bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonpharmacological pain relief for labour pain: knowledge, attitude, and barriers among obstetric care providers
Authors: Abdel-Fatah Ibrahim Heba , Alshahrani Majed Said , Al-Qinnah Amlak Jaber , Elgzar Wafaa Taha
Published: February 2024

Background

Labor pain is considered the worst pain in a woman’s life. Hence, pain control should be essential to labor management at any level. There is scarce information, and there are gaps regarding the knowledge, attitude, and barriers to the utilization of nonpharmacological approaches for pain relief in Saudi Arabia. Therefore, the current study aims to evaluate nonpharmacological pain relief (NPPR)-related knowledge, attitudes, and barriers among obstetric care providers in Najran, Saudi Arabia.

Methods

A cross-sectional analytical study was performed at maternity departments in Maternal and Children Hospital (MCH), Najran, Saudi Arabia, from April 1 to May 26 2023. The study involved 186 obstetric care providers (OPCs), physicians (19), nurses (144), and midwives (23). A structured self-reported questionnaire was used to collect data and involves five main sections: demographic data, work-related data, nonpharmacological pain relief-related attitude, perceived barriers, and knowledge quiz. The adjusted odds ratio (AOR) along with 95% CI was estimated to determine the factors associated with nonpharmacological pain relief-related knowledge and attitude using multivariate analysis in the binary logistic regression.

Results

Over three-quarters (79%) of obstetric care providers had adequate knowledge of nonpharmacological pain relief methods. The majority (85.5%) of the participants had a positive attitude toward NPPR in labour pain management, with the mean scores ranging from 3.55–4.23 for all sub-items. Obstetric care providers acknowledged that patient belief, lack of time, and workload were the strongest barriers to offering nonpharmacological pain relief methods for labour pain 67.6%, 64.5%, and 61.3%, respectively. In binary logistic regression analysis, the in-service training related to nonpharmacological pain relief (AOR = 5.871 (2.174–15.857), p = 0.000), (AOR = 3.942 (1.926–11.380), p = 0.013) and years of work experience (AOR = 1.678 (1.080–2.564), p = 0.019), (AOR = 1.740 (1.188–2.548), p = 0.003) were significantly associated with obstetric care providers’ knowledge and attitudes regarding nonpharmacological pain relief (p ≤ 0.05).

Conclusion

Although most OPCs have adequate knowledge and a positive attitude regarding NPPR, they need motivational strategies to enhance their utilization. In addition, an effort should be made to decrease OPCs’ workload to provide more time for NPPR application and patient education. Training courses and in-service training can play an important role in enhancing NPPR knowledge and attitude and, consequently, its application. Also, in each working unit, the policymakers should provide clear guidelines and policies that enhance and control the utilization of NPPR.
Condições de saúde bucal e acesso aos serviços odontológicos dos quilombolas: uma revisão integrativa
Authors: Lopes Carolyne Brito , Dos Santos Pedro Henrique Barros , Moimaz Suzely Adas Saliba , Saliba Tânia Adas
Published: February 2024

A população quilombola enfrenta diversas desigualdades que podem afetar diretamente sua saúde e qualidade de vida. O objetivo deste trabalho foi realizar uma pesquisa de revisão integrativa de literatura sobre saúde bucal e acesso aos serviços odontológicos da população quilombola. A metodologia empregada consistiu na busca de estudos nas bases de dados Biblioteca Virtual em Saúde, Medline, Web of Science e Scopus, utilizando os descritores "Quilombolas", “Black or African American”, "Quilombola Communities", "Saúde bucal", "Oral Health" e "Salud Bucal". A pesquisa resultou em 410 títulos identificados, sendo que 16 atenderam aos critérios de elegibilidade. Destes, 13 foram realizados no Brasil, 2 nos Estados Unidos e 1 na Colômbia. Os trabalhos incluídos analisaram a prevalência de doenças bucais, autopercepção de saúde bucal, hábitos de higiene e acesso aos serviços odontológicos em comunidades quilombolas. Em todos os estudos, os quilombolas apresentaram alta prevalência de cárie, doença periodontal, edentulismo, hábitos precários de higiene bucal, autopercepção negativa de saúde bucal e acesso irregular aos serviços odontológicos. Concluiu-se que é necessário desenvolver políticas públicas de saúde bucal, considerando as particularidades culturais das comunidades quilombolas. Estratégias devem ser implementadas para promover o acesso equitativo aos serviços odontológicos, visando melhorar a qualidade de vida dessas populações.
Collaural Fistula on the Left-side of the Face: A Case Report
Authors: Gaurkar Sagar , Singh Chandraveer , Kaur Jasleen , Deshmukh Prasad T
Published: February 2024

Collaural fistula, also known as cervico-aural fistula, is a rare anomaly that accounts for less than 8% of all branchial cleft anomalies. These fistulas result from incomplete closure of the first branchial cleft during embryonic development. A 21-yearold female exhibited depression on the left-side of her face since childhood, accompanied by a fistula. She had a history of intermittent discharge and a tuft of hair emerging from it. Additionally, the patient reported left ear discharge and an opening in the floor of the External Auditory Canal (EAC). Diagnostic imaging, including X-ray fistulogram, ultrasound, and Magnetic Resonance Imaging (MRI) neck fistulogram, played a crucial role in confirming the diagnosis of a collaural fistula. Surgical intervention involved excision of the fistulous tract and reconstruction of the EAC floor. The patient received antibiotic therapy, resulting in successful postoperative outcomes. The present case highlights the importance of recognising and appropriately managing collaural fistulas. When these fistulas are discovered early, along with a solid grasp of regional embryology and anatomy, there is a chance for better treatment outcomes.
Mycoplasma and Neisseria Prevalence in Asymptomatic Women and Investigation of their Susceptibility to Tamarindus indica and Syzygium aromaticum Aqueous Extracts
Authors: Djinou Hermann Joe Djeukoua , Yemele Didiane Mefokou , Nodem Steve Francky Sohanang , Talom Benjamin Tangue , Newe Rosine Dicko , Yangai Sodea , Fodouop Simeon-Pierre Chegaing
Published: February 2024

Neisseria and Mycoplasma are two prevalent bacteria in the female urogenital tract leading to gynecological infections and infertility. The aim of this study was to assess the antibacterial activity of Tamarindus indica and Syzygium aromaticum extracts against Neisseria and Mycoplasma isolates. It was a cross cectional study on 60 asymptomatic women at the Protestant and Regional Hospital of Ngaoundere. For this reason, a large consecutive sample of patients with several well-defined characteristics was assembled and urine and cervical-vaginal swab were collected using standard procedures. After being isolated on a specific medium, several strains of Neisseria and Mycoplasma were identified based on their morphological and biochemical characteristics. The antibacterial activity of the plant extracts was tested using the agar-well diffusion method. It was discovered that 70% of asymptomatic women had overall infection, with varying prevalence rates. The prevalence rates of Neisseria and Mycoplasma were 14.29% and 85.71% respectively. The aqueous extracts of Syzygium aromaticum against Neisseria produced inhibitory diameters of 43 mm, 40 mm and 32 mm at doses of 20 mg/mL, 10 mg/mL, and 5 mg/mL, respectively while, at the same doses, the aqueous extract of Tamarindus indica produced inhibitory diameters of 16 mm, 14 mm and 13 mm, respectively. The combined extract of Syzygium aromaticum and Tamarindus indica exhibits inhibitory diameters of 25, 23, and 30 mm at 20 mg/mL, 10 mg/mL, and 5 mg/mL, respectively.  Syzygium aromaticum extract alone showed efficacy against Mycoplasma, with diameters of 16.5 mm, 13 mm and 10.5 mm at concentrations of 20 mg/mL, 10 mg/mL, and 5 mg/mL respectively. Inhibition diameters of 18 mm (for fosfomycin and ofloxacin), 22 mm (for chloramphenicol and ceftriaxone) and 26 mm (for levofloxacin) were found using Neisseria isolates. The only drugs that demonstrated efficacy against Mycoplasma were Minocycline and Josamycin.  Given these findings, extracts of Syzygium aromaticum and Tamarindus indica can be investigated and used to treat infections caused by Niesseria and Mycoplasma.
Representation of Racism in the Film "Till"
Authors: Asrita Stara , Alreswara Muhammad Syah Tegar , Rhizky Deani Prionazvi
Published: February 2024

The purpose of this study is to answer how signifier and signified are displayed in the film "Till" and what are the representations of racism contained in the film "Till". The data studied is in the form of pieces of images, sounds, text or sounds contained in the film "Till". This study is using Saussure's semiotics. It will help analyse media texts with a set of signs that have various meanings. The result of this study shows that the film "Till" contains acts of racism based on Henry and Tator's theory in the form of Individual Racism and Systemic Racism. This theory is divided from Internalized Racism, Interpersonal Racism, Institutional Racism and Structural Racism. Researchers examine each component of the types of racism such as reactions, associations, perspectives, identity, labelling, stereotypes, separatism, and discrimination.
Instagram as A Digital Media to Build Self-Acceptance
Authors: Putri Gavrilla Syahriani , Pratiwi Mutia Rahmi
Published: February 2024

One of the effects of Broken Home children is the difficulty in accepting their condition, which causes their Self-Acceptance. Children who find it challenging to build Self-Acceptance will affect their self-concept, so they continue to think negatively. This research focuses on analyzing the content of digital media Instagram that is present as a communication tool to provide educational information for generation Z related to specific problems, especially about Self-Acceptance. The presence of social media in the digital era can now be used as a means of long-distance communication and help educate its users. The social media that is popular among Generation Z is Instagram. The object of this research is the Instagram account @Behome.id. The data is obtained from content from the @Behome.id account, which has information about Self-Acceptance. This research uses a qualitative content analysis method with the theory of self-acceptance and new media. This research concludes that Behome.id provides tips such as a friend who embraces another friend by using a casual style of language, mapping broken home children tend to have low self-acceptance due to lack of self-disclosure to others, giving many tips to broken home children to be more can get their emotions out in order to express themselves.
Storytelling untuk Pembelajaran Keuangan Sejak Usia Dini
Authors: Anastasia Njo , Linawati Nanik
Published: February 2024

Storytelling pada anak usia dini melalui dongeng merupakan salah satu teknik pembelajaran yang dapat menggugah minat anak-anak. Karakter pemeran dalam dongeng dapat diceritakan dengan lebih menarik melalui intonasi suara dan gerakan untuk menggugah imajinasi anak-anak sehingga dapat lebih mudah dipahami. Permasalahnya, anak usia dini sulit memahami pengetahuan keuangan. Oleh karena itu, kegiatan pengabdian masyarakat ini bertujuan untuk menanamkan dan meningkatkan literasi keuangan pada anak usia dini melalui storytelling untuk menanamkan pendidikan karakter. Secara tidak langsung, dongeng dapat membentuk tabiat, sikap serta perilaku anak-anak salah satunya perilaku keuangan. Literasi keuangan yang disampaikan terkait kegiatan menabung serta menanamkan kata-kata penting tentang keuangan seperti hemat dan boros. Metode pelaksanaan dimulai dari pendataan sekolah yang diajak kolaborasi, mempersiapkan materi dan media pembelajaran sesuai karakteristik sekolah serta melakukan evaluasi akhir kegiatan. Media pembelajaran menggunakan cerita dongeng, boneka peraga, dan games. Kegiatan ini dilakukan lebih kurang satu semester pada tiap bulan sesuai jadwal yang ditentukan di sepuluh sekolah Taman Kanak-Kanak di Surabaya. Mahasiswa dan dosen turut berkolaborasi sebagai pemain karakter dalam dongeng yang diceritakan di kelas. Hasil kegiatan pengabdian masyarakat ini menunjukkan anak-anak antusias mengikuti aktivitas kelas saat sesi dongeng, merasa menjadi tokoh penting dalam dongeng sehingga ingin bertindak sesuai tokoh yang diperankan dengan membagikan atau menerapkan hal-hal baik pada teman dan keluarga. Anak-anak juga antusias bercerita kembali pada orang tua tentang kegiatan yang dilakukan saat sesi dongeng. Secara tidak langsung, orang tua turut dilibatkan untuk ikut membimbing anaknya terkait pembentukan karakter dan pengetahuan keuangan sejak usia dini. Pada akhirnya, kegiatan ini memberikan manfaat pada anak-anak di sekolah Taman Kanak-Kanak, dosen dan mahasiswa yang berkolaboasi untuk mengenalkan dan menanamkan pemahaman literasi keuangan sejak dini sehingga terbentuk generasi bijak keuangan.
Statistical Approaches to the Design of Toxicology Studies
Authors: Gad Shayne C.
Published: June 2004

Application of statistics to toxicological studies involves hypothesis testing, model fitting, and reduction of dimensionality. This unit reviews statistical approaches to data (i.e., descriptive statistics, experimental design, outliers and rounding of numbers, and specific applications) and includes a set of decision trees to assist in choosing the appropriate methods.
Transgenic Animals in Toxicology
Authors: Ladiges Warren C. , Ware Carol B.
Published: June 2004

Transgenic animals are a powerful tool in the exploration of the interaction of specific genes with the environment and/or toxins to understand the relation between biochemical and metabolic pathways and toxic effect. This overview discusses DNA construct design, methods for production of transgenic animals, including pronuclear fusion, gene targeting and chimera production, animal husbandry, and colony management.
Detection of Covalent Binding
Authors: Bruno Mary K. , Cohen Steven D.
Published: June 2004

Immunochemical detection of xenobiotics covalently bound to cellular proteins can provide information about toxic mechanism and is more specific than the alternative radiochemical studies. Both immunoblotting and immunohistochemical methods are used to pinpoint the target protein(s) and to identify the tissue targets. Also included in this unit are protocols for synthesizing artificial antigens, immunizing suitable host species, and using noncompetitive and competitive ELISA assays to characterize the antibodies produced.
Mutagenesis Assays in Mammalian Cells
Authors: Klein Catherine B. , Broday Limor , Costa Max
Published: June 2004

Mutagenesis assays in mammalian cells are frequently used to complement bacterial mutagenesis assays. This unit describes a mutagenesis assay using either Chinese hamster V79 cells or V79‐derivative gpt transgenic cell line to assess the effects of chemical agents on mammalian cells.
Cell Transformation Assays
Authors: Costa Max , Sutherland Jessica E.
Published: June 2004

Morphological transformation of mammalian cells following exposure to a chemical agent is frequently used as a preliminary screen for the carcinogenic potential of the agent. This unit describes a protocol using Syrian hamster embryo (SHE) cells to assay transformation.
Measurements of UDP‐ Glucuronosyltransferase (UGT) Activities
Authors: Coffman Birgit L. , Rios Gladys R. , Tephly Thomas R.
Published: June 2004

Mammalian UDP‐glucuronosyltransferases are a family of isoenzymes that catalyze the reaction of endobiotics and xenobiotics with glucuronic acid resulting in the formation of hydrophilic glucuronides. This pathway is an important step in the metabolism and subsequent excretion of many compounds that would otherwise have toxic effects. This unit describes three methods for measuring UGT activity. Thin layer chromatography is a powerful screening method and may be used to analyze multiple substrates simultaneously. The Sep‐Pak C18 cartridge extraction method has been developed to specifically separate opioid glucuronides from UDP‐glucuronic acid. Finally, the ethyl acetate extraction method is used to separate the glucuronides of bilirubin, sterols, and vile acids from UDP‐glucuronic acid. These methods may be applied to a microsomal fraction or to cultured cells transformed with cDNA for UGT.
Detection of Metabolites Using High‐Performance Liquid Chromatography and Mass Spectrometry
Authors: McClure Thomas D.
Published: June 2004

This unit provides a discussion of high‐pressure liquid chromatography‐atmospheric pressure ionization mass spectrometry (LC‐API‐MS) for separation, detection, and identification of products from xenobiotic metabolism. It includes an introduction and important technical information for electrospray and atmospheric pressure chemical ionization methods as well as scan modes. Emphasis has been placed on each method as related to phase I and phase II metabolite identification and there are listings of specific applications (and references) to reduce method development time.
Isolated Perfused Porcine Skin Flap
Authors: Baynes Ronald E.
Published: June 2004

The isolated porcine skin flap is used to determine the rate and extent of absorption of chemicals through skin. This ex vivo model system allows cutaneous toxicology and pharmacology studies to be conducted in viable skin that has a normal structure and intact microvasculature. Porcine skin is also anatomically and physiologically similar to human skin, therefore data from studies using this ex vivo system can in some cases to predict dermatopharmacokinetics and dermotoxicity to test compounds in humans.
Porcine Skin Flow‐Through Diffusion Cell System
Authors: Baynes Ronald E.
Published: June 2004

Porcine Skin Flow‐Through Diffusion Cell System (Ronald E. Baynes, North Carolina State University, Raleigh, North Carolina). Porcine skin can be used in a diffusion cell apparatus to study the rate and extent of absorption of topically applied chemicals through the skin. Although the skin of a number of animals can be used in this system, that of the pig most closely approximates human skin anatomically and physiologically.
Measurement of Glutathione Transport
Authors: Lash Lawrence H.
Published: June 2004

This unit provides methods for analyzing uptake in rat kidney proximal tubule cells and rat kidney cortical mitochondria, preparation of proximal tubule cells and cortical mitochondria, and HPLC analysis of glutathione and related compounds.
Analysis of Glutathione‐Related Enzymes
Authors: Esworthy R.S. , Chu F.‐F. , Doroshow J.H.
Published: June 2004

Glutathione peroxidase activity can be measured using a variety of substrates to define total glutathione peroxidase activity or the activity of subsets of the family of enzymes. The assays for these activities are spectrophotometric assays, and the specificity depends on the choice of substrate and/or prior treatment of the samples by chromatography or immunoprecipitation. This unit provides the assays and support protocols for synthesizing substrates and calculating specific activity.
Overview of Heme Degradation Pathway
Authors: Maines Mahin D.
Published: June 2004

Heme oxygenase 1 and 2 activities are responsible for initiating most of the degradation of heme, although other enzyme pathways play a role as well. The degradation pathway also includes biliverdin reductase, the activity of which is coupled to oxidation of NADH and NADPH. This overview discusses the pathways and enzymes involved in heme degradation.
Detection of Heme Oxygenase 1 and 2 Proteins and Bilirubin Formation
Authors: McCoubrey William K.
Published: June 2004

It is possible to detect changes in the level of expression of the two isozymes of heme oxygenase (HO). The sequence differences allow the production of isozyme‐specific antibodies that can be used in immunoblot analysis. Total HO activity in tissue microsomal fractions or extracts of bacteria transformed with an HO gene can be measured as described in this unit using a coupled assay employing biliverdin reductase, which converts the biliverdin product to bilirubin. Bilirubin is then quantified with a scanning spectrophotometer.
An HPLC Method to Detect Heme Oxygenase Activity
Authors: Ryter Stefan W. , Tyrrell Rex M.
Published: June 2004

This unit presents a method to calculate heme oxygenase enzymatic activity from the formation of bilirubin equivalents [biliverdin‐Ixalpha (BV) and bilirubin‐IXalpha (BR)]. The BV and BR generated in the reaction are separated by reversed‐phase HPLC and detected using visible absorbance spectroscopy. Since both metabolites of heme degradation are directly quantifiable, the assay eliminates the requirement for biliverdin reductase supplementation.
Measurement of cGMP and Soluble Guanylyl Cyclase Activity
Authors: Southam Eric
Published: June 2004

Soluble guanylyl cyclase is responsible for synthesis of guanosine 3'5'‐cyclic monophosphate. The enzyme is activated by NO, so measurement of tissue levels of cGMP and the activity of guanylyl cyclase is important in studies of NO‐cGMP signal transduction. The method of choice for measuring tissue levels of cGMP is a radioimmunoassay, but it can also be measured using a scintillation proximity assay. Guanylyl cyclase activity is determined by measuring the conversion of GTP to cGMP using a radioimmunoassay or by following the conversion of [32P]GTP to [32P]cGMP, with separation of the product by precipitation and chromatography on an alumina column.
Principles of Electrophysiology: An Overview
Authors: Narahashi Toshio
Published: June 2004

This overview gives brief accounts of the mechanism of nerve excitation, receptor/channel functions, cable properties of neurons, and voltage clamp and patch clamp principles. Among various aspects of electrophysiology, emphasis is placed on the mechanistic studies of receptors/channels. A few sample records of membrane currents are illustrated to aid reader's comprehension. Finally some useful reference books are given for more comprehensive information.
Electrophysiological Studies of Neurotoxicants on Central Synaptic Transmission in Acutely Isolated Brain Slices
Authors: Yuan Yukun , Atchison William D.
Published: June 2004

Effects of neurotoxic chemicals on central synaptic function can be assessed using electrophysiological recording techniques in freshly isolated brain slices. The three most commonly used electrophysiological recording methods in brain slices are extracellular microelectrode recording, intracellular microelectrode recording, and whole‐cell patch clamp recording. This unit presents the basic procedures and applications of extra‐ and intracellular recordings in hippocampal slices and whole‐cell recording in cerebellar slices.
Isolation of Liver Kupffer Cells
Authors: Froh Matthias , Konno Akira , Thurman Ronald G.
Published: June 2004

Isolation of Liver Kupffer Cells (Matthias Froh, Akira Konno, Ronald G. Thurman, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina). Kupffer cells, the resident macrophages of the liver, play a major role in the pathogenesis of several diseases. This unit contains an easy‐to‐follow procedure for effective isolation of liver Kupffer cells from rats and mice. The protocol provides viable Kupffer cells in large amounts that can be used for further investigations.
Embryonic Stem (ES) Cell Culture Basics
Authors: Schmidt Jennifer V.
Published: June 2004

This unit describes the procedures to grow and maintain embryonic stem (ES) cells in culture. Growth‐inactivated mouse embryo fibroblasts (MEF) are required as a feeder layer for the ES cells; their isolation and culture is described as are the additional protocols required for generating a mouse with a particular targeted gene electroporation, cloning and selection for homologous recombinants, transfection with Cre, and preparation of cells for injection.
Overview of the Physical State of Proteins within Cells
Authors: Petty Howard R.
Published: June 2004

To understand how proteins work it is necessary to understand their physical state within the cell. This unit reviews the classification of proteins, how that is related to the hydropathicity of the protein, other factors that affect the heterogeneity of proteins, protein assemblies, methods for altering the solubility of proteins, and limitations of in vitro manipulations of proteins.
Computational Methods for Protein Sequence Analysis
Authors: Michaels George , Garian Robert
Published: June 2004

This unit is presented as a guide to addressing the issue of what to do with a protein sequence once it is obtained. A theoretical background for protein sequence analysis is provided first, followed by a discussion of matrix methods for sequence comparison (). Sequence similarity searching is then presented, including the BLAST and FASTA databases. Other aspects of protein sequence analysis covered here are alignment methods, scoring matrices, multiple alignments, cluster methods and trees, and identification of functional sites.
Internet Basics
Authors: Baxevanis Andreas D. , Ouellette B.F. Francis
Published: June 2004

With the explosion of sequence and structural information available to researchers, the field of bioinformatics is playing an increasingly large role in the study of fundamental biomedical problems. The challenge facing computational biologists will be to aid in gene discovery and in the design of molecular modeling, site‐directed mutagenesis, and experiments of other types that can potentially reveal previously unknown relationships with respect to the structure and function of genes and proteins. This challenge becomes particularly daunting in light of the vast amount of data that has been produced by the Human Genome Project and other systematic sequencing efforts to date. This unit begins with a review of the Internet and its terminology, also discussing major classes of Internet protocols, without becoming overly engaged in the engineering minutiae underlying these protocols. Matters of connectivity, ranging from simple modem connections to digital subscriber lines (DSL) are also discussed. Finally, one of the most common problems that has arisen with the proliferation of Web pages throughout the world is addressed‐‐i.e., finding useful information on the World Wide Web.
Protein Tertiary Structure Modeling
Authors: Guex Nicolas , Schwede Torsten , Peitsch Manuel C.
Published: June 2004

Insights into the 3D‐structure of a protein have proven useful during experiment design. Experimentally elucidated structures are often not available, but comparative protein modeling provides a viable alternative in many cases. This unit presents comparative protein modeling and how to use the highly sophisticated but easy‐to‐use free software available on the Internet.
Overview of Protein Expression in Pichia pastoris
Authors: Higgins David R.
Published: June 2004

Pichia pastoris is a methylotrophic yeast and can be used as a heterologous expression system. This microorganism is as easy to manipulate as Escherichia coli, but has many of the advantages of eukaryotic expression (e.g., protein processing, folding, and post‐translational modifications), and it is faster, easier, and cheaper to use than other eukaryotic expression systems, such as baculovirus or mammalian tissue culture. It also generally yields higher expression levels. This overview discusses important considerations for the use of Pichia pastoris, including strains for expression, expression plasmids, transformation by integration, and post‐translational modifications. Examples of expression are given and finally, legal issues regarding patent rights for heterologous protein expression in Pichia pastoris are described.
Production of Recombinant Proteins in Mammalian Cells
Authors: Chen Su , Gray David , Ma Johnny , Subramanian Shyamsundar
Published: June 2004

The best strategy for consistent production of larger quantities of pure protein is stable expression. Popular hosts for stable expression are Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK‐21) cells, myeloma cells, and the transformed kidney cell line 293. Protocols for stable production in CHO cells are described in this unit. Typical methods for transfection using commercially available plasmid expression vectors are described, along with methods to select for stable expression and methods for amplifying the expression level in the transfected cell. Following this, procedures are presented for efficient cell growth to obtain significant amounts of protein product. Support protocols describe freezing of cells, determination of growth rates, determination of specific productivity of cells, preparing samples for assay, and setting up a 10‐day shaker‐flask growth curve.
Characterizing Recombinant Proteins Using HPLC Gel Filtration and Mass Spectrometry
Authors: Begg Gillian E. , Harper Sandra L. , Speicher David W.
Published: June 2004

Recombinant proteins are subject to many forms of heterogeneity, including aggregation, proteolytic degradation, chemical modification, mutation, and incorrect translation. This unit describes methods for the detection and identification of these problems using analytical HPLC gel filtration and MALDI‐MS. Preliminary characterization of recombinants is necessary before the structure or function of the protein can be investigated.
Hydroxylapatite Chromatography
Authors: Broadhurst Anne V.
Published: June 2004

Hydroxylapatite (also called hydroxyapatite), a form of calcium phosphate, can be used as a matrix for the chromatography of both proteins and nucleic acids. Protocols are provided for both standard low‐pressure chromatography of a protein mixture using a hydroxylapatite column prepared in the laboratory, and an HPLC method, applicable to proteins and nucleic acids, that uses a commercially available column. Alternate protocols describe column chromatography using a step gradient or batch binding and step‐gradient elution.
Lectin Affinity Chromatography
Authors: Freeze Hudson H.
Published: June 2004

This unit describes the use of lectins for preparative glycoprotein purification. Con A‐Sepharose and WGA‐agarose are used for convenience and availability. Instructions are given for a small‐scale pilot procedure to test for lectin binding and to determine elution conditions. There are many variations on the basic procedure in the literature, but all use the same principles: bind the protein to immobilized lectin through its sugar chain, wash away unbound protein, and elute bound protein with a simple sugar that resembles the sugar ligand of the bound protein.
Metal‐Chelate Affinity Chromatography
Authors: Petty Kevin J.
Published: June 2004

Recombinant proteins engineered to have six consecutive histidine residues on either the amino or carboxyl terminus can be purified using a resin containing nickel ions (Ni2+) that have been immobilized by covalently attached nitrilotriacetic acid (NTA). This technique, known as metal‐chelate affinity chromatography (MCAC), can readily be performed with either native or denatured protein. This unit discusses techniques for creating a fusion protein consisting of the protein of interest with a histidine tail attached. A procedure for xpression of histidine‐tail fusion proteins and their purification in native form by MCAC is described, and two alternate protocols describe purification of histidine‐tail fusion proteins by MCAC under denaturing conditions and their renaturation by either dialysis or solid‐phase renaturation. Support protocols are provided for analysis of the purified product and regeneration of the NTA resin. All of these protocols are easily adaptable to any protein expression system.
Immunoaffinity Chromatography
Authors: Springer Timothy A.
Published: June 2004

This unit describes the isolation of soluble or membrane‐bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. This technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. Specifically, antibodies are coupled to Sepharose (an insoluble, large‐pore‐size chromatographic matrix). High‐molecular‐weight antigens pass freely into and out of the pores and bind to antibodies covalently bound to the matrix. To elute the bound antigen from the immunoaffinity matrix, the antibody‐antigen interaction is destabilized by brief exposure to high‐ or low‐pH buffer. Batch purification of antigens is provided as an alternate procedure that shortens the column loading time. The detergent octyl β‐D‐glucoside can be used instead of Triton X‐100 for elution. Because octyl β‐D‐glucoside has a high critical micelle concentration (CMC), a protocol is provided for its removal by dialysis. The procedure for covalently linking an antibody to Sepharose using the cyanogen bromide activation method is given in a support protocol.
Removal of N‐Terminal Blocking Groups from Proteins
Authors: Fowler Elizabeth , Moyer Mary , Krishna Radha G. , Chin Christopher C.Q. , Wold Finn
Published: June 2004

Two enzymatic methods commonly used in N‐terminal sequence analysis of blocked proteins are presented in this unit; one uses pyroglutamate aminopeptidase for Nα‐pyrrolidone carboxyl‐proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl‐peptide hydrolase for Nα‐acyl‐proteins blocked with other acyl groups. A describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl‐peptide hydrolase must include fragmentation of the protein before unblocking can be carried out, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N‐terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis. Protocols are also provided for unblocking N‐terminally blocked proteins using acid‐catalyzed hydrolysis or methanolysis, hydrazinolysis, and β‐elimination after acid‐catalyzed N‐O shift. Alternate protocols describe chemical removal of acetyl and longer‐chain alkanoyl groups, as well as formyl groups to open the cyclic imide of pyrrolidone carboxylate.
C‐Terminal Sequence Analysis
Authors: Bergman Tomas , Cederlund Ella , Jörnvall Hans , Fowler Elizabeth
Published: June 2004

Carboxy‐terminal (C‐terminal) sequence analysis is used for direct confirmation of the C‐terminal sequence of native and expressed proteins, for detection and characterization of protein processing at the C‐terminus, for identification of post‐translational proteolytic cleavages, and for obtaining partial sequence information on N‐terminally blocked protein samples in order to facilitate design of oligonucleotide probes for gene cloning. This unit describes an automated chemical method and a manual enzymatic (carboxypeptidase digestion) method for determining C‐terminal sequence information. Carboxypeptidase digestion requires only a standard amino acid analysis method.
Detection of Phosphorylation by Enzymatic Techniques
Authors: Shenolikar Shirish
Published: June 2004

This unit presents protocols for examining the functional effects elicited by nonspecific acid or alkaline phosphatases that dephosphorylate many phosphoproteins in vitro. Additional protocols describe digestion of phosphoproteins with a protein serine/threonine phosphatase and protein tyrosine phosphatase. A support protocol has been included to identify the radiolabel as 32PI based on its ability to form a complex with ammonium molybdate.
Analysis of Disulfide Bond Formation
Authors: Braakman Ineke , Hebert Daniel N.
Published: June 2004

In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein is then chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS‐PAGE with and without prior reduction. The difference in mobility observed between the gels with unreduced and reduced samples is due to disulfide bonds in the unreduced protein.
Analysis of Protein Acylation
Authors: Jackson Caroline S. , Magee Anthony I.
Published: June 2004

Protein acylation is the covalent attachment of fatty acids to a protein; the most commonly added fatty acids are myristate (14:0) and palmitate (16:0). In this unit, protocols describe the use of radiolabeled fatty acids to label eukaryotic cells in vitro. The radiolabeled material produced can then be analyzed by the various methods described here: determination of the type of fatty acid linkage, checking for interconversion by determining the nature of the protein‐bound label, and identification of the protein‐bound fatty acid.
Analysis of Oxidative Modification of Proteins
Authors: Yan Liang‐Jun , Sohal Rajindar S.
Published: June 2004

Protein oxidation has been implicated in a variety of degenerative diseases as well as in the aging process. This unit describes techniques for the quantification of various protein oxidation products, including protein carbonyls, loss of protein thiol groups, dityrosine and nitrotyrosine, and isoaspartate formation. Such oxidatively modified products may also be used as biomarkers for the assessment of oxidative stress during aging and/or disease.
Searching Sequence Databases Over the Internet: Protein Identification Using MS‐Tag
Authors: Jiménez C.R. , Huang L. , Qiu Y. , Burlingame A.L.
Published: June 2004

Although peptide mass mapping is fast and simple, its success can be compromised by the purity of the protein in one gel spot, errors in the sequence database, and the detection of too few peptides in the MS map of a given protein to permit reliable database matching. In those cases, partial amino acid sequence that is determined using post‐source decay (PSD) analysis or tandem mass spectrometry is required to establish the correct protein identification among possibilities suggested by MS‐Fit. Post‐source decay (PSD) involves the detection of the fragmentation product ions (metastable ions) of a selected mass value window containing the precursor ion chosen, that occur in the field‐free region between the ion source and the reflectron. This unit covers some of the practical issues in obtaining and analyzing a typical PSD spectrum using MS‐Tag, a database interrogation program that enables the identification of proteins with a limited number of peptide fragment ions by matching existing database sequences. It is a user‐friendly program that simultaneously considers a variety of ion types, but still allows single‐mismatch‐tolerant searching. Although MALDI‐PSD database searching is a widely used tool, an alternative method also described in this unit is analysis by MALDI collision‐induced dissociation (MALDI‐CID).
Overview of Protein Structural and Functional Folds
Authors: Sun Peter D. , Foster Christine E. , Boyington Jeffrey C.
Published: June 2004

This overview provides an illustrated, comprehensive survey of some commonly observed protein‐fold families and structural motifs, chosen for their functional significance. It opens with descriptions and definitions of the various elements of protein structure and associated terminology. Following is an introduction into web‐based structural bioinformatics that includes surveys of interactive web servers for protein fold or domain annotation, protein‐structure databases, protein‐structure‐classification databases, structural alignments of proteins, and molecular graphics programs available for personal computers. The rest of the overview describes selected families of protein folds in terms of their secondary, tertiary, and quaternary structural arrangements, including ribbon‐diagram examples, tables of representative structures with references, and brief explanations pointing out their respective biological and functional significance.
Disulfide Bond Formation in Peptides
Authors: Chen Lin , Annis Ioana , Barany George
Published: June 2004

The formation of disulfide bridges is often a crucial final stage in peptide synthesis. There is compelling evidence that the disulfide pattern can be critical in the folding and structural stabilization of many natural peptide and protein sequences, while the artificial introduction of disulfide bridges into natural or designed peptides may often improve biological activities/specificities and stabilities. This unit provides a highly selective, albeit state‐of‐the‐art, menu of procedures that can be performed to establish intramolecular or intermolecular disulfide bridges in targets of varying complexities.
Overview of the Quantitation of Protein Interactions
Authors: Philo John S.
Published: June 2004

The biological function of many proteins involves reversible interactions with other proteins, nucleic acids, or other non‐protein ligands. Such interactions play many different roles in a wide range of cellular processes. A few examples are: (1) storing or transporting key metabolites (e.g., O2 storage by myoglobin); (2) forming and maintaining the quaternary structure of multi‐subunit enzymes; (3) specific binding and recognition events (antigen‐antibody, hormone‐receptor, transcription factor‐promoter); and (4) self‐assembly of large structures (microtubules, chromatin). Thus, the quantitative characterization of such interactions represents an important part of understanding the function of such proteins and their role in these cellular events. This unit sets the tone for the rest of the chapter, and gives important information necessary to understand some of the topics that will be covered in future supplements, such as sedimentation equilibrium (analytical and micro‐preparative), surface plasmon resonance (SPR), size‐exclusion chromatography (SEC) with on‐line light scattering, and chemical cross‐linking.
Measuring Protein Interactions by Optical Biosensors
Authors: Schuck Peter , Boyd Lisa F. , Andersen Peter S.
Published: June 2004

This unit gives an introduction to the basic techniques of optical biosensing for measuring equilibrium and kinetics of reversible protein interactions. Emphasis is given to the description of simple and robust approaches that will provide reliable results. How to avoid the most commonly encountered problems and artifacts is also discussed.
An Overview of Serine Proteases
Authors: Hedstrom Lizbeth
Published: June 2004

This unit summarizes the families of serine proteases and their mechanism of catalysis. Methods for assays and determining substrate specificity are briefly described. The mode of action of commonly available inhibitors is also included.
Assaying Proteases in Cellular Environments
Authors: Wang Kevin K.W.
Published: June 2004

To better study protease activation, regulation, and inhibition physiologically or pharmacologically, it is often advantageous to use cell‐based systems. The introduction of the cellular environment poses additional challenges for the assay of the protease of interest. In this unit, various methods for monitoring protease activity in cell‐based systems are discussedctivity is discussed. The scope of the unit is restricted to mammalian cell system. It is also stressed that specific considerations or requirements for a particular protease or peptidase are often needed in order to optimize the assay.
Monitoring Metalloproteinase Activity Using Synthetic Fluorogenic Substrates
Authors: Troeberg Linda , Nagase Hideaki
Published: June 2004

Fluorogenic synthetic substrates are commonly used to monitor the activity of peptidases in vitro. This unit presents a representative protocol that employs (7‐methoxycoumarin‐4‐yl)acetyl‐Pro‐Leu‐Gly‐Leu‐(3‐[2,4‐dinitrophenyl]‐L‐2,3‐diaminopropionyl)‐Ala‐Arg‐NH2 (Mca‐Pro‐Leu‐Gly~Leu‐Dpa‐Ala‐Arg‐NH2) as a substrate to assay matrix metallopeptidases (MMPs). This substrate was first described for the assay of MMP‐1, ‐2 and ‐3 and it is now widely used as a general MMP substrate. Protocols are given for both stopped‐time assays (suitable for assaying MMP activity in a large number of samples) and continuous assays (commonly used when establishing an assay protocol or investigating kinetic aspects of enzyme behavior). Other fluorogenic peptides and protein substrates, together with non‐fluorogenic alternatives, are also discussed.
Receptors as Drug Targets
Authors: Williams Michael
Published: June 2004

This overview introduces the basic terminology and theory of receptor‐ligand interactions. Receptors are envisaged as cell surface recognition sites for endogenous hormones, neurotransmitters, and neuromodulators. Receptor selectivity, binding saturability and reversibility, and functionality are discussed in this unit, with special emphasis placed on receptors being targets for drug development. The term Receptor is now used in a broad sense to describe any recognition site for a compound, the latter being defined as an exogenous chemical or biological substance that acts through a binding site or receptor in tissues. Thus, enzymes, uptake sites, ligand‐binding proteins, voltage‐gated ion channels, and intracellular targets are also envisaged as receptors. The use of recombinant receptor systems, receptor mutations and chimeras and allosteric ligands are also described in the setting of drug discovery and development.
Characterization of Opioid Receptors
Authors: DeHaven Robert N. , DeHaven‐Hudkins Diane L.
Published: June 2004

This unit presents two convenient radioligand binding assay methods for opioid receptors. These are applicable to all three of the opioid receptors that have been cloned to date (the mu, kappa and delta receptors), and can be used with numerous commercially available radiolabeled ligands. Moreover, they can serve as reasonable starting points in the development of new assays. A method for determining the binding of radioligand to cloned opioid receptors expressed on the surface of cultured cells is detailed along with a similar method to study radioligand binding to receptors from tissue homogenates. Also included is a procedure for titrating inhibitors of opioid receptor‐ligand binding, along with guidelines for analysis of the resulting data.
Characterization of Potassium Channel Binding
Authors: Scott Victoria E.S.
Published: June 2004

This unit provides an introduction to the binding of specific ligands to K+channels that are expressed in a number of different preparations. The assays described can be used for screening a large number of compounds for interaction at specific binding sites and provides, in selected cases, details of expected results for unlabeled ligands that are known to bind to certain K+channels.
Characterization of Histaminergic Receptors
Authors: Hancock Arthur A. , Esbenshade Timothy A.
Published: June 2004

Histamine mediates vascular smooth muscle relaxation and bronchial smooth muscle contraction, but also acts as a neurotransmitter, mediating gastric acid release and modulating the release other neurotransmitters in addition to histamine. In this unit, radioligand binding protocols are described for each of the known subtypes of histamine receptors, namely, H1, H2, and H3. These receptors demonstrate low amino acid sequence homology at the protein level and have divergent pharmacological characteristics such that the conditions described for assaying each receptor are dissimilar.