bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural features and gel-forming activity of zosteran-pectin of marine phanerogams
Authors: Ovodov Yu S , G.Ovodova R , D.Sorochan V
Published: November 2023

Phanerogams of Zosteraceae (eelgrasses) are widespread in the sublittoral of the World Ocean especially in the coastal zone of the Sea of Japan. Eelgrasses may be successfuly used as commercial reserves. The peculiar systematism and ecology of these unusual plants indicated interesting features of their chemical composition. The polysaccharide obtained was named zosteran. The gel-forming properties of Zosteran have been discovered. Zosteran was suggested utilization. for industrial.
Characterisation of carrageenan from various red seaweeds
Authors: Ahmad Fasihuddin B , Williams Peter A
Published: November 2023

Five spesies offed seaweed i. e. Acanthophora sp., Halymenia sp., Hypnea sp. 1, Hypnea sp. 2 and Laurencia sp have been studied to characterize the carrageenan content. The percentage yield varied between 23.0 to 55.2% when extracted in water at 90 100°C for 3 hours. Higher yield were observed when the samples were extracted in 0.2% NaCl, 0.2% NaOH or 0.2% Ca(OH\. Intrinsic viscosity for the samples studied varied between 635 to900 ml/g andcarrageenan extracted fromAcanthophora sp gaves the highest intrinsic viscosity. The 3, 6-anhydrogalactose content for all the carrageenan samples studied varied between 5.0 to 35.0% while the ester sulphate content varied between 16.1 to 30.0%. Carrageenan extracted fromLaurencia sp. gave the lowest 3, 6an hydrogalactose content while carrageenan extracted fromHalymenia sp. gaves the highest 3, 6anhydrogalactose and lowest ester sulphate content. Based on the 3, 6-anhydrogalactose, ester sulphate andinfraredspectrumsit can be concluded thatHalymenia sp. andHypnea sp. gives almost pure kappa carrageenan, while Acanthophora sp. andLaurencia sp gives lambda-carrageenan with some mixture of kappa and iota fraction. Gel strength of the samples were also studied and the result showed thatcarrageenanextractedfromHalymenia sp. can gives gel as strong as gel prepared from carrageenan extracted from Eucheuma cottonii and Chondrus crispus.
The sedimentation of a single particle in xanthangalactomannan gum mixtures
Authors: Luyten H , Kloek W , Vliet T Van
Published: November 2023

A combination of xanthan with a galactomannan can give a dispersion an (apparent) yield stress. The measured viscosity of such systems at stresses higher than the yield stress depended on the method used. We observed that at the same stresses, the Stokes’ sedimentation viscosity was much larger than the viscosity measured with an apparatus (table 1 and (1)). Differences could be up to a few orders of magnitude.
Viscoelastic properties of PVA/Alginate snake-cage hydrogels and interpenetrating hydrogels
Authors: Yonese Masakatsu , Baba Kazuhiko , Kishimoto Hiroshi
Published: November 2023

Viscoelastic properties of blend gels composed of poly(vinyl alcohol) (PVA) and alginate (Alg) were studied by measuring tensile stresses. From PVA and Alg, four kinds of snake-cage and interpenetrating hydrogels were prepared. The interpenetrating gels, i. e. both polymers forms networks, showed greater elastic moduli and ultimate tensile strength than the snake-cage gels, i. e. the network of one polymer traps other polymer chains. Furthermore, rapid increase in the tensile stress was shown concomitant with the transition from the snake-cage to the interpenetrating hydrogel.
Influence of sucrose and pulp contents on non-oral texture of strawberry gels
Authors: M.Fiszman S , Costell E , Duran L
Published: November 2023

Non-oral sensory evaluation of texture of strawberry gels of different composition was performed by a ten members trained panel. Two series of 15 samples were prepared using kappa carrageenan (C) and the mixture of C plus locust bean gum (LBG) ( 1: 1), with strawberry pulp and sucrose added in different proportions. Eight parameters were evaluated in two steps: compression between fingers up to rupture and resistance to cut with a knife. ANOVA applied to sensory data for each series showed significant effects (p:S 0.05) of both pulp and sucrose additions to gel samples on most of the analysed parameters. Presence of pulp generally produced weakening of gels. Increasing addition of sucrose up to about 50 degrees Brix produced no effect on the firmness of gels; however, further increase of sugar level resulted in a fall of scores corresponding to compression between fingers parameters. Additionally, MANOVA applied to each series confirmed the influence of the two composition factors and their interaction on non-oral texture of these gellified products.
Effect of carrageenan on low fat, low salt and phosphate free meat emulsion systems
Authors: Hoegh L
Published: November 2023

The effects of adding 0.4 % or 0.8 % Iota carrageenan (IC) KappaCarrageenan (KC), Kappa-Carrageenan + Locust Bean Gums (KLB) and a mixture of Kappaand Iota-carrageenan and Locust Bean Gum (MIX) to low fat, low salt and phosphate free frankfurters and mortadella and low fat liver paste sausages were investigated. Texture and sensory evaluation indicated that 0.8 % MIX added to low fat frankfurters (10 % fat), mortadella (10 % fat) and liver paste (20 % fat) were acceptable as control sausages (30, 20 and 40 % fat, respectively). KC and KLB developed to dry textures and IC to soft texture. 0.6 % and 0.7 % MIX added to low salt and phosphate free mortadella and frankfurters, respectively, resulted in sensoric acceptable products comparable to control sausages.
The role of crystallisation in the process of pectin/glycerine solution gelation
Authors: A.Sheyenson V , I.Khomutov L , A.Lashek N , Ptitchkina N .M
Published: November 2023

The methods of differential thermal analysis, X-ray phase analysis and thermo mechanics were used to study the nature of the gel-like state of the systems the polymeric component of which was pectin. The experimental data obtained show that the processes of macromolecule micro crystallization play an important role in the gelation mechanism.
The effect of proteins on emulsion stability
Authors: R.Fisher3 L , E.Mitchell E
Published: November 2023

Modern methods of surface analysis have largely confirmed the traditional picture of an adsorbed protein film at the oil/water interface as consisting of a tightly packed, virtually irreversibly adsorbed monolayer of partially unfolded molecules, with which some less tightly bound, reversibly bound protein may also be associated. They have added to this picture the information that a proportion of the protein is mobile in the plane of the interface and that occasional .loops or tails of the polypeptide chain may protrude from the interface into the aqueous phase. Competitive exchange studies have revealed some detail of the chances of displacement of adsorbed protein by small molecule surfactants or other proteins.
Interfacial properties of xanthan gum
Authors: Takigami S , A.Williams P , O.Phillips G
Published: November 2023

The adsorption characteristics of xanthan gum onto a model substrate, i. e. monodisperse, spherical polystyrene latex, have been studied. Xanthan gum adsorbed onto the polystyrene latex from both aqueous and 0.5 M NaCl solutions and low affinity isotherms were obtained in the both cases. The configuration of the adsorbed polymer was studied using the nitro oxide spin label technique and, at the surface coverages studied, the results suggested that xanthan gum adsorbed onto the latex surface in the form of trains from both water and NaCl solution. Colloid stability monitored by turbidity measurements revealed that xanthan gum did not affect the stability of polystyrene latex in aqueous solution in the absence of electrolyte. In NaCl solution, the latices flocculated at low polymer surface coverage due to a bridging mechanism but were stabilized at high polymer surface coverage through steric repulsive forces. At even higher xanthan concentrations flocculation was observed which was attributed to depletion phenomena.
Chemically modified guar gum (galactomannan) having emulsion stabilising properties
Authors: Mathur R , K.Mathur N
Published: November 2023

The polysaccharide guar gum which is naturally hydrophilic haves rise to thickening in aqueous systems, but it is not compatible to water immiscible organic-liquids and does not produce stable emulsions. Incorporating fatty acyl or medium sized alkoxyl groups in its molecule produces a derivatized gum which prod ceshigh viscosity in solutions containing lower alcohols and mulsifying oils. Hence these can find food applications.
An enzyme linked immunosorbent assay (ELISA) for Acacia senegal gum exudate (gum arabic)
Authors: R.Menzies A , E.Osman M , O.Phillips G , A.Williams P
Published: November 2023

Although gum arabic has been an article of commerce for over 4,000 years\ no definitive assay for this heterogeneous food additive exists. Using immunoassay technology a sensitive, rapid and specific ELISA for Acacia senegal gum has been developed. The assay can differentiate Acacia seneKal gum from other Acacia gum species and also from other commonly used food hydrocolloids including other tree exudates such as gums ghatti, tragacanth and karaya.
The lymphomas and chronic lymphoproliferative disorders
Authors: Harrison C J
Published: November 2023

Lymphomas and chronic lymphoproliferative disorders represent two highly heterogeneous groups of malignant disease. Both arise as a result of neoplastic proliferation of lymphatic cells. Far fewer specific chromosomal rearrangements associated with a particular pathological type have been described in these diseases than any of the other groups of haematological disorders.
The role of cytogenetics in the assessment of haematological disorders
Authors: Bain B J
Published: November 2023

There are three important reasons for undertaking cytogenetic analysis in patients with haematological diseases. First, such analysis may be of immediate relevance to patient management. This is so when such investigations are performed as an aid to the diagnosis or classification of a haematological disorder or when cytogenetic analysis is expected to yield information of relevance to prognosis, to treatment, or to both. Second, the haematologist may wish for cytogenetic analysis in order to have a better understanding of the nature of the condition he is dealing with, even though he does not expect that patient management will be altered by the results. Third, cytogenetic analysis may be undertaken for research purposes, either for basic research or for clinical studies. Thus, the researcher may be attempting to identify new disease entities or to identify the likely sites of putative oncogenes or cancersuppressing genes. He may wish to gain information which, although it will not affect the patient being studied, may be useful in designing treatment protocols for future patients. Cytogenetic analysis may be required for patients entered into trials. This may be in order to ensure that patients being investigated form a homogeneous group with a clearly defined disease. For example, a trial of a new therapy for chronic myeloid leukaemia would be likely to require cytogenetic analysis with patients who were found to be Ph-negative either being excluded or being assessed separately. Conversely, an investigation of essential thrombocythaemia would be likely to require that Ph-positive patients be excluded. A trial might also require cytogenetic analysis to enable the prognostic import of certain karyotypic abnormalities to be assessed or, if this were already known, in order to stratify patients according to prognosis with treatment being assigned on the basis of the prognostic group.
Mutagen-induced chromosome damage in human lymphocytes
Authors: Tawn E J , Holdsworth D
Published: November 2023

The study of induced chromosome changes in human lymphocytes is wellestablished for monitoring the genotoxic effects of radiation and chemicals. Cytogenetic studies can be used to monitor populations for a range of environmental and occupational clastogens (1, 2) and, for radiation, can provide an estimate of individual dose (3, 4). They also form part of the battery of tests used in assessing chemicals for mutagenicity (5-9). Various endpoints can be studied. These include structural chromosome aberrations, sister chromatid exchanges (SCE), and micronuclei. All require short-term cultures of peripheral blood lymphocytes with modifications to allow the manifestation of the different types of genetic changes.
Microdissection of human metaphase chromosomes
Authors: Lillington D M , Cotter, F E , Riddle P N
Published: November 2023

The detailed analysis of the genomes of higher eukaryotes is currently being performed by a variety of techniques such as chromosome walking, in situ hybridization, somatic cell hybridization, pulse field gel electrophoresis and flow cytometry in conjunction with efficient cloning strategies. However, the complexity and abundance of repetitive sequences in higher eukaryotes has made chromosome walking difficult.
Malaria
Authors: Chiodini Peter L , Clark Sheila
Published: November 2023

More than 1.6 billion people live in malaria endemic areas. Diagnosis of malaria by microscopy is a vital laboratory procedure in endemic areas. In addition, given the substantial amount of travel from the developed world to the tropics there will remain a need to diagnose this imported infection in patients returning from the tropics to the temperate zone. Imported malaria is a common diagnosis in many developed countries. In 1991 there were 2332 reported cases of malaria in the UK, 1268 due to Plasmodium falciparum, with 12 deaths (11 due to P. falciparum).
Pneumocystis carinii
Authors: Deery Alastair
Published: November 2023

A preliminary description of this unicellular eukaryotic organism was given by Chagas in 1909 and then Carini in 1910. Both thought the observed cysts to be a stage in the life cycle of Trypanosomes. Delanoes in 1912 correctly identified that the organism was associated with a distinct infection in rats not infected with Trypanosomes and christened it Pneumocystis carinii, after the earlier Brazilian worker. The organism is an opportunistic pathogen causing disease exclusively in immunocompromised persons and pre-eminently in those with the acquired immunodeficiency syndrome (AIDS). Other groups of patients sporadically affected include those with congenital immunodeficiency states including cellular immune impairment, immunosuppressive therapy after organ transplantation, chemotherapy for neoplasia whether for ‘solid’ tumours or for leukaemias and lymphomas, and corticosteroid therapy for ‘autoimmune disease’ and various illnesses.
The use of synthetic oligonucleotides as specific hybridization probes in the diagnosis of genetic disorders
Authors: Thein Swee lay , Ehsani Ali , wallace R Bruce
Published: November 2023

This chapter deals with the use of synthetic oligonucleotides as probes in the detection of DNA sequence variations and point mutations. For practical purposes, point mutations can be considered to be single base substitutions, minor insertions, or deletions. To detect these changes in the DNA sequence directly, one exploits the principle that a perfectly matched hybrid formed between the target sequence and an oligonucleotide probe is thermally more stable than one with a single base pair (bp) mismatch. In this respect the design of the allele-specific oligonucleotide (ASO) is critical in that it has to be long enough so that the sequence detected is unique in the human genome and yet short enough so that the hybridization is sensitive to minor differences between probe and template.
Pulsed-field gel electrophoresis in the analysis of genomic DNA and YAC clones
Authors: den Dunnen J T , Grootscholten P M , van Ommen G J B
Published: November 2023

Since the first appearance of this book in 1986 pulsed-field gel electrophoresis (PFGE) has evolved from a newly developed technique (1) to a fundamental research tool indispensable to a modem human genetics laboratory. Initially, the technique was mainly used to separate very large DNA molecules, but more recently its potential for separating smaller DNA molecules and even its application in DNA sequencing (2) became evident. Applications of PFGE include physical mapping (3–6), size selection (7–9), the study of genetic rearrangements causing cancer and genetic disease (10–12) or underlying immunological variation (13), yeast artificial chromosome cloning (YAC) technology (7, 8, 14, 15), the separation of DNA molecules with specific structures, and applications in human and veterinary microbiology and parasitology (16, 17). To cope with the fast developments, especially of the technology, we had to completely revise the chapter published in the first edition (18).
Regulation of gene expression in neuronal cell lines
Authors: Lillycrop Karen A , Dent Carolyn L , Latchman David S
Published: November 2023

Ideally, the application of molecular techniques to the study of gene regulation in a particular tissue requires the isolation of large amounts of a single-cell type in which these processes can be analysed and into which DNA constructs derived from regulated genes can be introduced. Unfortunately, virtually none of these criteria are fulfilled in the case of neuronal cells in vivo. Thus, neurons from anatomically-defined structures can only be obtained in relatively small amounts whilst brain tissue, although available in relatively large amounts is highly heterogeneous containing many different neuronal and non-neuronal cell types. Moreover, even if neurons are identified, isolated and studied in culture, they are often difficult to transfect with DNA constructs so that the processes regulating the activity of a particular gene promoter cannot be studied in this way.
Xyloglucan Oligosaccharides
Authors: Fry Stephen C
Published: November 2023

Xyloglucan (XG) is a polysaccharide found in the primary cell walls of higher plants. Its main role is architectural, probably limiting cell expansion (1-3). In addition, xyloglucan oligosaccharides (XGOs), enzymically released from XG, are potent plant growth regulators at nanomolar to micromolar concentrations, i.e. they are ‘oligosaccharins’ (4). They are not elicitors. XGOs are formed from XG by the action of cellulase (EC 3.2.1.4), which hydrolyses the polysaccharide backbone at Glc residues that lack side-chains.
Strategies for Cloning Plant Disease Resistance Genes
Authors: Michelmore Richard W , Kesseli Richard V , Francis David M , Fortin Marc G , Paran, Ilan , Yang Chang-Hsien
Published: November 2023

Many single genes determining resistance to diverse pathogens have been identified by geneticists and plant breeders. Despite the wide use of these genes in agriculture, they have so far proved recalcitrant to molecular isolation, with one exception (see Section 5.3.1). The molecular bases and biochemical modes of action of their products remain one of the major unknowns in plant biology. Neither the pattern of expression nor the nature of resistance gene products are known. It is suspected that they may be constitutively expressed at a low level; therefore, many of the strategies used to clone more highlyexpressed or induced plant genes are inappropriate. Successful cloning strategies will probably utilize the best characterized feature of resistance genes which is their Mendelian nature. Many genes induced during the resistance response have already been cloned and their function in resistance is being determined. A distinction must be made between resistance genes identified at the genetic level and genes induced during the resistance response. For the purposes of this chapter, resistance genes are those that determine whether an interaction between a plant and potential pathogen is compatible (susceptible plant and virulent pathogen) or incompatible (resistant plant and avirulent pathogen). Response genes are those that are capable of being expressed in both resistant and susceptible plants and which may be involved in limiting invasion by the pathogen. A similar range of response genes seems to be induced regardless of the challenging pathogen (1, 2). Therefore, such genes are unlikely to be the determinants of intra-specific incompatibility between host and pathogen. Cloning and characterization of disease resistance genes will have profound fundamental and applied consequences. Therefore, the efforts of several laboratories are increasingly focused on isolating resistance genes.
Viral DNA replication
Authors: Stow Nigel D , Hay Ronald T
Published: November 2023

During the last decade considerable interest has focused upon the mechanisms by which the genomes of DNA animal viruses are replicated. These studies provide information relevant not only to our understanding of viral growth and potential strategies for intervention, but also to processes occurring during the replication of eukaryotic chromosomes. The genomes of DNA viruses provide attractive models for the study of cellular DNA synthesis because they are relatively simple, readily manipulated, accumulate to high levels during lytic infection, and, in some cases, can be faithfully replicated in cell-free systems. Some viruses (for example Epstein-Barr virus and bovine papilloma virus (BPV)) additionally establish latent infections in dividing cells during which genome replication is likely to be regulated by mechanisms similar to those operating upon the host chromosomes.
Poliovirus antigen chimeras
Authors: Evans David J
Published: November 2023

Poliovirus is the causative agent of paralytic poliomyelitis, a disease largely eradicated in the developed world by the widespread use of the killed (Salk) and live attenuated (Sabin) vaccines. The oral poliovirus vaccine (OPV) developed by Albert Sabin in the 1950s contains derivatives of each of the three serotypes (1, 2, and 3) of poliovirus generated by multiple passage in culture of wild type (WT) strains of the virus (2). The resulting live attenuated strains exhibit the following characteristics that have resulted in their establishment as extremely effective vaccines. 1. They induce good, long-lasting levels of immunity in recipients. 2. They are relatively inexpensive to manufacture. 3. They are easy to administer-on a sugar lump or as a drop on the tongue.
Detection of polypeptide growth factors: application of specific bio-assays and PCR technology
Authors: Zoelen E J J Van , Delaey B , Van Der Burg B , Huylebroeck D
Published: November 2023

The term ‘growth factor’ is used to describe a polypeptide produced by a cell or isolated from a tissue, and having a growth-regulatory role on other cell types. This activity is usually discovered by application of in vitro bio-assays which quantitate the dose-dependent factor-mediated growth modulation of a homogeneous culture of a particular target cell. However, some peptide growth factors both stimulate and inhibit cell proliferation and some may have biological effects which are unrelated to cell growth, such as the differentiation of certain cells. The first phenomenon can be due to the identity of the target cell used in the bio-assay, the concentration of the factor tested (growth-stimulatory versus growth-inhibitory concentrations), and the presence in the growth factor preparation of other polypeptides which may act synergistically or antagonistically with the growth factor under study (see Table 1, Chapter 1).
Detection and characterization of growth inhibitory factors: epidermal inhibitory pentapeptide (EIP)
Authors: Robinson Philip , Whitehead Paul , Hume William
Published: November 2023

Many growth factors have been described which have stimulatory effects on cell proliferation; fewer growth inhibitors have been described. In this chapter we describe methods for detecting inhibitory factors and illustrate their characterization with a family of small oligopeptide inhibitory factors. In 1982, Laerum and Paukovits isolated a pentapeptide, haemoregulatory pentapeptide (HP), from human granulocytes, which inhibited the prolifera tion of myelopoietic cells (1). In 1984, Elgjo and Reichelt isolated a pentapeptide from mouse backskin, epidermal inhibitory pentapeptide (EIP), which inhibited the proliferation of epithelial cells in mouse backskin (2). The primary structure of these inhibitors is similar (HP = pEEDCK, and EIP = pEEDSG. See Appendix 3 for single letter amino acid code). It was proposed that they may be acting as endogenous cell proliferation inhibitors, and may be involved in the control of normal tissue homeostasis.
Molecular methods
Authors: Savage David , Baxter-Lowe Lee Ann , Gorski, Jack , Middleton Derek
Published: November 2023

The application of molecular techniques to tissue typing has led to a fundamental change in methodology for HLA class II typing. The first part of this chapter describes the use of Southern blot (1) techniques which permit the identification of restriction fragment length polymorphisms (RFLPs) which correlate with known serological (HLA-DR/DQ) and cellular (HLA-Dw) defined specificities (2). The latter part of the chapter describes the enzymatic amplification of the second exon of HLA-DR/DQ class II genes using the polymerase chain reaction (PCR) (3) and analysis of the product with alleleand sequence-specific oligonucleotides (4). Compared to serotyping (see Chapter 3), DNA typing techniques provide more information on genetic variation. Other advantages include: • cell type, cell viability, or cell surface expression are generally unimportant • homozygosity can be confirmed
HLA class I gene and protein sequence polymorphisms
Authors: Little Ann-Margaret , Parham Peter
Published: November 2023

The unique feature of the HLA-A, -B, -C class I proteins is their polymorphism. Differences between class I proteins can be detected by alloantisera in the classical serological tissue typing assay, and it is this method which is most widely used to define and distinguish class I polymorphism (see Chapter 3). Comparison of class I sequences, together with the 3D structure of HLAA2 (1) has shown the majority of polymorphic residues are located within and around the peptide binding cleft (2). It is likely that certain residues found deep within this cleft are not directly accessible to allo-recognition mechanisms and go undetected by alloantisera. Additional techniques have been applied to define class I polymorphisms undetected by serology, these include analysis of variants recognized by cytotoxic T lymphocytes (CTL), electrophoretic heterogeneity of immuno precipitated class I molecules, and molecular structure determination via nucleotide sequencing. Collectively these techniques show that many more class I alleles exist than are detected at present by serology.
In vivo assays for multipotential and marrow repopulating cells
Authors: Lord B I
Published: November 2023

Until comparatively recently, the functional status of the bone marrow has been assessed purely on qualitative criteria; primarily the absolute and differential cellularities. The existence of a stem cell population, responsible for the generation of all the recognizable haemopoietic cells, had been assumed but only about 0.5% of cells in the marrow are multipotent. Morphological assessment was clearly out-functional assays were necessary (see Appendix 1). The advent of radiation for military, peaceful and clinical purposes made objective assessment of haemopoietic tissue essential since haemopoiesis appeared to be the process most critically affected by it. Thus was spawned a major offensive in radiation biology. Jacobson and his colleagues (1, 2) reported a circulating factor that could prevent haemopoietic death and accelerate haemopoietic recovery. This was explained (3) on the basis of a repopulating cell. It was found that haemopoietic recovery (at least in mice) could be anticipated by the appearance of splenic nodules (4) the significance of which was recognized by Till and McCulloch (5).
Enrichment of progenitor cell populations from murine and human haemopoietic tissue
Authors: Lord B I , Marsh J C W
Published: November 2023

The progenitor cells exist in very small numbers, the pluripotent cells representing about 0.4% of the total bone marrow cells and the more restricted committed progenitors being perhaps 3% of the population (see Appendix 1). To study direct effects of growth factors on progenitor cells, to assess their performance, or to carry out biochemical studies, therefore, it is desirable to enrich the concentrations of the appropriate cells. Historically cells are separated on physical parameters--density and size-and these properties are incorporated into the modern techniques which employ electronic sorting of cells tagged with fluorescent molecules, or a process of continuous flow centrifugal elutriation. It is these two processes that will be outlined. Numbers of animals and/or cells used will be stated merely as a guide to convenient and practical handling of a cell sort. They may of necessity vary with specific practical requirements. For all procedures involving the use of mice, local and/or national legislation governing their use must be observed.
In vitro clonal assays for murine multipotential and lineage restricted myeloid progenitor cells
Authors: Heyworth C M , Spooncer E
Published: November 2023

The first experiments describing the clonal growth of mouse haemopoietic progenitor cells immobilized in a soft-gel matrix in vitro were reported in the mid-sixties (1, 2). The significance of these observations was readily apparent to research workers in haematology and their pioneering studies allowed quantification of primitive progenitor cells that possess an ability to proliferate, differentiate, and develop into phenotypically and functionally mature myeloid cells. Indeed, this early work, along with the spleen colonyforming assays developed by Till and McCulloch in 1961 ((3) and Chapter 1), forms the basis of modern haematology and has culminated in the clinical use of the colony-stimulating factors.
Development of haemopoietic cells in liquid culture
Authors: Whetton O Kan A D , Heyworth C M
Published: November 2023

The development of haemopoietic stem and progenitor cells to form mature cells can be regulated by soluble peptide growth factors, or alternatively via specific interactions with other cell types (such as bone marrow stromal cells). The original work which defined the target cells for the various haemopoietic growth factors used soft-gel assays which permitted the clonal development of haemopoietic cells from several distinct lineages to be assessed (see Chapter 3, Appendix I). This approach demonstrated that the progenitor cells require haemopoietic growth factors for survival as well as proliferation with accompanying development. Whilst these soft-gel assays have permitted the identification and characterization of novel growth factors and specific progenitor cell populations, they do not readily lend themselves to a study of the molecular mechanisms activated by haemopoietic growth factors to stimulate proliferation and development.
Analysis of growth factor binding to haemopoietic cells
Authors: Walker F , Heyworth C M
Published: November 2023

Haemopoietic cells must receive and act upon information from other parts of the body or from the local environment. Both mature haemopoetic cells and also their precursor cells in the bone marrow receive some of this information in the form of messenger molecules known as cytokines. The type and concentration of these cytokines presented to the cells governs the response observed. The cytokines which govern haemopoietic cell proliferation (haemopoietic growth factors) are believed to elicit their effects through cell surface receptors which display specific high affinity, reversible binding of these growth factors (1). The expression of the appropriate receptors on the cell surface is a prerequisite generally required for the cell to be capable of responding to the growth factor in question. Similarly cells which are incapable of responding to a specific growth factor, e.g. FDCP-1 cells (see Chapter 6), which cannot proliferate in response to M-CSF, can be transfected with the M-CSF receptor and acquire the ability to proliferate in response to M-CSF (2).
Retroviral gene transfer into haemopoietic cells
Authors: Fairbairn L J , Spooncer E
Published: November 2023

The transfer and expression of foreign genes in haemopoietic cells is emerging as a powerful and important tool for the study of the effects of oncogene products and other biologically active molecules. In the past five to ten years, a plethora of gene transfer methods have become available, including calcium phosphate precipitation, scrape loading, and lipofection. However, although these methods have been used successfully with adherent cell lines, (e.g. 3T3 mouse fibroblasts), haemopoietic cells appear less susceptible to gene transfer using these techniques. Electroporation has been successfully used to transfer DNA to haemopoietic cells by some groups, but often involves timeconsuming optimization of conditions coupled with extensive cell death and results in a low frequency of DNA transfer. In addition, techniques like electroporation generally lead to incorporation of multiple copies of the transferred gene in the recipient genome, which can cause problems of stability of transfer and expression.
Assays of the haemopoietic microenvironment
Authors: Friedenstein A J , E. Luria, , Molineux G ,
Published: November 2023

Prolonged blood cell production both in vivo and in vitro depends upon a complex of haemopoietic and non-haemopoietic stromal cell types and extracellular matrix molecules which are collectively called the haemopoietic microenvironment (HM, (1)). They provide both the physical framework within which haemopoiesis occurs, and probably also play some role in directing the processes by synthesizing, sequestering, or presenting haemopoietic growth factors. The precise role of each of the cell types composing the HM has yet to be defined and quantitative assays of their individual components are only available for the fibroblastic cells. In the absence of any specific functional assays for the various components of the HM several approaches have been devised to measure the functional capacity of the entire complex.
Visualization of haemopoietic and stromal cell types and their interactions in bone marrow culture
Authors: Allen T D , Wynter E A De
Published: November 2023

The growth of both haemopoietic and stromal cells in tissue culture conditions is a well established practice (see Chapters 4, 5, and 11). The methods outlined in this chapter will be restricted to liquid culture systems, as agar and methylcellulose-based milieu do not readily lend themselves to in situ morphological examination at the ultrastructural level.
Molecular analysis of clonality and transplantation in haemopoiesis
Authors: Marsh J C W , Hampson I N , Cowling G J
Published: November 2023

In this chapter Southern analysis of genomic DNA (1) is used to define

the clonality of abnormal cells found in some haemopoietic diseases

the fate of transplanted haemopoietic cells

In the first of these applications the phenomenon of X-chromosome inactivation, coupled with the analysis of DNA polymorphisms at X-linked loci, is used to determine whether haemopoietic cells in females are of monoclonal or polyclonal origin. Such methods were first described by Vogelstein et al. (2), who analysed the clonality of tumours in various diseases including acute and chronic non-lymphocytic leukaemias, uterine leiomyomas, and certain monoclonal solid tumours. In the second application, Southern analysis of Y-chromosome linked loci is used to distinguish between male donor and female recipient cells following allogeneic bone marrow transplantation in animal models (3).
Structural characterization of mucin-type O-linked oligosaccharides
Authors: Piller Friedrich , Piller Veronique
Published: November 2023

O-glycosylation is one of the most common post-translational modifications of proteins. O-glycans are found on a variety of proteins with widely different functions. O-linked saccharides are classified according to the sugar involved in the O-glycosidic linkage with a hydroxyamino acid: GalNAcαl→SerfThr: found in secreted and membrane proteins (1) Xylβ1→Ser: encountered in proteoglycans and in some membrane glyco-proteins (2) Galβ1→Hyl/Hyp: present in collagen and in complement Clq (3) GlcNAcβ1→SerfThr: discovered in nuclear and cytoplasmic proteins (4) Fuc/Glcβ1→Serffhr: found in some serum proteins (5, 6)
Characterization of nuclear and cytoplasmic glycoproteins
Authors: Berlin William K , Hanover John A
Published: November 2023

Traditionally, the O-linked and N-linked glycoproteins were thought to be synthesized within the lumen of the endoplasmic reticulum and Golgi apparatus and ultimately delivered to the cell surface via the secretory pathway. Work carried out in the past few years has revealed that glycoproteins also exist in the cytoplasm and the nucleoplasm (that part of the cytoplasm surrounded by the nuclear envelope), and are not restricted to the membrane bound organelles as biosynthetic intermediates. Nuclear and cytoplasmic proteins modified by O-linked N-acetylglucosamine are now known to comprise a major class of glycoproteins, with strong evidence that other classes of glycoproteins and proteoglycans are present in the nuclear and cytoplasmic compartments of the eukaryotic cell (1–3).
T cell antigen receptor genes
Authors: Davis Mark M , Chien Yueh-Hsiu
Published: November 2023

The characteristics of T cell recognition and the nature of their antigen receptors has long been a difficult and controversial area for immunologists. However, the development of cloned T cell lines and hybrids (as reviewed in ref. 1), the genera tion of antisera (2) and monoclonals (3-7) enabling the identification of polymor phic T cell receptor (TCR)-heterodimer-CD3 complexes (8-11) and the isolation of four different TCR genes (o:, 13, -y, 8) (12-18) has generated a large amount of information in the past decade. It has also shifted the focus of this area from the question of 'What molecules mediate T cell recognition?' to 'How do they work?' and 'How are they selected in the thymus?' Although antibodies and their genes (see Chapter 1) are a natural touchstone for understanding some aspects of TCRs, the emphasis in this chapter will be on the unique features of the different T cell antigen receptors and their possible biological significance.
T lymphocyte signal transduction
Authors: Terhorst Cox , Spits Hergen , Staal Frank , Exley Mark
Published: November 2023

That the immune system combines an extraordinary specificity of recognition with extremely complex control mechanisms has been amply demonstrated by the studies of antigen recognition by T lymphocytes. In response to antigen recogni tion, resting T lymphocytes are required to undergo a complex series of events collectively known as T cell activation. Although much has been learned about T cell activation in the last years, an orderly array of the molecular events involved in the whole process remains to be established. There is heterogeneity in the effector cells within the T cell compartment comprising T helper (TH) and cytotoxic T lymphocyte (CTL) subsets.
Structure and function of MHC class I and class II antigens
Authors: Jewell David A , Wilson Ian A
Published: November 2023

The major histocompatibility complex (MHC) is a contiguous set of genes that code for polymorphic cell surface receptors involved in antigenic peptide presentation (reviewed in ref. 1). Although originally observed in transplant rejection (2), it is now clear that the biological role of MHC proteins is to initiate cell-mediated immune responses. The co-ordinated activities of MHC proteins act to rid the body of xenobiotic infections (3). In order to combat a wide spectrum of pathogens, the class I and class II antigens have evolved to act as indicators of foreign intrusion at the surface of the host cell. The MHC antigens bind to peptide fragments, derived from the pathogen's proteins, while within the cell, and thereby direct peptides to the cell surface for presentation to immune cells. Antigenic peptide fragments (4, 5) tend to bind with high-affinity to the MHC molecules by virtue of their 'bound' shape and chemical complementary to the MHCs peptide binding groove. The lifetime of these MHC-peptide complexes on the cell surface is an important parameter of MHC restriction. Stable or long-lived peptide-MHC com plexes help to increase the probability that the immune system will detect the presence of foreign antigens.
Flow cytometric selection of responsive subclones and fluorimetric analysis of intracellular Ca2+ mobilization
Authors: Ransom John T , Dunne John F , Sharif, N A
Published: November 2023

With commonly utilized cell lines of clonal origin, we have observed that a substantial percentage of the population often fails to respond to a particular stimulus while a smaller percentage responds with a clearly detectable response. This suggests that, with multiple culture passages over time, genetic variants have developed that exist in the population and which exhibit heterogeneous response phenotypes that may not be morphologically apparent.
Confortnational studies on oligosaccharides
Authors: Homans Steven W
Published: November 2023

Since the initial demonstration by Burnet that the influenza virus requires a specific carbohydrate signal on the erythrocyte for attachment and infectivity (1), oligosaccharides have been implicated as recognition signals in a variety of biological processes. Indeed, in the field of cell adhesion, we now have knowledge of the oligosaccharide ligands and a detailed knowledge of their protein receptors has given rise to the term 'selectins' for this class of membrane proteins (reviewed in references 2, 3, see also Chapter 4). As techniques for the purification and sequence analysis of oligosaccharides improve in efficiency and sensitivity, we can anticipate the characterization of the precise oligosaccharide ligand and its receptor in other important biological processes.
Strategies for using gene probes
Authors: Lamb Richard F , Birnie George D
Published: November 2023

One of the most common questions, asked both by newcomers and experienced researchers alike, in designing a strategy for nucleic acid hybridization, is ‘which is the “best” nucleic acid probe for a particular experimental application?’ Since both the number of possible applications of nucleic acid hybridization and the choice of possible labelling and detection systems available have increased dramatically over the last ten years, it is useful before embarking upon a particular strategy to ask a few basic questions of the detection system to be employed in order to maximize the chance of success. The most critical parameters which have to be considered in designing a strategy for using a particular gene probe successfully are those outlined in Table 1. The first and most important question is, how sensitive does the detection system need to be? The answer to this question is often unknown, but can usually be guessed at intelligently. Moreover, the answer very often dictates both the choice of probe and the method of labelling used. For example, in situations requiring maximum sensitivity, such as the detection of single copy genes in mammalian DNA where sequences equivalent to about 10 fg per µ,g of DNA must be revealed, long DNA or RNA probes randomly labelled throughout their length with 32P are likely to be successful in most filter hybridization experiments.
Synthesis of riboprobes
Authors: Durrant Ian , Cunningham Martin
Published: November 2023

RNA probes (riboprobes) have been used in all the applications traditionally associated with DNA probes including Southern and Northern blotting and in situ hybridization. They have several potential advantages over DNA probes that have enabled them to become well established for particular applications such as the detection of mRNA by in situ hybridization.
| The discovery and significance of mobile genetic elements
Authors: Shapiro James A
Published: November 2023

One of the most intriguing facts about the history of mobile genetic elements is that no-one set out to discover them. In every case, they were initially found by geneticists studying other problems: chromosome breakage and rejoining in maize, transfer of genetic information between bacterial cells, lysogeny, regulation of gene expression, or antibiotic resistance. Is there a message in this repetitive serendipity? I think so. The message appears to concern the power that unspoken assumptions hold over our thinking (in this case, the idea that the genetic material is basically stable) and the need to be alert for those unexpected findings which overturn those assumptions and suddenly open a whole new universe for scientific exploration. Such major reorientations in thinking are inevitable in science Genetics has travelled a long conceptual road from the Constant Genome paradigm of the 1940s, when McClintock first described transposable controlling elements in maize, to the Fluid Genome paradigm that reigns today. There is an even longer and more fascinating voyage ahead as we come to terms with the implications of mobile genetic elements and molecular discoveries about genome organization and reorganization. This historical chapter affords a rare opportunity to trace the outlines of the intellectual journey already completed as well as to attempt some projections of where we might be going. The emphasis here will be on new ways of looking at genetic problems, on key observations, and on the unexpected convergence of discoveries in separate experimental systems. Accounts of the history of particular systems with a more mechanistic focus can be found in the first Mobile Genetic Elements book (2).
| Transposable elements as tools for molecular analyses in bacteria
Authors: Berg Claire M , Berg Douglas E
Published: November 2023

The transposable elements of bacteria vary in size and structure, and in the ways they move. Some half-dozen elements and their engineered derivatives have been exploited as research tools since the late 1970s. Their use in conjunction with recombinant DNA methods has revolutionized the practice of microbial genetics. The value of transposons as molecular genetic tools stems from their ability to move as discrete DNA segments to new locations where they disrupt the target DNA. Natural and engineered transposons can serve as mobile sources of a variety of phenotypic or physical markers, including selectable (or sometimes counter-selectable) genes, predetermined restriction sites, reporter genes, promoters, and replication origins.
Analysis of protein expression by immunocytochemistry
Authors: Brown Dennis
Published: November 2023

Immunocytochemistry is a major technique in unravelling the roles of proteins expressed by cells. This is true whether the protein is endogenous, or whether it is expressed as a result of transfection of a cell with a specific cDNA. Remarkable insights into the complex physiology and cell biology of cells, tissues, and organs have been obtained by combining immunocytochemical observations with the powerful tools of molecular biology, and with measurements of physiological and cellular function. In view of the increasing use of such detection techniques in multidisciplinary studies, it becomes important to understand the strengths and weaknesses of the many methods that are available, in order to choose the correct procedure for each particular antigen.
Cosmid cloning of yeast DNA
Authors: Stucka Rolf , Feldmann Horst
Published: November 2023

After in vitro packaging into premature phage heads, recombinant cosmids can easily be introduced into Eschericha coli cells and propagated with high efficiency like plasmids. Depending on the vector, genomic DNA fragments in the size range 35–45 kb can be accommodated: 78–105% of the length of wild-type 11. DNA (38–55 kb) can be packaged efficiently. The in vitro packaging systems give an approximately 10-fold higher yield of transformants per µg of DNA than the standard CaC12 transformation procedure.