈ architecture, whereas GH-16 family members fold as a β-jelly roll. Barley β-d-glucan exohydrolase, a member of the GH-3 family has an N-terminal TIM-barrel domain and a C-terminal 6-stranded β-sandwich. Notably, glucanases from these families are retaining enzymes. The newly resolved structure of Lam55A, an inverting enzyme in GH-55 family (13), has two β-helical domains, separated by a long linker region, that form a ribcage-like structure. To date, no structure has been reported for GH-64 and GH-81, and thus detailed information on the mode of catalysis is lacking.Laminaripentaose-producing β-1,3-glucanase (LPHase) cleaves a long-chain polysaccharide, β-1,3-glucan, including laminarin, into a specific pentasaccharide oligomer “laminaripentaose” (14, 15). Of interest, β-1,3-glucans such as laminarin, which constitute the cell walls of plants and fungi have interesting biological roles in immune modulation (8, 16, 17). Biochemical characterization of LPHase from Streptomyces matensis DIC-108 showed that the enzyme belongs to the GH-64 family and is an inverting enzyme (14, 15). This enzyme is unique that it releases mainly laminaripentaose as the end product, as compared with that exo-type enzymes produce much smaller sugars (monosaccharides or disaccharides) (18–20) while endo-type enzymes yield heterogeneous forms of oligosaccharides. This atypical product specificity, to our knowledge, has not been reported for other glucanases. Here we report the three-dimensional structures for the apo and complex LPHase of S. matensis. Structural analysis, modeling, and mutagenesis results revealed a novel crescent-fold structure containing Glu¹⁵⁴ and Asp¹⁷⁰ involved in the cleavage of a long-chain oligosaccharide from the reducing end.     

Mapping the genetic and tissular diversity of 64 phenolic compounds in Citrus species using a UPLC–MS approach

Background and Aims Phenolic compounds contribute to food quality and have potential health benefits. Consequently, they are an important target of selection for Citrus species. Numerous studies on this subject have revealed new molecules, potential biosynthetic pathways and linkage between species. Although polyphenol profiles are correlated with gene expression, which is responsive to developmental and environmental cues, these factors are not monitored in most studies. A better understanding of the biosynthetic pathway and its regulation requires more information about environmental conditions, tissue specificity and connections between competing sub-pathways. This study proposes a rapid method, from sampling to analysis, that allows the quantitation of multiclass phenolic compounds across contrasting tissues and cultivars.Methods Leaves and fruits of 11 cultivated citrus of commercial interest were collected from adult trees grown in an experimental orchard. Sixty-four phenolic compounds were simultaneously quantified by ultra-high-performance liquid chromatography coupled with mass spectrometry.Key Results Combining data from vegetative tissues with data from fruit tissues improved cultivar classification based on polyphenols. The analysis of metabolite distribution highlighted the massive accumulation of specific phenolic compounds in leaves and the external part of the fruit pericarp, which reflects their involvement in plant defence. The overview of the biosynthetic pathway obtained confirmed some regulatory steps, for example those catalysed by rhamnosyltransferases. The results suggest that three other steps are responsible for the different metabolite profiles in ‘Clementine’ and ‘Star Ruby’ grapefruit.Conclusions The method described provides a high-throughput method to study the distribution of phenolic compounds across contrasting tissues and cultivars in Citrus, and offers the opportunity to investigate their regulation and physiological roles. The method was validated in four different tissues and allowed the identification and quantitation of 64 phenolic compounds in 20 min, which represents an improvement over existing methods of analysing multiclass polyphenols.     

CstF-64 supports pluripotency and regulates cell cycle progression in embryonic stem cells through histone 3′ end processing

Embryonic stem cells (ESCs) exhibit a unique cell cycle with a shortened G₁ phase that supports their pluripotency, while apparently buffering them against pro-differentiation stimuli. In ESCs, expression of replication-dependent histones is a main component of this abbreviated G₁ phase, although the details of this mechanism are not well understood. Similarly, the role of 3′ end processing in regulation of ESC pluripotency and cell cycle is poorly understood. To better understand these processes, we examined mouse ESCs that lack the 3′ end-processing factor CstF-64. These ESCs display slower growth, loss of pluripotency and a lengthened G₁ phase, correlating with increased polyadenylation of histone mRNAs. Interestingly, these ESCs also express the τCstF-64 paralog of CstF-64. However, τCstF-64 only partially compensates for lost CstF-64 function, despite being recruited to the histone mRNA 3′ end-processing complex. Reduction of τCstF-64 in CstF-64-deficient ESCs results in even greater levels of histone mRNA polyadenylation, suggesting that both CstF-64 and τCstF-64 function to inhibit polyadenylation of histone mRNAs. These results suggest that CstF-64 plays a key role in modulating the cell cycle in ESCs while simultaneously controlling histone mRNA 3′ end processing.