Optical Properties of Noncontinuous Gold Shell Engineered on Silica Mesosphere

The optical properties of individual noncontinuous shells with different gold coverage are investigated by the single-particle dark field scattering measurements and single-particle surface-enhanced Raman scattering (SERS) measurements at different excitation wavelengths. By controlling the growth of gold seeds, multi-metallic nanogaps/crevices with different optical responses are assembled on silica mesospheres forming noncontinuous shells that can be confirmed through the transmission electron microscope images. We find the surface plasmon resonance of single shell red-shifts from 510 to 680 nm with the increase of gold coverage. At the excitation of 532 and 785 nm, the best enhancements about 2.0?×?10⁵ and 1.1?×?10⁷ are obtained on spheres with ～60 and 83 % gold coverage, respectively. The weak polarization-dependent SERS indicates that the enhancement is from the multi-gaps on single noncontinuous shell. This optical tunable and SERS active noncontinuous gold shell can be applied in biosensing, ultra trace detection, and molecule analysis needing multi-wavelengths excitation.     

Mapping the functional domain of the prion protein.

Prion diseases such as Creutzfeldt-Jakob disease are possibly caused by the conversion of a normal cellular glycoprotein, the prion protein (PrPc) into an abnormal isoform (PrPSc). The process that causes this conversion is unknown, but to understand it requires a detailed insight into the normal activity of PrPc. It has become accepted from results of numerous studies that PrPc is a Cu-binding protein and that its normal function requires Cu. Further work has suggested that PrPc is an antioxidant with an activity like that of a superoxide dismutase. We have shown in this investigation that this activity is optimal for the whole protein and that deletion of parts of the protein reduce or abolish this activity. The protein therefore contains an active domain requiring certain regions such as the Cu-binding octameric repeat region and the hydrophobic core. These regions show high evolutionary conservation fitting with the idea that they are important to the active domain of the protein.     

Identification of a human valacyclovirase: biphenyl hydrolase-like protein as valacyclovir hydrolase

Valacyclovir is the 5'-valyl ester prodrug of acyclovir, an effective anti-herpetic drug. Systemic availability of acyclovir in humans is three to five times higher when administered orally as the prodrug. The increased bioavailability of valacyclovir is attributed to carrier-mediated intestinal absorption, via the hPEPT1 peptide transporter, followed by the rapid and complete conversion to acyclovir. The one or more human enzymes responsible for in vivo activation of the prodrug to the active drug and its conversion sites, however, have not been identified. In this report, we describe the purification, identification, and characterization of a human enzyme that activates valacyclovir to acyclovir. A protein with significant hydrolytic activity toward valacyclovir, the 5'-glycyl ester of acyclovir, and the 5'-valyl ester of zidovudine (AZT), was purified from Caco-2 cells derived from human intestine. Using a non-redundant data base search, the N-terminal 19-amino acid sequence of the purified 27-kDa, basic protein revealed a perfect match within the N terminus of a serine hydrolase, Biphenyl hydrolase-like (BPHL, gi:4757862) protein, previously cloned from human breast carcinoma. Recombinant BPHL exhibited significant hydrolytic activity for both valacyclovir and valganciclovir with specificity constants (kcat/Km), 420 and 53.2 mm-1.s-1, respectively. We conclude that BPHL may be an important enzyme activating valacyclovir and valganciclovir in humans and an important new target for prodrug design.     

Duality of polynucleotide substrates for Phi29 DNA polymerase: 3'-->5' RNase activity of the enzyme

Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3'-->5' DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3' to 5' polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is approximately 10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection.     

Principal components analysis of protein sequence clusters

Sequence analysis of large protein families can produce sub-clusters even within the same family. In some cases, it is of interest to know precisely which amino acid position variations are most responsible for driving separation into sub-clusters. In large protein families composed of large proteins, it can be quite challenging to assign the relative importance to specific amino acid positions. Principal components analysis (PCA) is ideal for such a task, since the problem is posed in a large variable space, i.e. the number of amino acids that make up the protein sequence, and PCA is powerful at reducing the dimensionality of complex problems by projecting the data into an eigenspace that represents the directions of greatest variation. However, PCA of aligned protein sequence families is complicated by the fact that protein sequences are traditionally represented by single letter alphabetic codes, whereas PCA of protein sequence families requires conversion of sequence information into a numerical representation. Here, we introduce a new amino acid sequence conversion algorithm optimized for PCA data input. The method is demonstrated using a small artificial dataset to illustrate the characteristics and performance of the algorithm, as well as a small protein sequence family consisting of nine members, COG2263, and finally with a large protein sequence family, Pfam04237, which contains more than 1,800 sequences that group into two sub-clusters.     

Influence of amino acid side-chain modification on the uptake system for beta-lactam antibiotics and dipeptides from rabbit small intestine

The influence of chemical modification of functional amino acid side-chains in proteins on the H(+)-dependent uptake system for orally active alpha-amino-beta-lactam antibiotics and small peptides was investigated in brush-border membrane vesicles from rabbit small intestine. Neither a modification of cysteine residues by HgCl2, NEM, DTNB or PHMB and of vicinal thiol groups by PAO nor a modification of disulfide bonds by DTT showed any inhibition on the uptake of cephalexin, a substrate of the intestinal peptide transporter. In contrast, the Na(+)-dependent uptake systems for D-glucose and L-alanine were greatly inhibited by the thiol-modifying agents. With reagents for hydroxyl groups, carboxyl groups or arginine the transport activity for beta-lactam antibiotics also remained unchanged, whereas the uptake of D-glucose and L-alanine was inhibited by the carboxyl specific reagent DCCD. A modification of tyrosine residues with N-acetylimidazole inhibited the peptide transport system and did not affect the uptake systems for D-glucose and L-alanine. The involvement of histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics and small peptides (Kramer, W. et al. (1988) Biochim. Biophys. Acta 943, 288-296) was further substantiated by photoaffinity labeling studies using a new photoreactive derivative of the orally active cephalosporin cephalexin, 3-[phenyl-4-3H]azidocephalexin, which still carries the alpha-amino group being essential for oral activity. 3-Azidocephalexin competitively inhibited the uptake of cephalexin into brush-border membrane vesicles. The photoaffinity labeling of the 127 kDa binding protein for beta-lactam antibiotics with this photoprobe was decreased by the presence of cephalexin, benzylpenicillin or dipeptides. A modification of histidine residues in brush-border membrane vesicles with DEP led to a decreased labeling of the putative peptide transporter of Mr 127,000 compared to controls. This indicates a decrease in the affinity of the peptide transporter for alpha-amino-beta-lactam antibiotics by modification of histidine residues. The data presented demonstrate an involvement of tyrosine and histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics across the enterocyte brush-border membrane.     

Activation of phospholipase A2 by Hsp70 in vitro

We recently suggested a novel mechanism for the activation of phospholipase A2 (PLA2), with a (catalytically) highly active oligomeric state, which subsequently becomes inactivated by conversion into amyloid. This process can be activated by lysophosphatidylcholine which promotes both oligomerization and amyloid activation/inactivation. The heat shock protein 70 (Hsp70), has been demonstrated to be able to revert the conversion of α-synuclein and Alzheimer β-peptide to amyloid fibrils in vitro. Accordingly, we would expect Hsp70 to sustain the lifetime of the active state of the enzyme oligomer by attenuating the conversion of the enzyme oligomers into inactive amyloid. Here we show that Hsp70 activates PLA2 in vitro, in a manner requiring ATP and Mg(2+).     

Benchmarking protein–protein interface predictions: Why you should care about protein size

A number of predictive methods have been developed to predict protein–protein binding sites. Each new method is traditionally benchmarked using sets of protein structures of various sizes, and global statistics are used to assess the quality of the prediction. Little attention has been paid to the potential bias due to protein size on these statistics. Indeed, small proteins involve proportionally more residues at interfaces than large ones. If a predictive method is biased toward small proteins, this can lead to an over‐estimation of its performance. Here, we investigate the bias due to the size effect when benchmarking protein‐protein interface prediction on the widely used docking benchmark 4.0. First, we simulate random scores that favor small proteins over large ones. Instead of the 0.5 AUC (Area Under the Curve) value expected by chance, these biased scores result in an AUC equal to 0.6 using hypergeometric distributions, and up to 0.65 using constant scores. We then use real prediction results to illustrate how to detect the size bias by shuffling, and subsequently correct it using a simple conversion of the scores into normalized ranks. In addition, we investigate the scores produced by eight published methods and show that they are all affected by the size effect, which can change their relative ranking. The size effect also has an impact on linear combination scores by modifying the relative contributions of each method. In the future, systematic corrections should be applied when benchmarking predictive methods using data sets with mixed protein sizes. Proteins 2014; 82:1444–1452. © 2014 Wiley Periodicals, Inc.     

用嫁接活性位点方法设计新的功能蛋白质

将一个蛋白质的活性位点,嫁接到另一个分子量较小但是稳定的蛋白质（骨架蛋白）上,从而形成一种新的功能蛋白质,这是蛋白质设计中一种很有效的方法,在我们发展的异型自洽系综最优化方法的基础上,结合３Ｄ－模体搜索等工具,实现了这种位点嫁接的设计。并以在卡律蝎毒素分子骨架上嫁接碳酸酐酶Ｂ的Ｚｎ＾２＋结合位点的设计为例进行检验,表明此设计系统是可行的和有效的。     

Probing the evolution of hydroxyisourate hydrolase into transthyretin through active-site redesign

5-Hydroxyisourate hydrolase (HIUase) and transthyretin (TTR) are closely related phylogenetically and structurally, while performing quite different functions. The former catalyzes the hydrolysis of 5-hydroxyisourate within the urate degradation pathway, and the latter is a carrier protein involved in the extracellular transport of thyroid hormones and in the cotransport of retinol. The evolution of HIUase into TTR represents a remarkable example of adaptation of a new function by active-site modification of an enzyme. On the basis of phylogenetic reconstructions and structural comparison of HIUase and TTR, two mutations (Y116T and I16A) were likely to be crucial events in order to induce, after a gene duplication event, the conversion of the enzyme into a binding protein. By rational reshaping of the active sites of HIUase and functional analyses of its mutant forms, we have provided insights into how its neofunctionalization could be achieved. We show here that the two mutations at the active sites of HIUase open up the two ends of the channel that transverses the entire tetrameric protein, generating two cavities accessible to the thyroxine molecule and abrogating, at the same time, the enzymatic activity. Our data indicate that a small number of critical mutations affecting the active site of an enzyme may be sufficient to generate a drastically different function, while a large number of additional mutations may be required for the fine-tuning of the structural and functional features of new proteins.     

Liver carboxylesterase cleaves surfactant protein (SP-) B and promotes surfactant subtype conversion

Conversion of the biophysically active large surfactant aggregate subtype of alveolar surfactant into the less surface active small surfactant aggregates occurs in vitro and in vivo, possibly in dependency of a carboxylesterase, entitled surfactant convertase. The substrate has yet not been safely identified. Utilizing the in vitro cycling assay we investigated conversion of an organic rabbit lavage extract reconstituted with SP-A. Porcine liver carboxylesterase, which is closely related to surfactant convertase, induced subtype conversion to a similar degree as compared with native lavage fluid containing endogenous convertase. In addition, we asked for cleavage products of SP-B and identified a approximately 12 kDa band upon cycling with liver carboxylesterase, having the same N-terminus as mature SP-B. A band of same molecular weight was found in native lavage fluid after in vitro conversion mediated by the endogenous convertase. We conclude that SP-B plays a pivotal role during subtype conversion and represents the substrate for surfactant convertase.     

Genetic enhancement of fatty acid synthesis by targeting rat liver ATP:citrate lyase into plastids of tobacco

ATP:citrate lyase (ACL) catalyzes the conversion of citrate to acetyl-coenzyme A (CoA) and oxaloacetate and is a key enzyme for lipid accumulation in mammals and oleaginous yeasts and fungi. To investigate whether heterologous ACL genes can be targeted and imported into the plastids of plants, a gene encoding a fusion protein of the rat liver ACL with the transit peptide for the small subunit of ribulose bisphosphate carboxylase was constructed and introduced into the genome of tobacco. This was sufficient to provide import of the heterologous protein into the plastids. In vitro assays of ACL in isolated plastids showed that the enzyme was active and synthesized acetyl-CoA. Overexpression of the rat ACL gene led to up to a 4-fold increase in the total ACL activity; this increased the amount of fatty acids by 16% but did not cause any major change in the fatty acid profile. Therefore, increasing the availability of acetyl-CoA as a substrate for acetyl-CoA carboxylase and subsequent reactions of fatty acid synthetase has a slightly beneficial effect on the overall rate of lipid synthesis in plants.     

Microsatellite analyses reveal fine-scale genetic structure in grey mouse lemurs (Microcebus murinus)

Information on genetic structure can be used to complement direct inferences on social systems and behaviour. We studied the genetic structure of the solitary grey mouse lemur (Microcebus murinus), a small, nocturnal primate endemic to western Madagascar, with the aim of getting further insight on its breeding structure. Tissue samples from 167 grey mouse lemurs in an area covering 12.3 km2 in Kirindy Forest were obtained from trapping. The capture data indicated a noncontinuous distribution of individuals in the study area. Using 10 microsatellite markers, significant genetic differentiation in the study area was demonstrated and dispersal was found to be significantly male biased. Furthermore, we observed an overall excess of homozygotes in the total population (F(IT) = 0.131), which we interpret as caused by fine-scale structure with breeding occurring in small units. Evidence for a clumped distribution of identical homozygotes was found, supporting the notion that dispersal distance for breeding was shorter than that for foraging, i.e. the breeding neighbourhood size is smaller than the foraging neighbourhood size. In conclusion, we found a more complex population structure than what has been previously reported in studies performed on smaller spatial scales. The noncontinuous distribution of individuals and the effects of social variables on the genetic structure have implications for the interpretation of social organization and the planning of conservation activities that may apply to other solitary and endangered mammals as well.     

A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity

Pharmacological chaperone therapy is an emerging counterintuitive approach to treat protein deficiencies resulting from mutations causing misfolded protein conformations. Active-site-specific chaperones (ASSCs) are enzyme active-site directed small molecule pharmacological chaperones that act as a folding template to assist protein folding of mutant proteins in the endoplasmic reticulum (ER). As a result, excessive degradation of mutant proteins in the ER-associated degradation (ERAD) machinery can be prevented, thus restoring enzyme activity. Lysosomal storage disorders (LSDs) are suitable candidates for ASSC treatment, as the levels of enzyme activity needed to prevent substrate storage are relatively low. In addition, ASSCs are orally active small molecules and have potential to gain access to most cell types to treat neuronopathic LSDs. Competitive enzyme inhibitors are effective ASSCs when they are used at sub-inhibitory concentrations. This whole new paradigm provides excellent opportunity for identifying specific drugs to treat a broad range of inherited disorders. This review describes protein misfolding as a pathophysiological cause in LSDs and provides an overview of recent advances in the development of pharmacological chaperone therapy for the diseases. In addition, a generalized guidance for the design and screening of ASSCs is also presented.     

Preparation, properties and metabolism of retro-3-dehydroretinyl acetate

1. Treatment of 3-dehydroretinyl acetate with aqueous hydrobromic acid resulted in the formation of retro-3-dehydroretinyl acetate, which, on alkaline hydrolysis, gave the corresponding alcohol. 2. retro-3-Dehydroretinyl acetate was isomerized to 3-dehydrovitamin A when fed to vitamin A-deficient rats. 3. When retro-3-dehydroretinyl acetate was administered orally, it was hydrolysed to retro-3-dehydroretinol in the rat intestine, isomerized to 3-dehydroretinol and esterified before being transported to the liver for storage. 4. When administered intraperitoneally, both 3-dehydrovitamin A and retro-3-dehydrovitamin A were accumulated in liver and other tissues, whereas after enterectomy 3-dehydrovitamin A was not detected anywhere in the body. 5. The small intestine was shown to be the major site of conversion of retro-3-dehydrovitamin A into 3-dehydrovitamin A. 6. The extent of conversion of retro-3-dehydroretinyl acetate into 3-dehydrovitamin A was much smaller than that of the conversion of retro-retinyl acetate into vitamin A. 7. The biological potency of retro-3-dehydroretinyl acetate, determined by the rat-growth assay, was 2.6% of that all-trans-retinyl acetate, when given orally.     

Antidiabetic features of AdipoAI,a novel AdipoR agonist

Adiponectin is an antidiabetic endogenous adipokine that plays a protective role against the unfavorable metabolic sequelae of obesity. Recent evidence suggests a sinister link between hypoadiponectinemia and development of insulin resistance/type 2 diabetes (T2D). Adiponectin's insulin-sensitizing property is mediated through the specific adiponectin receptors R1 and R2, which activate the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR) α pathways. AdipoAI is a novel synthetic analogue of endogenous adiponectin with possibly similar pharmacological effects. Thus, there is a need of orally active small molecules that activate Adipoq subunits, and their downstream signaling, which could ameliorate obesity related type 2 diabetes. In the study we aim to investigate the effects of AdipoAI on obesity and T2D. Through in-vitro and in-vivo analyses, we investigated the antidiabetic potentials of AdipoAI and compared it with AdipoRON, another orally active adiponectin receptors agonist. Our results showed that in-vitro treatment of AdipoAI (0–5 µM) increased adiponectin receptor subunits AdipoR1/R2 with increase in AMPK and APPL1 protein expression in C2C12 myotubes. Similarly, in-vivo, oral administration of AdipoAI (25 mg/kg) observed similar effects as that of AdipoRON (50 mg/kg) with improved control of blood glucose and insulin sensitivity in diet-induced obesity (DIO) mice models. Further, AdipoAI significantly reduced epididymal fat content with decrease in inflammatory markers and increase in PPAR-α and AMPK levels and exhibited hepatoprotective effects in liver. Further, AdipoAI and AdipoRON also observed similar results in adipose tissue. Thus, our results suggest that low doses of orally active small molecule agonist of adiponectin AdipoAI can be a promising therapeutic target for obesity and T2D.     

Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide.

beta-Lactamase with the -20 to -1 region of the leader peptide deleted (almost complete deletion of the leader peptide) [delta(-20,-1) beta-lactamase] was released from Escherichia coli cells by osmotic shock. Fractionation of the cells by conversion to spheroplasts and protease accessibility experiments further indicated that a portion of the protein may be bound to the cytoplasmic membrane and be partially exposed in the periplasmic space. Expression of delta(-20,-1) beta-lactamase conferred a 25-fold increase in the 50% lethal dose for ampicillin relative to that for controls, thus confirming that a small amount (about 2%) of the active protein is completely exported from the cytoplasm. These results suggest that even in the absence of a leader peptide, mature beta-lactamase is able to interact with the cytoplasmic membrane and be translocated into the periplasmic space, albeit with a low efficiency.     

A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.     

Semiautomated cell-free conversion of prion protein: applications for high-throughput screening of potential antiprion drugs

Transmissible spongiform encephalitis (TSE) is a lethal illness with no known treatment. Conversion of the cellular prion protein (PrP(C)) into the infectious isoform (PrP(Sc)) is believed to be the central event in the development of this disease. Recombinant PrP (rPrP) protein folded into the amyloid conformation was shown to cause the transmissible form of prion disease in transgenic mice and can be used as a surrogate model for PrP(Sc). Here, we introduced a semiautomated assay of in vitro conversion of rPrP protein to the amyloid conformation. We have examined the effect of known inhibitors of prion propagation on this conversion and found good correlation between their activity in this assay and that in other in vitro assays. We thus propose that the conversion of rPrP to the amyloid isoform can serve as a high-throughput screen for possible inhibitors of PrP(Sc) formation and potential anti-TSE drugs.