bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis and material properties of syndiotactic polystyrene/organophilic clay nanocomposites
Authors: Ho Kim Mun , Park Cheon Il , Choi Won Mook , Lee Jin Woo , Lim Jae Gon , Park O Ok , Kim Jung Min
Published: March 2004

Syndiotactic polystyrene (sPS)/organophilic clay nanocomposites were fabricated by direct‐melt intercalation method. To overcome the thermal instability of organophilic clay at high‐melt processing temperatures of sPS, an organophilic clay modified by alkyl phosphonium was adopted, which is known to be thermally stable. By using the newly synthesized clay, we could fabricate sPS intercalated nanocomposites. The microstructures of nanocomposites were confirmed by X‐ray diffraction (XRD) and transmission electron microscopy (TEM). The crystallization rate of nanocomposites investigated by differential scanning calorimetry (DSC) does not increase despite the presence of clay, which may be due to the physical hindrance of organic modifiers in the clay dispersion. Nanocomposites exhibited enhanced mechanical properties such as strength and stiffness relative to the virgin polymer. In addition, thermal stability was confirmed to be improved by thermogravimetric analysis (TGA). © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 2144–2150, 2004
13C‐NMR study of Dipteryx Alata Vogel starch
Authors: Tavares Maria Inês B. , Bathista André Luis B. S. , Silva Emerson O. , Costa Paula M. , Filho Nicolau P. , Nogueira José S.
Published: March 2004

Natural starches obtained by fruit seeds have been a subject of studies because of their applications, as a consequence of their chemical properties. As it is known, the fruits depend on the geographic region. Because of this, several factors affect the seed starches nature, such as granules size, morphology, components, and composition. Thus, to better understand starch properties, it is necessary to investigate the chemical behavior of these factors. This work focused on the chemical components and molecular dynamic behavior of the Dipterxy Alata Vogel (cumbaru) starch, because this fruit is very popular and is used in abundance for people because they think that it is good for some kinds of diseases. The main chemical components and the molecular dynamics of starch from the Dipterxy Alata Vogel were studied by applying solid‐state 13C nuclear magnetic resonance spectroscopy (NMR). © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 2151–2154, 2004
Structural characterization of reactor blends of polypropylene and ethylene‐propylene rubber
Authors: Pires Márcia , Mauler Raquel S. , Liberman Susana A.
Published: March 2004

Blends of isotactic polypropylene (PP), ethylene‐propylene rubber copolymer (EPR), and ethylene‐propylene crystalline copolymer (EPC) can be produced through in situ polymerization processes directly in the reactor and blends with different structure and composition can be obtained. In this work we studied the structure of five reactor‐made blends of PP, EPR, and EPC produced by a Ziegler‐Natta catalyst system. The composition of EPR was related to the ratio between ethylene and propylene used in the copolymerization step. The ethylene content in the EPR was in the range of 50–70 mol %. The crystallization behavior of PP and EPC in the blends was influenced by the presence of the rubber, and some specific interactions between the components could be established. By preparative temperature rising elution fractionation (P‐TREF) analysis, the isolation and characterization of crystalline EPC fractions were made. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 2155–2162, 2004
Free‐radical grafting of acrylic acid onto isotactic polypropylene using styrene as a comonomer in supercritical carbon dioxide
Authors: Dong Qingzhi , Liu Ying
Published: March 2004

Free‐radical grafting of acrylic acid (AAc) onto isotactic polypropylene (iPP) using styrene (St) as a comonomer in supercritical carbon dioxide (SCCO2) medium was studied. The effects of temperature and pressure of reaction on functionalization degree (grafting degree of AAc) of the products were analyzed. The increase of reaction temperature increases the diffusion of monomers and radicals in the disperse reaction system of SCCO2. In addition, the increase of temperature accelerates the decomposition rate of 2,2′‐azobisisobutyronitrile (AIBN), thus promoting grafting reaction. It was also observed that functionalization degree of the products decreases with the increase of pressure of SCCO2 in the range of experiment. The effects of comonomer St on the functionalization degree of the products were investigated. The AAc graft degree of the resulting polymer was drastically higher in the present of St. It reached a maximum when the mass ratio of St and AAc was about 0.7 : 1. Because AAc is not sufficiently reactive toward iPP macroradicals, it would be helpful to use a second monomer that can react with them much faster than AAc. St preferentially reacts with the iPP macroradicals to form more stable styrene macroradicals, which then copolymerize with AAc to form branches. The highest functionalization degree was obtained when the AIBN was 0.75 wt %. When the initiator was used excessively, the functionalization degree decreased because of severe chain degradation of the iPP backbone. The morphologies of pure iPP and grafted iPP are different under the polarizing optical microscope. The diameter of the pure iPP spherulites is 20–38 μ and that of the grafted iPP spherulites is reduced with the increase of the functionalization degree of the products. This is proposed to be because the polar grafts formed during the reaction would have a tendency to associate in the hydrophobic PP environment. This might preserve some of the local crystalline order that existed during the reaction in the swollen iPP phase. It can be proven by a DSC cooling investigation that the crystallization temperature increased as the functionalization degree increased. This is proposed to be because the side‐chain of grafting polymer helps to bring about the heterogeneous nucleation in grafting polymer. Therefore, a large number of nuclei can emerge to a lesser supercooling degree. It can be also proven that the percent crystallization decreased as the functionalization degree increased, probably due to the grafted branches, which disrupted the regularity of the chain structure and increased the spacing between the chains. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 2203–2210, 2004
Rheological, electrical, and microwave properties of polymers with nanosized carbon particles
Authors: Kotsilkova R. , Nesheva D. , Nedkov I. , Krusteva E. , Stavrev S.
Published: March 2004

The rheology, dc‐conductivity, and microwave properties of acrylic, polyurethane, and epoxy composites containing 0–15 vol % of nanosized carbon particles have been investigated. Carbon nanoparticles (1–3 nm) are produced by a shock wave technology. Steady‐state shear and oscillatory flow tests are applied to investigate the rheological properties of dispersions; dc‐conductivity and MW absorption/reflection are investigated for solid composite films. Rheological characteristics are used for the evaluation of agglomeration processes of nanoparticles in dispersions, as controlled by volume fraction and processing technology. The percolation threshold is interpreted as a structural transition from a dispersed to an agglomerated state and it is found to depend significantly on the type of the matrix polymer. Above the percolation threshold, the presence of carbon nanoparticles produces a strong increase in the viscosity of dispersions as well as of the electrical conductivity and microwave properties of solid composites. A good correlation between the three characteristics is found for the systems in a wide range of carbon volume fractions. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 2220–2227, 2004
Comparative morphology of the eye (with particular attention to the retina) in various species of cardinal fish (Apogonidae, Teleostei)
Authors: Fishelson Lev , Ayalon Guy , Zverdling Adi , Holzman Roi
Published: March 2004

Various parameters of the eye dimension and structure have been compared in 15 species of cardinal fish (Apogonidae), including both nocturnal and diurnal forms, mostly inhabiting rocky habitats in tropical and subtropical regions. In general, in the nocturnal forms the eye and retina are larger than in the diurnal fish of similar dimensions. In the nocturnal species, eye diameter to body length is ca. 12–13%, whereas in the diurnal species it is less than 10%. Retina size in adult fish of the various species varies from 20 mm2 to 183 mm2. Cytological examination of the studied retinas revealed that they are composes, additional to rods (20–40 μm), of both bulbous and slender double cones, as well as single cones. These cones form a mosaic comprising four double with one single in the center, a pattern that is less prominent at the periphery of the retina and more so in the fundus. The rod ellipsoids reveal normal mitochondria, whereas the cones bear ellipsoids featuring opaque and unusual, ellipsosome‐like mitochondria. The number of rods in a retinas varies from 15 to 128 million, and the number of cones from 460,000 to 5,673,000. As revealed in cardinal fish of similar dimensions, the number of visual cells found in the retina is much higher in the nocturnal than in the diurnal species. The ecological and developmental aspects of the observed phenomena are discussed. Anat Rec Part A 277A:249–261, 2004. © 2004 Wiley‐Liss, Inc.
Osteoblasts and osteocytes express MMP2 and ‐8 and TIMP1, ‐2, and ‐3 along with extracellular matrix molecules during appositional bone formation
Authors: Hatori Kouki , Sasano Yasuyuki , Takahashi Ichiro , Kamakura Shinji , Kagayama Manabu , Sasaki Keiichi
Published: March 2004

Our previous studies suggested that a part of bone extracellular matrix (ECM) molecules are degraded and remodeled during embryonic bone formation. In contrast, little is known about ECM remodeling in postnatal appositional bone formation. The present study was designed to investigate expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during experimentally initiated appositional bone formation in rats. Expressions of ECM molecules, MMPs, and TIMPs were examined using in situ hybridization. Osteoblasts and osteocytes expressed MMP2 and ‐8, TIMP1, ‐2, and ‐3, as well as type I collagen, osteopontin, and osteocalcin in the course of the appositional bone formation, while they showed few transcripts of MMP13. The results indicated that while osteoblasts and osteocytes in the apposed bone produce ECM molecules, they degrade ECM molecules with MMPs and regulate the degradation by inhibiting the activity of MMPs using TIMPs. Osteoblasts and osteocytes may reorganize the ECM composition to mature the bone matrix in appositional bone formation. Anat Rec Part A 277A:262–271, 2004. © 2004 Wiley‐Liss, Inc.
Expression of the liver X receptor α and β in embryonic and adult mice
Authors: Annicotte Jean‐Sébastien , Schoonjans Kristina , Auwerx Johan
Published: March 2004

We characterized the expression pattern of the nuclear receptors liver X receptor (LXR) α and β during mouse embryonic development and in adulthood by in situ hybridization experiments. LXRα and LXRβ are detected in the liver starting at 11.5 days postcoitum. Later, LXRα expression remains high in organs involved in lipid homeostasis, such as liver, intestine, and brown adipose tissue, whereas LXRβ is more ubiquitously expressed and enriched in tissues of neuronal and endocrine origin. Anat Rec Part A 277A:312–316, 2004. © 2004 Wiley‐Liss, Inc.
Capillary‐to‐fiber ratio of hind limb muscles in the male Syrian golden hamster
Authors: Swisher Anne K. , Alway Stephen E. , Yeater Rachel
Published: March 2004

The hamster has been the accepted model of emphysema since the 1970s, demonstrating disease‐related effects on respiratory skeletal muscle. However, there is scant information available about the model's ability to replicate the peripheral skeletal muscle changes seen in human disease, such as alterations in capillarity. The present study described the capillary‐to‐fiber ratio (C/F) of normal hamster plantaris, gastrocnemius, and soleus muscles in eight animals. C/F was 1.72 ± 0.38 for plantaris, 1.95 ± 0.40 for gastrocnemius, and 2.22 ± 0.43 for soleus. C/F of soleus was significantly greater (P < 0.01) than plantaris. The C/F of hamster hindlimb muscles varies from those seen in rat species, and having baseline data on hamsters makes it possible to determine the effects of emphysema on C/F in this model. Anat Rec Part A 277A:272–274, 2004. © 2004 Wiley‐Liss, Inc.
Tight junctions in differentiating ameloblasts and odontoblasts differentially express ZO‐1, occludin, and claudin‐1 in early odontogenesis of rat molars
Authors: João Silvia M.A. , Arana‐Chavez Victor E.
Published: March 2004

Little is known about the expression of associated proteins during the assembly of tight junctions (TJs). We studied the distribution of ZO‐1, occludin, and claudin‐1 between differentiating ameloblasts and odontoblasts in molar tooth germs from 1‐ to 3‐day‐old rats by confocal laser scanning microscopy. Immunoreactivity for ZO‐1 was strong at proximal and distal junctional complexes of differentiating ameloblasts, while it was weak and punctuate at the distal region of differentiating odontoblasts. Occludin was immunoreactive at distal and proximal complexes of early differentiating ameloblasts and at distal regions of differentiating odontoblasts. However, in more advanced stages, occludin was only evident at the proximal complex of ameloblasts. Claudin‐1 was strongly detected at the proximal complex but it was weak at distal complex of late differentiating ameloblasts. Thus, our results showed that ZO‐1, occludin, and claudin‐1 are differentially expressed as TJs assemble for regulating polarity and/or paracellular permeability in differentiating ameloblasts and odontoblasts. Anat Rec Part A 277A:338–343, 2004. © 2004 Wiley‐Liss, Inc.
Development of multiorgan pathology in the wpk rat model of polycystic kidney disease
Authors: Gattone Vincent H. , Tourkow Benjamin A. , Trambaugh Chad M. , Yu Alexander C. , Whelan Shelly , Phillips Carrie L. , Harris Peter C. , Peterson Richard G.
Published: March 2004

Rodent models of polycystic kidney disease (PKD) have provided valuable insight into the cellular changes associated with cystogenesis in humans. The present study characterizes the morphology of renal and extrarenal pathology of autosomal recessive PKD induced by the wpk gene in Wistar rats. In wpk−/− rats, proximal tubule and collecting duct cysts develop in utero and eventually consume the kidney. Increased apoptosis, mitosis, and extracellular tenascin deposition parallel cyst development. Extrarenal pathology occurs in the immune system (thymic and splenic hypoplasia) and central nervous system (CNS; hypoplasia to agenesis of the corpus callosum with severe hydrocephalus). Severity of hydrocephalus varied inversely with size of the corpus callosum. In wpk−/− rats, the corpus callosum exhibits relatively few axons that cross the midline. This CNS pathology is similar to that described in three human renal cystic syndromes: orofaciodigital, genitopatellar, and cerebrorenal‐digital syndromes. Collecting duct and ventricular ependymal cilia appear morphologically normal. To determine if rodent background strain and the presence of modifier genes affect severity of the disease, we crossed the Wistar‐wpk rat with Brown Norway (BN) and Long Evan (LE) rats and found the degree of renal and cerebral pathology was diminished as evidenced by lower kidney weight as a percent of body weight and serum urea nitrogen concentration in cystic rats on LE or BN strains as well as less prominent cranial enlargement. Crosses with BN rats allowed us to localize the wpk gene on chromosome 5 very close to the D5Rat73 marker. The wpk gene lies within a chromosomal region known to harbor a PKD modifier locus. In summary, the types of renal and cerebral pathology seen in the Wistar wpk rat are a unique combination seen only in this rodent model. Anat Rec Part A 277A:384–395, 2004. © 2004 Wiley‐Liss, Inc.
Postnatal development of the eye in the naked mole rat (Heterocephalus glaber)
Authors: Nikitina Natalya V. , Maughan‐Brown Bronwen , O'Riain M. Justin , Kidson Susan H.
Published: March 2004

The naked mole rat (Heterocephalus glaber) is a subterranean rodent whose eyes are thought to be visually nonfunctional and as such is an ideal animal with which to pursue questions in evolutionary developmental biology. This report is the first in‐depth study on the development and morphology of the naked mole rat eye. Using standard histological analysis and scanning and transmission electron microscopy, we describe the structural features of the eye. We further report on the morphological changes that accompany the development of this eye from neonate to adult and compare them with those that occur during mouse eye development. We observed numerous abnormalities in the shape and cellular arrangement of the structures of the anterior chamber, with notable malformations of the lens. Cell proliferation and cell death assays were conducted to investigate the possible causes of lens malformation. We found that neither of these processes appeared abnormal, indicating that they were not responsible for the lens phenotype of the mole rat. In order to investigate the process of lens differentiation, we analyzed the expression of γ‐crystallins using Western blots and immunocytochemistry. At birth, levels of γ‐crystallin appear normal, but soon thereafter, the γ‐crystallin expression is terminated. Absence of detectable γ‐crystallins in adults suggests that there is a gradual degradation and loss of these proteins. The evolutionary factors that could be responsible for the eye morphology of the naked mole rat are discussed. A model for abnormal lens differentiation and the role it plays in the morphogenesis of the rest of the eye in the naked mole rats is proposed. Anat Rec Part A 277A:317–337, 2004. © 2004 Wiley‐Liss, Inc.
Association of early endosomal autoantigen 1 with macropinocytosis in EGF‐stimulated a431 cells
Authors: Hamasaki Masao , Araki Nobukazu , Hatae Tanenori
Published: March 2004

Association of early endosomal autoantigen 1 (EEA1) with macropinosomes was examined in EGF‐stimulated A431 cells by dual labeling with immunofluorescence of anti‐EEA1 and FITC‐dextran (FDx), a fluid‐phase endocytic marker. Addition of EGF to A431 cells drastically enhanced macropinosome formation. Newly formed macropinosomes labeled with 5‐min pulse of FDx were located at the cell periphery and labeled weakly for EEA1. After a 5‐min chase, these macropinosomes aggregated and frequently fused with each other. Immunofluorescence showed that EEA1 appeared on the membrane of FDx‐labeled macropinosomes at that time, suggesting that EEA1 functioned in homotypic macropinosome fusion. With longer chase (30–60 min), macropinosomes decreased in number and size, indicating that FDx was largely exocytosed via recycling compartments. A small amount of FDx‐labeled macropinosomes remained in the perinuclear region even at 60 min after pulse labeling. They were EEA1‐positive but negative for cathepsin D, a lysosomal enzyme. This indicates that macropinosomes do not mature to late endosomes or fuse with lysosomes. Instead, EEA1 continuously mediates homotypic fusion as long as the macropinosomes persist. Anat Rec Part A 277A:298–306, 2004. © 2004 Wiley‐Liss, Inc.
Gunther von Hagens and Body Worlds part 2: The anatomist as priest and prophet
Authors: Moore Charleen M. , Brown C. Mackenzie
Published: March 2004

Part 1 of this two‐part series highlighted tensions between the anatomical quest for scientific knowledge about the human interior and artistic representations of the anatomized body, contrasting the roles of Goethe's scientific Prosektor and humanistic Proplastiker—roles disturbingly fused in Gunther von Hagens. Part 2 first examines religious interpretations of the human body that fuel the tensions manifest in anatomy art. The body in Western cultures is a sacred text amenable to interpretation as handiwork of God, habitation for the soul, and vehicle for resurrection. As handiwork of God the body beckons the anatomist's scalpel, helping establish dissection as the hallmark of Western medicine. The body as divinely designed machine encompasses the idea of an indwelling soul expressing its will in actions mediated through the intricate network of muscles—an understanding reflected in the oft occurring muscle men of early anatomical textbooks. Interconnections of body and soul in medieval somatic spirituality are examined with reference to ideas of resurrection and their impact on anatomical illustration. Part 2 concludes with consideration of von Hagens as priest and prophet, culminating in the Promethean impulse that recognizes not God but ourselves as proper owners and molders of our destiny, embodied in the plastinator's visionary quest to create the superhuman. Anat Rec (Part B: New Anat) 277B:14–20, 2004. © 2004 Wiley‐Liss, Inc.
Loved one's body in the anatomy laboratory
Authors: McNeill Paul M. , Kerridge Ian H. , Purdon Tina
Published: March 2004

An Australian medical student recognized parts of her father's body during classes in an anatomy laboratory and suffered considerable trauma as a consequence. While the risk of a further case may be small, the potential for trauma could be minimized by adopting appropriate policy and procedures. Institutions that receive donations of bodies may benefit from considering suggestions for minimizing that risk and for responding appropriately should it ever occur. The article draws on the investigation into this case to offer some suggestions for minimizing the chance of recognition of bodies or body parts by students and academic or professional staff and for managing those circumstances should they ever occur. The suggestions extend to minimizing the risk of recognition of deceased persons or their body parts through photographic records, video material, and case studies presented in a number of different formats. Anat Rec (Part B: New Anat) 277B:10–13, 2004. © 2004 Wiley‐Liss, Inc.
Intellectual property conundrum for the biological sciences
Authors: Olds James L.
Published: March 2004

Policy regarding academically generated biomedical intellectual property (IP) has been shaped by two important events: the Vannevar Bush report to then President Roosevelt in 1945 and the Bayh‐Dole Act of 1980. This policy, which vests the intellectual property produced from federally funded biomedical research from the government to the academic institution, was designed to promote technology transfer and thus promote the health of the U.S. economy. However, the policy has led to significant challenges, particularly in implementation. Here it is argued that the difficulties are due to differences in the structure of motivations between biomedical scientists, institutional officials, and private sector entrepreneurs. Understanding these differences may lead to a review of policy with the goal of enhancing technology transfer for the future. Anat Rec (Part B: New Anat) 277B:5–9, 2004. © 2004 Wiley‐Liss, Inc.
A framework for understanding user requirements for an information service: Defining the needs of informal carers
Authors: Hepworth Mark
Published: March 2004

The aim of this research was to develop a conceptual framework that would help to collect and understand the information needs of a target community. Even though many information behavior frameworks already exist, they tend to focus on different aspects of the person and their interaction with information. It was proposed that a synthesis of the existing frameworks could lead to one comprehensive framework. Previous research was analyzed and an initial framework defined. It was piloted, adapted, and then applied to data on informal carers, who are people caring for another person, generally a relative, for more than 14 hours per week, and who are not paid for it. The data stemmed from 60 interviews that were transcribed and coded. This paper presents the data on informal carers and their information experience using the final framework. It serves to demonstrate how the framework sensitizes the researcher to certain types of significant data, enables the organization of the data, indicates the relationships between different types of data, and, overall, helps to provide a rich picture of the target community's information needs. In conclusion, the paper discusses the differences and advantages of the framework in relation to previous work, the limitations of the study, and possible further research.
Multiple viewpoints as an approach to digital library interfaces
Authors: French James C. , Chapin A. C. , Martin Worthy N.
Published: March 2004

We introduce a framework of multiple viewpoint systems for describing and designing systems that use more than one representation or set of relevance judgments on the same collection. A viewpoint is any representational scheme on some collection of data objects together with a mechanism for accessing this content. A multiple viewpoint system allows a searcher to pose queries to one viewpoint and then change to another viewpoint while retaining a sense of context. Multiple viewpoint systems are well suited to alleviate vocabulary mismatches and to take advantage of the possibility of combining evidence. We discuss some of the issues that arise in designing and using such systems and illustrate the concepts with several examples.
EBizPort: Collecting and analyzing business intelligence information
Authors: Marshall Byron , McDonald Daniel , Chen Hsinchun , Chung Wingyan
Published: March 2004

To make good decisions, businesses try to gather good intelligence information. Yet managing and processing a large amount of unstructured information and data stand in the way of greater business knowledge. An effective business intelligence tool must be able to access quality information from a variety of sources in a variety of forms, and it must support people as they search for and analyze that information. The EBizPort system was designed to address information needs for the business/IT community. EBizPort's collection‐building process is designed to acquire credible, timely, and relevant information. The user interface provides access to collected and metasearched resources using innovative tools for summarization, categorization, and visualization. The effectiveness, efficiency, usability, and information quality of the EBizPort system were measured. EBizPort significantly outperformed Brint, a business search portal, in search effectiveness, information quality, user satisfaction, and usability. Users particularly liked EBizPort's clean and user‐friendly interface. Results from our evaluation study suggest that the visualization function added value to the search and analysis process, that the generalizable collection‐building technique can be useful for domain‐specific information searching on the Web, and that the search interface was important for Web search and browse support.
Finding governmental statistical data on the Web: A study of categorically organized links for the FedStats topics page
Authors: Ceaparu Irina , Shneiderman Ben
Published: March 2004

More than 100 U.S. governmental agencies offer links through FedStats, a centralized Web site that facilitates access to statistical tables, reports, and agencies. This and similar large collections need appropriate interfaces to guide the general public to easily and successfully find information they seek. This paper summarizes the results of 3 empirical studies of alternate organization concepts of the FedStats Topics Web page. Each study had 15 participants. The evolution from 645 alphabetically organized links, to 549 categorically organized links, to 215 categorically organized links tied to portal pages produced a steady rise in successful task completion from 15.6 to 24.4 to 42.2%. User satisfaction also increased. We make recommendations based on these data and our observations of users.
Regulation of protein diversity by alternative pre‐mRNA splicing with specific focus on chondrogenesis
Authors: McAlinden Audrey , Havlioglu Necat , Sandell Linda J.
Published: March 2004

Analysis of the human genome has dramatically demonstrated that the majority of protein diversity is generated by alternative splicing of pre‐mRNA. This powerful and versatile mechanism controls the synthesis of functionally different protein isoforms that may be required during specific stages of development from a single gene. Consequently, ubiquitous and/or tissue‐specific RNA splicing factors that regulate this splicing mechanism provide the basis for defining phenotypic characteristics of cells during differentiation. In this review, we will introduce the basic mechanisms of pre‐mRNA alternative splicing, describe how this process is regulated by specific RNA splicing factors, and relate this to various systems of cell differentiation. Chondrogenesis, a well‐defined differentiation pathway necessary for skeletogenesis, will be discussed in detail, with focus on some of the alternatively‐spliced proteins known to be expressed during cartilage development. We propose a heuristic view that, ultimately, it is the regulation of these RNA splicing factors that determines the differentiation status of a cell. Studying regulation at the level of pre‐mRNA alternative splicing will provide invaluable insights into how many developmental mechanisms are controlled, thus enabling us to manipulate a system to select for a specific differentiation pathway. Birth Defects Research (Part C) 72:51–68, 2004. © 2004 Wiley‐Liss, Inc.
Heparan sulfate proteoglycans: Coordinators of multiple signaling pathways during chondrogenesis
Authors: Kirn‐Safran Catherine B. , Gomes Ronald R. , Brown Anissa J. , Carson Daniel D.
Published: March 2004

Heparan sulfate proteoglycans are abundantly expressed in the pericellular matrix of both developing and mature cartilage. Increasing evidence indicates that the action of numerous chondroregulatory molecules depends on these proteoglycans. This review summarizes the current understanding of the interactions of heparan sulfate chains of cartilage proteoglycans with both soluble and nonsoluble ligands during the process of chondrogenesis. In addition, the consequences of mutating genes encoding heparan sulfate biosynthetic enzymes or heparan sulfate proteoglycan core proteins on cartilage development are discussed. Birth Defects Research (Part C) 72:69–88, 2004. © 2004 Wiley‐Liss, Inc.
Hyaluronan and morphogenesis
Authors: Spicer Andrew P. , Tien Janet Y. L.
Published: March 2004

In the past decade, there has been an explosion of interest in hyaluronan, an often misunderstood, biochemically simple, yet functionally complex carbohydrate polymer that is a resident of many extracellular matrices. Previously thought of as a passive, space‐filling component of the extracellular matrix, the so‐called “goo” concept, hyaluronan has risen to a much higher regard in recent years, even being called “magic glue” in a recent perspective. Hyaluronan is likely to be the common thread in many morphogenetic processes, including condensation events and epithelial‐to‐mesenchymal transformation. Hyaluronan is comparatively unique as a component of the extracellular matrix as it is solely composed of carbohydrate. In order to truly understand this biopolymer, one must first understand its biosynthesis, then understand its uptake and turnover, then identify its binding proteins and receptors. Major advances have been made in all of these arenas within the past decade. Hyaluronan synthases, hyaluronidases, and the hyaladherins have been molecularly identified and cloned. Furthermore, many have now been inactivated, employing gene targeting strategies, to create mice deficient in the respective gene product function. Collectively, huge strides have been made in our understanding of the diverse biological functions for this fascinating molecule. Hyaluronan appeared in metazoans immediately prior to the arrival of the vertebrates, and may be required for the differentiation, development, and/or function of most cell lineages, structures, and tissues that we associate with vertebrates, such as the neural crest, the skeleton, including the teeth, skin, and hair, and the chambered heart. In this review, we will update the reader on the advances of the past decade and provide insight into those morphogenetic processes through which hyaluronan regulates vertebrate development. Birth Defects Research (Part C) 72:89–108, 2004. © 2004 Wiley‐Liss, Inc.
Aqueous solubility and membrane interactions of hydrophobic peptides with peptoid tags
Authors: Tang Yan‐Chun , Deber Charles M.
Published: March 2004

Lysine tagging of hydrophobic peptides of parent sequence KKAAALAAAAALAAWAALAAAKKKK‐NH2 has been shown to facilitate their synthesis and purification through water solubilization, yet not impact on the intrinsic properties of the hydrophobic core sequence with respect to its insertion into membranes in an α‐helical conformation. However, due to their positively charged character, such peptides often become bound to phospholipid head groups in membrane surfaces, which inhibits their transbilayer insertion and/or prevents their transport across cellular bilayers. We sought to develop more neutral peptides of membrane‐permeable character by replacing most Lys residues with uncharged peptoid [N‐(R)glycyl] residues, which might similarly confer water solubility while retaining membrane‐interactive properties of the hydrophobic core. Several “peptoid‐tagged” derivatives of the parent peptide were prepared with varying peptoid content, with five of the six Lys residues replaced with peptoids Nala and/or Nval. Conformations of these peptides measured by circular dichroism spectroscopy demonstrated that these water‐soluble peptides retain the α‐helix structure in micelles (lysophosphatidylcholine and sodium dodecyl sulfate) notwithstanding the known helix‐breaking capacity of the peptoid tags. Blue shifts in Trp fluorescence spectra and quenching experiments with acrylamide confirmed that peptoid‐tagged peptides insert spontaneously into micellar membranes. Results suggest that upon introduction of uncharged tags, the interaction between the membrane and the peptides is dominated by the hydrophobicity of the peptide core rather than the electrostatic interactions between the Lys and the head groups of the lipids. The overall findings indicate that peptoid residues are effective surrogates for Lys as uncharged water‐solubilizing tags and, as such, provide a potentially valuable feature of design of membrane‐interactive peptides. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Synthesis of fluorine‐containing bioisosteres corresponding to phosphoamino acids and dipeptide units
Authors: Otaka Akira , Mitsuyama Etsuko , Watanabe Junko , Watanabe Hideaki , Fujii Nobutaka
Published: March 2004

It has been shown that fluorinated analogues of naturally occurring biological active compounds including amino acids often exhibit unique physiological activity. Among wide varieties of fluorine‐containing amino acids, nonhydrolyzable phosphoamino acids possessing a substituent of the difluoromethylene (CF2) unit for the phosphoryl ester oxygen are of value in the medicinal and biological fields. We have engaged in the synthesis of these classes of nonhydrolyzable phosphoamino acids corresponding to pTyr 3, pSer 4, and pThr 5 with their incorporation into peptides using newly developed deprotecting procedures. In this article, stereoselective synthesis of the CF2‐substituted pThr mimetics and development of a two‐step deprotecting methodology for the nonhydrolyzable analogues are reviewed. In the course of the above synthetic study, we found that γ,γ‐difluoro‐α,β‐enoates were reduced to γ‐fluoro‐β,γ‐enoates by organocopper reagents and then applied to the synthesis of (Z)‐fluoroalkene dipeptide isosteres, which have served as potential dipeptide mimetics having structural as well as electrostatic similarity to the parent peptide bonds. Furthermore, mechanistic investigation of the organocopper‐mediated reduction led us to development of a SmI2‐mediated approach toward the synthesis of the fluoroalkene isosteres. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Recognition and fluorescence sensing of specific amino acid residue on protein surface using designed molecules
Authors: Ojida Akio , Miyahara Yoshifumi , Kohira Takahiro , Hamachi Itaru
Published: March 2004

Many biological processes are mediated by surface recognition between proteins. Small molecules that recognize and bind a specific region of a protein surface may be promising agents for disrupting certain protein–protein surface interactions, which consequently leads to regulation of cellar functions. This article describes our recent efforts toward the development of the designed small molecules, which can recognize histidine or phosphorylated amino acid residues on peptide surfaces in a sequence‐selective manner. These results demonstrate that cooperative metal–ligand interaction is powerful for tight and selective binding to the specific amino acid residues of proteins in aqueous medium. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Molecular spacers for physicochemical investigations based on novel helical and extended peptide structures
Authors: Toniolo Claudio , Crisma Marco , Formaggio Fernando , Peggion Cristina , Broxterman Quirinus B. , Kaptein Bernard
Published: March 2004

A proper understanding of the detailed nature and mechanism of physicochemical interactions depends heavily upon our ability to design and synthesize conformationally constrained 3D structures whose intercomponent geometry (either rigorously rigid or able to undergo destructuration, if required, but in all cases precisely tunable) would be well defined. To this end we have recently reported a few initial studies and we are currently working on the exploitation of stable, short, helical peptide spacers based on achiral and/or chiral Cα‐tetrasubstituted α‐amino acids. These building blocks are known to force the peptides either to predominantly fold into a 310‐helical structure or to adopt a fully extended, planar 2.05‐helix. The systems under investigation involve a donor and an acceptor moiety linked to the N‐ and C‐termini of the oligopeptide spacer main chain. By increasing the number of intervening residues the donor···acceptor separation can be easily modulated. This review highlights details of these two novel peptide secondary structures and their use as molecular spacers in physicochemical investigations. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004
Enhancing multiple disulfide bonded protein folding in a cell‐free system
Authors: Yin Gang , Swartz James R.
Published: March 2004

A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell‐free protein synthesis system. Due to the unstable and reducing environment in the initial E. coli‐based cell‐free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E. coli periplasmic chaperone, and expressing PA at 30°C increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 μg/mL of bioactive PA in a simple 3‐h batch reaction. © 2004 Wiley Periodicals, Inc.
Screening of large protein libraries by the “cell immobilized on adsorbed bead” approach
Authors: Freeman Amihay , Cohen‐Hadar Noa , Abramov Simona , Modai‐Hod Ronit , Dror Yael , Georgiou George
Published: March 2004

Screening of mutant libraries for in vitro enzyme evolution is carried out primarily by physical separation of the cells, followed by growth of individual clones and screening of biocatalytic activity on the basis of color or fluorescence signal development. Currently, most frequently employed methods are labor‐intensive or require robotic equipment, resulting in screening limited to a relatively small fraction of the potential inherent in a given library. In this study we present a design, development, and feasibility demonstration of a new screening approach, providing convenient handling of large libraries consisting of 106 to 107 clones and screening based on a simultaneous enzymatic assay with commercially available substrates. This new screening method is based on the “cell immobilized on adsorbed bead” approach: the cell population to be screened is mixed with an excess of medium pre‐equilibrated polyacrylamide beads, chemically derivatized to affect quantitative cell immobilization by adsorption. The resulting bead population, comprising of single cell on a bead or blank beads, is then immobilized on a solid glass support. After removal of the freely flowing liquid, the cells immobilized on the adsorbed beads are allowed to grow into microcolonies, utilizing the medium retained within the supporting hydrogel matrix. These colonies are subsequently equilibrated with chromogenic or fluorogenic substrate and screening is affected under a stereomicroscope, resulting in readily retrieved of the most active colonies. This technique may be particularly useful when the screened mutants are expressed and displayed on the cell surface, providing an active and homogeneous “naturally immobilized” enzyme population with minimal substrate diffusion limitations. © 2004 Wiley Periodicals, Inc.
A novel system for expressing recombinant proteins over a wide temperature range from 4 to 35°C
Authors: Nakashima Nobutaka , Tamura Tomohiro
Published: March 2004

Escherichia coli cells are the most commonly used host cells for large‐scale production of recombinant proteins, but some proteins are difficult to express in E. coli. Therefore, we tested the nocardioform actinomycete Rhodococcus erythropolis, which grows at temperatures ranging from 4 to 35°C, as an expression host cell. We constructed inducible expression vectors, where the expression of the target genes could be controlled with the antibiotic thiostrepton. Using these expression vectors, several milligrams of reporter proteins could be isolated from 1 liter of culture of R. erythropolis cells grown at a temperature range from 4 to 35°C. Moreover, we successfully purified serum amyloid A1, NADH dehydorogenase 1 α subcomplex 4, cytochrome b5‐like protein, apolipoprotein A‐V, cathepsin D, pancreatic Rnase, and HMG‐1 that are all difficult to express in E. coli. In the case of kallikrein 6, mouse deoxyribonuclease I and Kid1, which are also difficult to express in E. coli, the expression level of each protein increased when proteins were expressed at low temperature (4°C). Based on these results, we conclude that a recombinant protein expression system using R. erythropolis as the host cell is superior to respective E. coli systems. © 2004 Wiley Periodicals, Inc
New‐generation multicistronic expression platform: pTRIDENT vectors containing size‐optimized IRES elements enable homing endonuclease‐based cistron swapping into lentiviral expression vectors
Authors: Fux Cornelia , Langer Dominik , Kelm Jens M. , Weber Wilfried , Fussenegger Martin
Published: March 2004

Capitalizing on a proven multicistronic expression vector platform we have designed novel pTRIDENT vectors which (1) enable coordinated expression of three desired transgenes, (2) are size‐optimized, (3) take advantage of small highly efficient internal ribosome entry sites of the GTX or Rbm3 type, (4) harbor various sites specific for homing endonucleases facilitating promoter/multicistronic expression unit/polyadenylation site swapping as well as (5) straightforward integration into human HIV‐l‐based lentiviral expression vectors tailored to contain compatible homing endonucleases. Multicistronic expression profiles of novel pTRIDENT vectors engineered for different tricistronic expression configurations encoding human low‐molecular‐weight urokinase‐type plasminogen activator (u‐PALMW) or Bacillus stearothermophilus‐derived α‐amylase (SAMY), human vascular endothelial growth factor (hVEGF), and human placental secreted alkaline phosphatase (SEAP) have been quantified in Chinese hamster ovary cells (CHO‐K1), mouse fibroblasts (NIH/3T3), and/or human fibrosarcoma (HT‐1080) cells. In addition, a pTRIDENT‐derived SAMY‐VEGF‐SEAP expression cassette transferred into a compatible lentiviral expression vector enabled simultaneous high‐level transgene expression following transduction of transgenic lentiviral particles into primary human chondrocytes. © 2004 Wiley Periodicals, Inc.
Bulking sludge in biological nutrient removal systems
Authors: Martins Antönio M. P. , Heijnen Joseph J. , van Loosdrecht Mark C. M.
Published: March 2004

Bulking sludge problems are commonly reported in biological nutrient removal (BNR) systems. This has led to the general belief that intrinsic BNR conditions favor the growth of undesirable and excessive filamentous bacteria. The present study shows that other factors have a major role in bulking, and not the BNR conditions. These factors have been verified in well‐controlled, strictly anoxic–aerobic and strictly anaerobic–aerobic sequencing batch reactor systems. The experimental results show that conditions known to be responsible for bulking sludge in aerobic systems (i.e., low concentration of electron donor and/or electron acceptor) did not lead to bulking. Even when acetate was present at very low concentrations in the aerobic stage of an anaerobic–aerobic bio‐P system, the sludge settleability remained very good. This clearly demonstrates that good bio‐P activity can stabilize and improve sludge settleability. The presence of microaerophilic conditions in the anoxic stage of the anoxic–aerobic system was the only factor leading to worsening sludge settling characteristics. The results are discussed in light of our previous hypothesis about the importance of diffusion‐limited substrate uptake for the development of filamentous structures in biological flocs. The hypothesis is extended to anaerobic–aerobic and anoxic–aerobic conditions, typical of BNR‐activated sludge systems. Taking into account the effect of feeding patterns on biochemical rates and on the development of filamentous bacterial structures, we recommend the adoption of plug‐flow selector configurations, with strictly anaerobic and/or strictly anoxic conditions, wherein microaerophilic conditions are excluded, in order to maintain reliable and robust BNR performance. © 2004 Wiley Periodicals, Inc.
Catalytic antibody light chain capable of cleaving a chemokine receptor CCR‐5 peptide with a high reaction rate constant
Authors: Mitsuda Yukie , Hifumi Emi , Tsuruhata Kumi , Fujinami Hiroko , Yamamoto Naoki , Uda Taizo
Published: March 2004

A monoclonal antibody (MAb), ECL2B‐2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR‐5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B‐2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B‐2 could possess one or two catalytic triad(s), composed of Asp1, Ser27a (or Ser27e), and His93 (or His27d), in the light chain of ECL2B‐2. The three amino acid residues, Asp1, Ser27a, and His93, are identical to those of catalytic antibody light chains such as VIPase and i41SL1‐2. The light chain of ECL2B‐2 MAb degraded the antigenic peptide CCR‐5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (kcat) of 2.23 min−1, which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B‐2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR‐5 peptide. © 2004 Wiley Periodicals, Inc.