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Various techniques for isolating compounds from aqueous extracts of marine organisms are reviewed. The major isolation techniques involve separation by differences in molecular size, charge or adsorption properties. Successful strategies employed to isolate bioactive constituents from extracts which displayed activity in a variety of biological screens are discussed. The recent marine natural product literature is reviewed and the isolation procedures for compounds isolated from aqueous extracts are summarized.

A crude bacterial extract containing approximately 4 mg/ml protein, 25% of which was human growth hormone (hGH), was subjected to two alternative gel electrophoretic isolation procedures, designated I and II. Procedure I exploits the high electrophoretic net mobility (RM larger than 0.127) at pH 7.6, 0 degrees C, of the bacterial contaminants relative to hGH. This allows one to stack the contaminants at a protein load of 31.5 mg/cm2 of gel, using a "non-restrictive" gel concentration. Unstacked hGH is collected from the gel section between 0.3 and 0.6 of relative gel length and extracted electrophoretically as described previously. Alternatively, the unstacked hGH was concentrated on the gel by dispatching a second moving boundary behind the original stack ("re-stacking") and a gel section (relative gel length 0.45 to 0.6) between the two moving boundaries was excised and subjected to electrophoretic extraction. The yield of hGH ranged from 70 to 82%, and its purity (weight/Lowry) ranged from 86 to 115%. Procedure II exploits the high electrophoretic net mobility (RM larger than 0.064) at pH 10.5, 0 degrees C, of hGH relative to its bacterial contaminants at a gel concentration of 9 %T, 2 %CBis, at a protein load of 2.5 mg/cm2 of gel. The selectively stacked hGH is collected by preparative elution-PAGE, using an apparatus with 17.6 cm2 gel surface area. The yield of hGH was 90% and its purity ranged from 84-92%.

In the second edition of the book Neurochemistry, Rossiter (1962) stated, “Since pure ‘myelin’ is not available for direct chemical analysis, the neurochemist has been compelled to deduce the constituents of myelin from such analyses as are practicable.” The available analyses were extensive. Qualitative information had been accumulating from histological staining techniques since the latter half of the nineteenth century. Quantitative studies on brain constituents from which information on myelin could be deduced began shortly after the turn of the century. Thus in his chapter on “The Myelin Sheath, Metabolism of Myelin and Experimental Demyelination,” Rossiter was able to discuss myelin composition and metabolism with considerable assurance and accuracy. The direct analysis of myelin constituents was, however, not possible until techniques were developed for isolating myelin membranes essentially free of other subcellular structures. The first of such procedures were being developed and published in the same year Neurochemistry was published. In this chapter, I will discuss isolation procedures and the composition of purified myelin obtained by these procedures.

Publisher Summary Recent data from the laboratories demonstrated that a variety of intracellular organelles, as well as outer cell membranes of bovine corpora lutea, contain PGE and PGF 2α receptors. This chapter outlines the methodology and approach that can be used in obtaining data. Achieving a high degree of purity of various organelles is not only a function of good centrifugation strategy, but also critically depends on the care and handling during the procedures. To illustrate this point, one should remove organelles from the top sucrose interfaces first, not attempt to remove them to the extent that would overlap with other interfaces, and use fresh syringes and needles to remove different organelles. Purity is severely compromised despite an excellent centrifugation strategy if sufficient attention is not paid to the above and other seemingly minor handling details. The use of marker enzymes to assess the purity of subcellular organelles should be more sensitive than morphological assessment when they remain active during the isolation procedures. A prerequisite for using marker enzymes for assessment of purity is that the selected enzyme(s) should be found in only one organelle. In reality, this prerequisite may not be strictly met for all the enzymes, primarily because it is difficult to resolve unequivocally whether small amounts of enzymes found in inappropriate organelles are a reflection of low-level contamination or intrinsic.

Reactive dyes have found widespread interest as biomimetic ligands in numerous subjects of enzymology and protein chemistry, taking advantage of the principle of biorecognition. The use of Cibacron Blue F3G-A and other triazine dyes as ligands in affinity chromatography and in related techniques has been successfully demonstrated in diverse isolation procedures of enzymes and proteins. In the last few years affinity partitioning became attractive for studying dye-protein interaction because of its high sensitivity in the recognition of ligand-protein binding and of its methodical simplicity.

A family of acidic proteins has been isolated from human submandibular-sublingual saliva using Sephadex G-200 gel filtration, DE-52 cellulose chromatography, and immuno-affinity chromatography. These molecules were homogenous in size and by immunological criteria, but exhibited charge heterogeneity. The heterogeneity displayed was not due to gustatory stimulation or degradation during isolation procedures.

Publisher Summary This chapter discusses the analytical techniques for isolated metaphase chromosome fractions. Metaphase chromosomes represent reproducible precise subfractions of the total genome of an organism. The technology described in this chapter was developed to evaluate isolated chicken chromosomes in experiments designed to locate specific viral DNA sequences integrated into the chicken genome. In principle, the same approach can identify proteins peculiar to specific chromosomes. The chapter presents a discussion on the analysis of chromosome fractions. All chromosome isolation procedures require a hypotonic treatment step to disrupt any spindle remnants and to make the cell susceptible to shear disruption. Isolated chromosomes offer few advantages over isolated whole nuclei, unless some type of sorting of the individual kinds of chromosomes can be achieved. The techniques for fraction analysis are visual inspection and flow microfluorometry. Visual inspection of the various fractions with a phase-contrast microscope confirms the general impression that the chromosomes are distributed in the gradient according to size. In contrast to the qualitative data which visual examination provides, precise quantitative data are readily obtained by the particle analysis technique—flow microfluorometry (FMF)..

Conventional methods of isolating islets of Langerhans rely upon the differential sensitivity of the pancreatic acinar tissue vs islets to enzymatic dissociation by crude collagenase, however, the yield of intact islets obtainable with this technique is quite low. Higher yields of islets can be obtained with pharmacological or surgical methods which either destroy acinar cells or cause them to release their zymogen granules. However, because of the requirement to pretreat the donor, these methods cannot be scaled-up for potential clinical use. To overcome the limitations of the conventional isolation procedures, we exploited the differential sensitivity of acinar cells and islet cells to freezing damage. Using this approach we are able to isolate greater than 2500 islets from the pancreas of a single rat. Basically, we rapidly mince pancreatic tissue, subject the tissue to a short collagenase digestion, briefly freeze the tissue at -30 degrees C in the presence of glycerol, and immediately thaw it. Subsequent enzyme treatment digests the residual acinar tissue, collagen, DNA, and proteins. Preliminary results indicate that the islets are morphologically indistinguishable from islets isolated using conventional digestion techniques.

The membrane envelope of the milk fat globule (MFGM) was observed by freeze-fracture after secretion, in milk and cream and during isolation procedures. In fat globules this membrane reveals aspects of particle distribution which reflect, in part, the regional segregation of integral membrane components at the apical plasmalemma of lactating cells. Tearing, folding and sealing of membrane fragments were observed during isolation procedures: sharp membrane folding occurs at particle-rich areas; sealing of membrane fragments was observed between particle-rich membrane edges in a zipper-like fashion. MFGM preparations contain small vesicles and discoidal cisternae. Particles were observed in the vesicles and at the rim of the cisternae. Our observations suggest limits to the use of MFGM preparations as the structural equivalent of the acinar plasma membrane of mammary epithelial cells, but support this system as a model to study membrane structure and dynamics.

Procedures for the isolation of mitochondria from dipteran flight muscle have been investigated in an attempt to determine the extent and to identify the causes of deterioration associated with isolation. In the light of the results obtained isolation procedures have been improved by minimising mechanical damage, avoiding the development of anoxic conditions, and by the use of an isolation medium of a more physiological nature, containing the potassium salt of an organic anion as the principal osmoeffector, phosphate as the principal buffer, and low concentrations of free Mg2+. The oxidative capacity of mitochondria isolated by the improved method is adequate to support the in vivo requirements of the flight system.

Although short-term bioassays have usually been developed by testing individual chemicals, samples which need to be evaluated for potential human health hazard are often complex mixtures. These mixtures may contain many toxic substances, each present in small quantities. To detect such toxins in bioassays, sample preparation will involve concentration of the compounds of interest and removal of material which will interfere with the test system. If the toxicity is due to many compounds, isolation and concentration of the total biological activity will be useful either as a preliminary step for chemical analysis or as a method for characterizing some of the toxic and chemical properties of the mixture. Mutagenicity tests are useful for monitoring genotoxic activity during such isolation procedures, since these assays are relatively quick and simple. Moreover, a large number of carcinogens are mutagenic (McCann et al., 1975; Rinkus and Legator, 1979); therefore, isolating mutagenic activity may develop procedures which will be useful in isolating carcinogens. Using simple chemical separation techniques and mutagenicity testing, we have highly concentrated the mutagens in smokers’ urine and have elucidated some of their chemical characteristics.

The applications of affinity chromatography for the isolation and purification of physiological substances from urine have been reviewed. The use of affinity supports for the detection and measurement of various urinary components has also been briefly examined. These examples illustrate the usefulness of urine as a source of fine reagents and the versatility, simplicity and practicality of affinity isolation procedures.

Plasma glycosaminoglycans (GAG) were isolated from 10 ml of plasma by a modification of the method of Calatroni et al. (3). DE-52 anion-exchange cellulose was used in the isolation of the fraction operationally defined as free GAG. Chondroitin sulphate and heparin added to plasma were quantitatively recovered in this fraction. After proteolysis with papain the fraction operationally defined as bound GAG was isolated using anion-exchange resin AG 1 X 2. GAG were measured as hexuronate with the m-hydroxydiphenyl method of Blumenkrantz and Asboe-Hansen (7) which was superior to various modifications of the carbazole/borate carbazole procedures. In 15 healthy females and in 15 healthy males the concentrations of the free GAG (mean +/- S.D., expressed as microgram per 10 ml of plasma) were: 12.2 +/- 2.8 and 16.8 +/- 3.8 (P less than 0.001); of the bound GAG 40.4 +/- 7.7, and 40.2 +/- 11.6; and of the total GAG 52.7 +/- 9.0 and 57.0 +/- 10.4, respectively. With the isolation procedures used, plasma GAG were obtained in sufficient quantity for their electrophoretic characterization. Assay of plasma GAG can be performed with satisfactory accuracy and precision within two days by the present method. In clinical chemistry its application to the study of proteoglycan and GAG metabolism in various diseases may prove valuable.

Publisher Summary This chapter presents procedure for purification and assay of microtubule-associated proteins (MAPS). The procedures begin with microtubules assembled in vitro from either brain or tissue culture cells. The solution of microtubule proteins is usually carried through at least two to three complete assembly-disassembly cycles prior to beginning one of the following MAP isolation procedures. The strategies employed to isolate MAPs from the total microtubule protein preparation are based on several criteria: (1) net charge distribution affecting their behavior on ion exchange resins, (2) overall molecular size and axial ratio, which affect their elution from molecular sieve columns, and (3) varying stability to the effects of elevated temperatures (100°). One or more combinations of these properties can be used as the basis for the isolation procedures. Both the high molecular weight MAPs and Tau from brain and the MAPs from tissue culture cells have been assayed by their ability to promote the assembly of tubulin in vitro. Generally, these assays are carried out by determining whether the putative MAP will enhance the initial rate and total amount of assembly of a given concentration of PC or DEAE-purified brain tubulin.

In parallel studies the effects of FHL1 and PNL on plated melanoma cells from cell lines and short-term cultures were compared. FHL showed more frequent and also stronger cytotoxic and/or growth-inhibiting effects than PNL. On melanoma target cells from cell lines both FHL and PNL showed more frequent and stronger cytotoxic and/or growth-inhibiting effects than on melanoma target cells from short-term cultures. In the individual donors the percentage of monocytes and EAC-rosette-forming cells in FHL was significantly higher than in PNL. A significant correlation was found between multiplication of the melanoma target cells during the test period and an increased susceptibility towards lymphocytes from healthy donors. Melanoma target cells from cell lines were not more fragile, or more susceptible to unfavourable culture conditions than cells from short-term cultures, since non-lymphocytic „effector” cells showed much weaker cytotoxic and/or growth-inhibiting effects than lymphocytes from healthy donors. Cytotoxic effects of lymphocytes from healthy donors were also registered on target cells from a mammary carcinoma and an osteosarcoma cell line. No significant differences in the cytotoxic effects of lymphocytes from healthy donors were observed when tested on mycoplasma-contaminated melanoma cells and the same cells made mycoplasma-free. Mitomycin-C-treated lymphocytes retained their cytotoxic effects. Lymphocytes from a healthy donor tested on different occasions on the same melanoma cells from a short term culture showed an incidental cytotoxic reaction.

Publisher Summary Progress on vacuolar physiology is, however, still very much dependent on the isolation of intact, functional vacuoles, and so far successful isolation procedures have been developed for only a relatively few plant species. Any description of the methodology of transport in vacuoles depends entirely on the methodology of vacuole isolation suitable to the plant species of interest. Transport activities have been based on a series of measurements, such as protein, intravacuolar volume, vacuole counts, marker enzyme activity, and intravacuolar marker compounds (phenolics, alkaloids, and dyes). Although none of these means of measurement can be regarded as ideal, some are definitely invalid when a comparison with transport activities of other plant material is required. For instance, betaine or alkaloids are absent in most plants. Therefore, it is helpful to determine for each type of vacuole preparation the relationship among protein, intravacuolar volume, vacuole number, and intravacuolar marker compound.

Publisher Summary This chapter describes a method for the isolation of nuclei from animal cells in culture. The objective of the method for isolating nuclei is to obtain a preparation that consists solely of nuclear material and in which the nuclei are morphologically identical to those in undisrupted cells, with contents qualitatively and quantitatively identical to those of nuclei in vivo . The chapter discusses the factors of importance in nuclear isolation procedures and the criteria that used to assess the integrity and purity of the isolated nuclei. The chapter describes two methods for isolating nuclei as preliminary steps in the isolation of chromatin and chromatin constituents. Nuclear isolation procedures essentially consist of two steps: (1) the lysis of the cells in a suitable medium, and (2) the separation of the nuclei from the unbroken cells and cell debris. Examination of the isolated nuclei by high-resolution electron microscopy is the best method of assessing the integrity of the nuclei and the purity of the preparation. The methods for isolating nuclei include isolation with (1) citric acid, and (2) sucrose and detergent. The chapter also compares the products obtained from these methods.

Purification of proteins from PNS myelin began in the early 1970s. Isolation procedures were developed to obtain the basic proteins (Uyemura et al., 1970; London, 1971; Brostoff et al., 1972), and an early study (Brostoff et al., 1972) suggested that one of them was able to induce experimental allergic neuritis (EAN).

I would like to make a few comments about the earliest talks in the day. The later talks in the day concentrated on TS and the effects of dUMP, FdUMP, and various factors related to TS. What the earlier talks point out is that there is a real need to be looking at earlier steps. The transport of CF, for example, and the activation reaction, both have to be considered. As Dave Duch pointed out, for example, his particular cell line seems to be unable to be modulated because it does not activate CF and that could potentially be a mechanism of intrinsic resistance to this kind of modulation phenomenon and I think that should be looked at in more detail. In terms of the transport, the pharmacokinetic data show the 6R-isomer remains at very high levels in the plasma as the natural isomer falls, so that ratio becomes very high. There needs to be more work in looking at the effects of the 6R-isomer and Dr. Rustum, in fact, is interested in doing some of those studies as are some other groups. In terms of the activation, there were two pathways described and I think most people seem to think that the methenyltetrahydrofolate synthetase is the one primarily responsible for the effect of CF in these combinations, but the other pathway has not been ruled out yet. An interesting aspect to examine is whether both of those pathways are important or only one of them. In terms of a basic biochemical question, at least two groups reported the normal occurrence of 5-formyltetrahydrofolate which had previously been thought to be an artifact of isolation procedures. Since we don’t have a reaction that uses 5-formyltetrahydrofolate I think it would be interesting to determine the function of that pool. A storage role was postulated, but the levels are very low; it seems that if you were going to store something you’d store higher levels than the minor amount that was reported. The transport and the activation, the steps prior to TS, deserve more investigation.

A preparative procedure was developed for the isolation of human apohaemopexin from Cohn fraction IV or blood serum, based on bioaffinity chromatography on haeme-Sepharose. The isolation is carried out in the pH range 4-8; hence the possibility of degradation of the carbohydrate moiety of the glycoprotein in the acidic media used in other isolation procedures is decreased. Owing to the conditions of the separation and the good stability of the affinity support, the column can be used repeatedly for long periods without a significant loss of binding capacity. The reversibility of the conformational changes that haemopexin undergoes in acidic media was examined by hydrophobic chromatography. The original hydrophobic characteristics were restored only approximately 48 h after haemopexin had been brought into a neutral medium.

The purpose of this article was to introduce a new technique of isolating multiple adjacent teeth requiring endodontic therapy where the teeth to be isolated were not amenable to traditional isolation procedures. The article outlines a method for the custom fabrication of an acrylic retainer which is used in conjunction with a conventional rubber dam. Photographs of a model and a clinical case are used to illustrate the method and clinical applicability.

The human non-allergized K (killer) lymphocyte has been characterized using selective depletion and isolation procedures based on surface markers. It was found to be a non-thymus-dependent, Fc-receptor-bearing cell. In an area which is controversial, our findings also indicate that the cell has immunoglobulin on its surface but lacks the receptor for the C3 component of complement (mouse). This would suggest it to be an (Fc + C3––) B lymphocyte.

A quantitative study of the individual steps in the isolation of elastin from mature and fetal aortas of rabbits was carried out. The proportions of insoluble residue and acetone- and ether-soluble substances were determined. The yields of elastin and other associated components of connective tissue derived from the aortas by two different isolation procedures were compared, i.e. the classical Lansing treatment with hot alkali and the less drastic procedure described by Rasmussen et al. Results were found to be strongly influenced by the method used. The purity of the elastin preparations was evaluated by means of amino acid analyses for relative contents of desmosines and dicarboxylic amino acids. The possible reasons for the differences found are discussed.

Publisher Summary This chapter describes the methods for studying nucleocytoplasmic exchange of macromolecules and discusses the morphology of the nuclear envelope that represents the major physical barrier between the nucleoplasm and cytoplasm. The inherent difficulties in studying envelope permeability and the permeability concepts developed from studies using exogenous tracers are also discussed. A method that avoids many of the technical problems encountered using smaller cells is described for studying the uptake of endogenous proteins by oocyte nuclei. The oocytes are pulse labeled either by microinjection or incubation, and a cytoplasmic pool of labeled polypeptides is established. The rapid accumulation of large endogenous polypeptides could be because of the existence of specific transport processes or simple diffusion. Modifications of the labeling and nuclear isolation procedures should make it possible to investigate quantitative and qualitative changes in nuclear protein uptake during different stages of oogenesis.

• Umbilical and axillary skin cultures for bacteria and yeast were obtained within two hours of birth from 108 term infants born vaginally under sterile (52) and nonsterile (56) conditions. The skin of infants born under nonsterile conditions was more frequently colonized (91%) than that of infants born under sterile conditions (66%). The most common skin flora of both groups was Staphylococcus epidermidis , diphtheroids, and Escherichia coli. A significantly higher frequency of colonization with gram-positive cocci and diphtheroids was noted in infants born under nonsterile conditions. Cultures obtained on day 3 or 4 from infants born under sterile conditions revealed a higher frequency of colonization with E coli, enteric organisms, and potential pathogens than cultures obtained immediately after delivery. Cultures did not aid in clinical management, since in this sample no overt infection developed. Culture and isolation procedures in clinically well term infants born under nonsterile conditions are not warranted. ( Am J Dis Child 1983;137:351-353)

Publisher Summary The mitotic spindle consists of the chromatic and the achromatic figure. The chromatic figure consists of the chromosomes and includes their attachment points to the spindle, the kinetochores. The achromatic figure is more difficult to define. It consists of the transient spindle-like structure, and, where presents, the asters and the centrioles. This chapter discusses the biochemistry of the achromatic figure, the evolution of the mitotic spindle, the ultrastructure of the mitotic spindle, the molecules in the isolated mitotic apparatus, the characteristics of the various isolation procedures, the criteria used for evaluating them, and the composition of the isolated spindle. The chapter discusses the repetition of the mitotic process in vitro, using spindle proteins in spindle models. Microtubules are one of the prominent structures found in the spindle. They are composed of one major protein that was named “tubulin.” The two protomeric subunits are termed “α-tubulin” and “β-tubulin.”

An outbreak of shigellosis is described in an intermediate care hospital for children, where routine isolation procedures failed to halt the outbreak. Two weeks after the appearance of the first case, small numbers of patients be came symptomatic with fever and diarrhea, but remained culture-negative. One week later, two patients with positive cultures were identified, with many other symptomatic patients having negative cultures. Twenty-five days after identification of the first case, there were fourteen cases identified with shigellosis and many more symptomatic patients. It was at that time that hospital-wide measures of infection control were instituted, and treatment of all patients, infected and uninfected, was initiated with trimethoprim-sulfa methoxazole (TMP-SMX). No new cases of shigellosis were diagnosed after initiation of treatment, and symptoms abated within 72 hours.

Publisher Summary This chapter describes the classification of halobacteria. Recently, the halobacteria seemed to form a very homogeneous bacterial group, but this may have been largely becaue of uniformly utilized enrichment and isolation procedures. A major problem in the classification of halobacteria is the proper assignment of species names. This problem has led to a proliferation of new species names and to the dubious assignment of some new isolates to established species. In addition some strains have been given new species designations owing to mislabeling or to subjective decisions by the laboratories carrying the strain. Until accumulating taxonomic data permits a more definite species differentiation, one may be justified in regarding most species as just strain epithets. Accordingly no attempt is made to redefine species criteria, and descriptions of designated halobacteria strains are given for recognition purposes only.

This chapter discusses the amount and nature of aquatic humic substances in all types of natural waters. The chapter contains thirteen major divisions and is the largest chapter. The major divisions include: early studies of aquatic humus, overview of isolation procedures, amount of humic substances in water, elemental composition of aquatic humus, functional group analysis, molecular weight, characterization by spectroscopy, trace components of humic substances, characterization by chromatography, chemical degradation of humic substances, structure of aquatic humic substances, and theories of formation of soil and aquatic humic substances.

A non-toxigenic strain of Corynebacterium diphtheriae var. gravis was isolated from 31 boys during an outbreak of sore throats at a boarding school of 15 staff and 134 pupils. Despite the non-toxigenicity of the strain involved, conventional isolation procedures were observed and all boys at the school were treated with antibiotics. Restrictions on the school were only relaxed when no further carriers were detected. The rationale of such management of an episode involving a non-toxigenic strain is discussed.

Two cultural and two rapid Listeria isolation procedures used in the United States will be reviewed in this presentation.

Isocoumarins have been isolated from a wide variety of microbial, plant and insect sources and have been shown to possess an impressive array of biological activities. Since the review by Barry in 1963 (24), the number of known naturally occurring isocoumarins has increased dramatically. This increase is largely due to improvements in isolation procedures and structural analysis. Previous reviews have concentrated on fungal isocoumarins (293, 294) and mycotoxic isocoumarins (301). This review lists over 160 naturally occurring isocoumarins. Leading references on isolation, structure elucidation, biosynthesis and synthesis are given in the accompanying Tables.

Two procedures are described for the isolation of vitellogenin from plasma. Vitellogenin can be selectively precipitated from the plasma of estrogen-treated animals by Mg2+ in the presence of EDTA. The vitellogenin thus obtained is >99% pure and is relatively undegraded. In the second procedure, vitellogenin can also be isolated by DEAE-cellulose chromatography, resulting in preparations that contain

New techniques are described to improve the sensitivity and specificity of detection and isolation procedures for bacteria in epidemiological and quality control studies.

The general theme of the conference, “from biological activity to structure”, has prevailed in the biological research laboratory for decades. Although the methods used are now more sophisticated and the questions asked more detailed, the general approach to solving structural problems is much the same. Typically, biological activity is identified and the source and/or storage site of the material is located. Usually, an assay is developed to follow this activity through purification and isolation procedures. Chemical characterization is accomplished by a wide variety of techniques, including classical procedures as well as modern analytical methods in order to deduce the structure of the molecule responsible for this activity. It is often the case that from structure we return to biological activity in several ways. For example, this may be by chemical synthesis of the molecule in the laboratory in order to produce greater quantities of the compound at decreased cost, or by chemical modification of the original compound in order to increase its potency, to change its physical and chemical properties, or to decrease its side effects.

Summary Chromatin was prepared by two different methods from isolated nuclei of Gyrodinium cohnii ( Cryptothecodinium cohnii) and Peridinium trochoideum. These isolation procedures are different from those generally used to prepare eukaryote chromatin, because the latter do not work for dinoflagellate chromatin. The chemical composition of this chromatin is similar for both methods of preparation and both organisms. Dinoflagellate chromatin contains DNA, RNA, acid-soluble and acid-insoluble protein as does chromatin from higher plants and animals, but the amount of acid-soluble protein relative to DNA (0.02--0.08) is much lower than that of typical eukaryotes (about i). Evidence is presented to show that proteolytic degradation is unlikely to account for the low acid-soluble protein content in dinoflagellate chromatin. Exclusion chromatography of the chromatin on large-pore gels (Bio Gel A-15m or Sephadex G-200) indicates that the bulk of the protein present in the chromatin preparations migrates with the DNA. G. cohnii and P. trochoideum chromatin show an ultraviolet absorption spectrum, which is intermediate between DNA and typical eukaryote chromatin, and this is not significantly changed by gel exclusion chromatography. Preliminary results suggest that the dinoflagellate DNA-associated proteins do not stabilize the DNA against melting. Chromatin prepared from log-phase cells has more protein and RNA than chromatin from stationary-phase cells. The chemical composition of dinoflagellate chromatin is compared with that of prokaryotes and eukaryotes.

A peptide found in acetic acid extracts of porcine hypothalami and capable of stimulating the release of ACTH in vitro was isolated in pure state, structurally identified as Phe-Leu-Gly-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Pre-Pro-His-Phe and synthesized. This tetradecapeptide, which corresponds to amino acid residues no. 33–46 in the sequence of the α-chain of porcine hemoglobin, probably represents an artefact of extraction or isolation procedures. Since this peptide stimulates ACTH release from rat pituitary fragments and from monolayer cultures of pituitary cells, but not in vivo , caution must be exercised in interpreting the results of in vitro assays for corticotropin releasing factor.

In experimental neurobiology, it is often desirable to decrease the level of complexity presented by the intact adult mammalian nervous system by various isolation procedures. In this chapter, we will summarize evidence that transplantation to the anterior chamber of the eye of rats and other rodents is an efficient means of obtaining such a decrease in complexity while preserving the functional integrity of the grafted tissues. A transplant can be defined for our purposes as a tissue piece in a living organism that has been completely physically isolated from its normal environment at some point during its life. When placed back into the original type of environment, it is referred to as a homotopic transplant; when grafted to other locations, it is called a heterotopic transplant. Thus, grafts of neuron-containing tissues to the anterior chamber of the eye are always considered heterotopic.

INTRODUCI'ION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 1 LOCATION OF CRYSTALLINE LAYERS . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 2 ULTRASTRUCTURAL STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 14 ISOLATION PROCEDURES . . . . . . . . ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 CHEMICAL CHARACTERIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 SELF-ASSEMBLY, RECRYSTALLIZATION, AND REATTACHMENT . . . . . . . . . . . . . . . . 325 SYNTHESIS AND MORPHOGENESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 FUNCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 Functions Depending on Pore Size .. . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . .. . . . . . . . . . . . .... .. . .. . .. 331 Other Protective Functions 332 MorphogE'netic Functions 332 CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

Publisher Summary This chapter describes the isolation of surface membranes from mammalian cells. The preparation of purified surface membranes is an art not to be approached lightly. Methods are available to prepare membranes from most cell types. These methods produce whole membranes, large or small fragments, or vesicles. Cells performing different functions require variations of the isolation procedures. Therefore, the method to be used should be selected on the basis of the cell type as well as on the ability of the final membrane product. Two kinds of restrictions are placed on the interpretation of any data on isolated membranes. The possibility exists that not all of the cytoplasmic contents have been expelled, particularly when whole membranes are prepared. It may not be possible to separate completely all of the internal membranes from those of the surface, particularly when the method causes vesiculation of the membrane systems. As there are vast differences among cells, the criteria cannot be universal but should be categorized for particular cell types. In addition, when possible, similar methods should be used for a particular cell type or comparisons should be made with the other methods if there is a conflict.

Publisher Summary Protein C was originally isolated from bovine plasma" and more recently from human plasma. In the isolation procedures described in this chapter, dextran sulfate-agarose and preparative electrophoresis are key steps in the isolation of these proteins. Recently, a homogeneous preparation of bovine protein C has been described employing chromatography on heparin-agarose as a final purification step. Protein C concentration can be assayed quantitatively by immunochemical procedures/ or qualitatively by its ability to prolong the kaolin-cephalin clotting time of plasma following its activation by thrombin. One obvious drawback of the immunochemical method is that it requires a pure preparation of protein C for antibody production. The two-stage anticoagulant assay, described below, does not require purified protein C and appears to be a useful operational assay for evaluating the relative concentration of this protein in chromatographic eluants. It should be pointed out that with the anticoagulant assay, the prolongation of the clotting time by activated protein C is dramatically dependent on the use of plasma from which the protein C is derived.

Publisher Summary Glycophorins are relatively small and heavily glycosylated proteins that are found in plasma membranes of many, possibly all, cell types. This chapter describes the isolation procedures and properties of intact glycophorins, and simplified procedures to obtain preparative amounts of the glycosylated domains of the three glycophorin molecules. The isolation procedures involve preparation of the crude glycophorin fraction, preparation of reagents, LIS/Phenol extraction, isolation of Glycophorin A, isolation of Glycophorin B and Glycophorin C, and preparation of the glycosylated (cell surface) domains of Glycophorins. Glycophorins may be associated to form dimeric structures in the membrane. Glycophorin A is a transmembrane protein, and the linear arrangement of the three domains—extracellular, intramembranous, and cytoplasmic—is evident not only from studies on the primary structure, but also from experiments performed with cells. However, there is no information available on the mode of insertion or transmembrane nature of glycophorins B and C.

The antineoplastic constituents of the marine ascidian Aplidium californicum were found to be bryostatins 4 and 5. Bioassay-directed isolation procedures using the National Cancer Institute's P-388 lymphocytic leukemia system led to the bryostatins. The probable symbiotic relationship between A. californicum and the bryozoan Bugula neritina was discussed.

A detailed procedure is described for isolating chromatin from small amounts of tissue culture cells. The method utilizes the observation that it is possible to obtain a purified, concentrated, chromatin fraction by isopycnic gradient centrifugation in a low ionic strength solution containing 39% (wv) metrizamide. Such chromatin preparations appear to give good yields of DNA and have their normal complement of protein and RNA when compared with other chromatin isolation procedures. The procedure is simple to use, except for the isopycnic gradient centrifugation step, the only major equipment required is a clinical centrifuge and a light microscope. The method should be of value when a number of chromatin preparations are being assayed simultaneously.

Publisher Summary This chapter discusses isolation procedures for inside-out thylakoid vesicles. Inside-out vesicles from the thylakoid membrane is first obtained from spinach chloroplasts by a combination of mechanical fragmentation and separation by aqueous two-phase partition. The isolation of inside-out thylakoid vesicles can be divided principally into two steps: fragmentation and subsequent separation. The ionic conditions imposed during fragmentation have a profound influence on both the yield and the properties of the inside-out vesicles. Three different fragmentation procedures will be described. The first and original procedure gives rise to inside-out vesicles originating from the appressed thylakoid region, highly enriched in photosystem II, but relatively low in yield. The second procedure gives inside-out vesicles in a higher yield but somewhat lower photosystem II purity. The inside-out vesicles obtained by these two procedures are particularly suitable for the studies on the lateral distribution of thylakoid components and for the studies on the organization and function of photosystem II and its oxygen-evolving system. The third procedure gives inside-out vesicles with the same relative amounts of photosystem I and II as whole thylakoids, and is therefore, especially suited for the studies on transverse arrangements of all components of the thylakoid membrane.

Asymptomatic infection with either Entamoeba histolytica or Giardia lamblia was found in 60 per cent of the residents in an institution for the mentally retarded one year after eradication of these parasites. The residents were then segregated into infected and noninfected groups and drug therapy was again successfully undertaken. The two groups remained separated except during periods of play. A one-year follow-up showed that both study and control groups were equally infected. The failure of segregation was confounded by patient age and infection with nonpathogenic parasites.

Publisher Summary There has been considerable interest in the properties of tumor mitochondria during the past 25 years. This interest initially stemmed from Warburg's hypothesis, which predicted respiratory impairment in tumor cells. In recent years, interest in tumor mitochondria has advanced beyond the simple assessment of Warburg's hypothesis. It is now known that mitochondria participate in a variety of metabolic processes other than oxidative phosphorylation, they have the capacity to accumulate ions such as Ca 2+ and P i , and that they contain a genetic apparatus that codes for several inner membrane proteins. There have been reports that some types of mitochondria may play a role in virus production. These properties make the mitochondrion an attractive target for studies designed to detect differences between normal and neoplastic tissue. In order to assess such differences, it is necessary to have available reliable mitochondrial isolation procedures. The chapter describes two different procedures for the isolation of mitochondria from Morris hepatomas. Morris hepatomas are particularly suitable as a source of tumor mitochondria because of the considerable stability of each tumor cell line, the availability of cell lines with rapid, slow, and moderate growth rates, and the possibility of using normal and host livers as reasonable controls.