Synthesis of some analogs of estradiol

As part of a search for estradiol derivatives designed for conjugation to carboxyl or amine functions of anti-cancer agents or suitable derivatives thereof, estradiol analogs with side chains at the C-16 or -17 position were prepared for biological assay. These analogs include several which have a substituted nitrogenous function at C-17. The avidity of some of these analogs for binding to estrogen receptor was found to be of a low order.     

Aspects for the future of biotransformations

Biotransformations have gained extensive importance in practical use as a support for chemical synthesis or in the conversion of natural products. Biotransformations may present an enlargement, a sequential degradation or a specific modification of synthetic or natural compounds. The tools for biotransformations are principally mammalian, plant or microbial cells and their cell-free enzymes. In technical practice the biocatalysts are so far limited to the use of microorganisms and some cell-free enzymes of low cost. Although numerous microbial or enzymatical reactions were already developed for industrial processes, the capacities of biotransformations offer a broad field of inexhaustible possibilities for the future.

     

Tailing of survivor curves of clostridial spores heated in edible oils

Tailing of survivor curves was observed for Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores heated whilst suspended in edible oils, but not for the same spores suspended in buffer (pH 7.2) or mineral oil or for Bacillus cereus F4165/75 spores suspended in buffer or oils. The tailing cannot be ascribed to a genetic or developmental heterogeneity in the resistance of the spore population or to a heterogeneity of the treatment severity during heating. Heat adaptation due to the release of protective factor(s), to the selection for resistant spores or to the diffusion of oil constituents inside the spore protoplast to protect key molecules from heat denaturation was also ruled out. The tailing can be ascribed to spore clumping during the course of heating or to a heterogeneity in heat resistance of germination system(s) within spores, concurrently with the activation of a dormant germination system. It is probably caused by some oleic acid containing triglycerides.     

Tailing of survivor curves of clostridial spores heated in edible oils

Tailing of survivor curves was observed for Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores heated whilst suspended in edible oils, but not for the same spores suspended in buffer (pH 7˙2) or mineral oil or for Bacillus cereus F4165/75 spores suspended in buffer or oils. The tailing cannot be ascribed to a genetic or developmental heterogeneity in the resistance of the spore population or to a heterogeneity of the treatment severity during heating. Heat adaptation due to the release of protective factor(s), to the selection for resistant spores or to the diffusion of oil constituents inside the spore protoplast to protect key molecules from heat denaturation was also ruled out. The tailing can be ascribed to spore clumping during the course of heating or to a heterogeneity in heat resistance of germination system(s) within spores, concurrently with the activation of a dormant germination system. It is probably caused by some oleic acid containing triglycerides.     

Ion Dependence of Neurotransmitter Uptake: Inhibitory Effects of Ion Substitutes

Several ions commonly used as substitutes for Na+ or Cl- were found to inhibit directly the high-affinity uptake of norepinephrine, dopamine, serotonin, and gamma-aminobutyric acid, but not glutamate or glutamine. When Na+ was partially replaced by any of several different cations or sucrose the uptake of all neurotransmitters studied except that of serotonin was reduced more than could be accounted for by just the inhibitory effect of the cation substitute. In contrast, when Cl- was partially replaced by any of several anions only the uptake of dopamine was reduced more than could be accounted for by the inhibitory effect of the anion substitute. These results suggest that for most neurotransmitters the electrochemical potential for Na+, but not for Cl-, contributes to the uptake driving force. When either Na+ or Cl- was totally replaced by an ion substitute or by sucrose the high-affinity uptake was virtually abolished, an exception being that glutamate uptake was not affected when isethionate was substituted for Cl-. The lack of uptake in the absence of either Na+ or Cl- may reflect a specific role for these ions in either increasing the affinity between the substrate and the carrier, or facilitating the translocation process. Alternatively, the transport carriers may undergo a nonspecific conformational change to an inactive form in the absence of Na+ or Cl-. A partial substitution of Na+ with Li+ or sucrose differentially affected the kinetics of uptake in that replacement with Li+, but not sucrose, usually resulted in a marked increase in the Km values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of repertoires of surface antigens on leukemias using an antibody microarray

We have previously described a microarray of cluster of differentiation (CD) antibodies that enables concurrent determination of more than 60 CD antigens on leukocytes. This procedure does not require protein purification or labeling, or a secondary detection system. Whole cells are captured by a microarray of 10 nL antibody dots immobilized on a nitrocellulose film on a microscope slide. Distinct patterns of cell binding are observed for different leukemias or lymphomas. These haematological malignancies arise from precursor cells of T- or B-lymphocytic, or myeloid lineages of hematopoiesis. The dot patterns obtained from patients are distinct from those of peripheral blood leukocytes from normal subjects. This microarray technology has recently undergone a number of refinements. The microarray now contains more CD antibodies, and a scanner for imaging dot patterns and software for data analysis provide an extensive immunophenotype sufficient for diagnosis of common leukemias. The technology is being evaluated for diagnosis of leukemias with parallel use of conventional diagnostic criteria.     

Bioreactors for 3-dimensional high-density culture of human cells

A bioreactor was developed as an instrument to culture human or animal cells that require attachment in a large quantity or at a high density. The purpose for developing such a bioreactor is two-fold: to produce a large quantity of animal or human cells that have been modified by gene recombination technology to accommodate manufacture of physiologically-active substances or human proteins on an industrial scale; and for research to culture animal cells to form a high-density 3-dimensional structure as a morphological or functional tissue or organ entity. In the current report, the circulatory flow bioreactor and radial flow bioreactor (RFB) are introduced, in which the former can be scaled up. As a small bioreactor produced for the latter purpose, a rotary cell culture system and novel multicoaxial hollow-fiber bioreactor are introduced. Finally, a small RFB culture system that was scaled down by the present author and his collaborators for the study of a 3-dimensional high density culture system is described. The RFB can be readily scaled up for manufacturing or scaled down for research purposes. This is a cell culturing system that can induce the functions of human tissues by preparing a high density 3-dimensional organization of cells of human origin.     

Infectious features of atherosclerosis

As an inflammatory focus, the atherosclerotic plaque is viewed as a response to aggressions. Suppressing these causal injuries appears as the best means for preventing the disease. Infection is among the clues for answering the etiological challenge of atherosclerosis. Through direct or indirect, and specific or non specific pathways, some candidate viruses or bacteria are suspected to induce or stimulate plaque formation or complications. Yet, none of these working hypotheses has reached the level of proof required for establishing a valid concept. Although submitted to intensive investigations, anti-infectious drugs and antimicrobial vaccinations are still far-sighted expectations in the treatment and prevention of coronary artery disease.     

Quantitative analysis of diploid translocation heterozygotes: test of models and equations

Summary Equations have been derived for two different models of chromosome pairing and chiasmata distribution. The first model represents the normal condition and assumes complete synapsis of homologous bivalents and the arms of interchange quadrivalents. This is followed by a nonrandom distribution of chiasmata among bivalents and multivalents such that each bivalent or bivalent-equivalent always has at least one chiasma. Univalents occur only as part of a III, I configuration at diakinesis or metaphase I. The second model assumes that a hologenomic mutation is present in which all chromosomes of a genome are equally affected. Two different assumptions can be made for such a mutation, and both give the same results: (1) homologous or homoeologous chromosome arms may be randomly paired or unpaired, but synapsis always leads to a crossover; (2) homologous or homoeologous arms always pair, but chiasmata are randomly distributed among the arms. The meiotic configurations at diakinesis or metaphase I are the same for both assumptions. Meiotic configurations of normal diploid interchange heterozygotes show good agreement with numbers predicted by the equations for nonrandom chiasmata distribution among configurations. Inter-specific hybrids with supernumerary chromosomes produced meiotic configurations frequencies in agreement with predictions of equations for random chiasmata distribution, but a hybrid without supernumeraries fitted the nonrandom expectations.     

Complementary food resources of carnivory and frugivory affect local abundance of an omnivorous carnivore

A major unresolved question for omnivorous carnivores, like most species of bears, is to what degree are populations influenced by bottom–up (food supply) or top–down (human‐caused mortality) processes. Most previous work on bear populations has focused on factors that limit survival (top–down) assuming little effect of food resource supply. When food resources are considered, most often they consider only the availability/supply of a single resource, particularly marine‐subsidized or terrestrial sources of protein (carnivory) or alternately hard or soft mast (frugivory). Little has been done to compare the importance of each of these factors for omnivorous bears or test whether complementary resources better explain individual animal and population measures such as density, vital rates, and body size. We compared landscape patterns of digestible energy (kcal) for buffaloberry (a key source of carbohydrate) and ungulate matter (a key source of protein and lipid) to local measures in grizzly bear Ursus arctos abundance at DNA hair snag sites in west‐central Alberta, Canada. We tested support for bottom–up hypotheses in either single (carnivory [meat] versus frugivory [fruit]) or complementary (additive or multiplicative) food resources, while accounting for a well‐known top–down limiting factor affecting bear survival (road density). We found support for both top–down and bottom–up factors with complementary resources (co‐limitation) supported over single resource supplies of either meat or fruit. Our study suggests that the availability of food resources that provide complementary nutrients is more important in predicting local bear abundance than single foods or nutrients (e.g. protein) or simply energy per se. This suggests a nutritionally multidimensional bottom–up limitation for a low density interior population of grizzly bears.     

Proteomic analysis of striated muscle

The techniques collectively known as proteomics are useful for characterizing the protein phenotype of a particular tissue or cell as well as quantitatively identifying differences in the levels of individual proteins following modulation of a tissue or cell. In the area of striated muscle research, proteomics has been a useful tool for identifying qualitative and quantitative changes in the striated muscle protein phenotype resulting from either disease or physiological modulation. Proteomics is useful for these investigations because many of the changes in the striated muscle phenotype resulting from either disease or changes in physiological state are qualitative and not quantitative changes. For example, modification of striated muscle proteins by phosphorylation and proteolytic cleavage are readily observed using proteomic technologies while these changes would not be identified using genomic technology. In this review, I will discuss the application of proteomic technology to striated muscle research, research designed to identify key protein changes that are either causal for or markers of a striated muscle disease or physiological condition.     

Capillary Depletion Method for Quantification of Blood–Brain Barrier Transport of Circulating Peptides and Plasma Proteins

Recent studies indicate that circulating peptides or plasma proteins, such as insulin or transferrin, or modified proteins, such as cationized albumin, undergo receptor-mediated or absorptive-mediated transport through the brain capillary wall, i.e., the blood-brain barrier (BBB). Although morphologic studies such as autoradiography or immunoperoxidase labeling can demonstrate transport of blood-borne protein into brain, there is a need for a rapid, sensitive, and quantifiable physiology-based technique for comparing the relative rates of transport of several different blood-borne peptides or proteins into brain. Therefore, the present investigations describe a carotid arterial infusion technique coupled with a capillary depletion method for quantifying transport of blood-borne cationized albumin, cationized IgG, and acetylated low-density lipoprotein (LDL). Because differentiation of true transcytosis into the postcapillary compartment of brain parenchyma from binding and/or endocytosis to the brain microvasculature is important, the present studies use a dextran density centrifugation step to deplete brain homogenate of the vasculature. In addition, 3H-labeled native albumin is used as a vascular space marker to account for release of capillary contents into the postcapillary supernatant following homogenization of brain. This study demonstrates rapid transport of cationized IgG or cationized albumin into brain, as these compounds achieve a volume of distribution of 20-30 microliters/g within 10 min of arterial perfusion. Conversely, acetylated LDL, although rapidly bound by cerebral microvasculature, is shown not to undergo transport into the post-capillary compartment of brain parenchyma. These studies provide the basis for a sensitive, quantifiable technique for studying transport of radiolabeled blood-borne peptides and proteins across the BBB of anesthetized animals.     

Construction of an artificial cell membrane anchor using DARC as a fitting for artificial extracellular functionalities of eukaryotic cells

The need to functionalize cell membranes in a directed way for specific applications as single cell arrays or to force close cell-to-cell contact for artificial intercellular interaction and/or induction concerning stem cell manipulation or in general to have a tool for membrane and cell surface-associated processes, we envisaged a neutral inactive membrane anchor for extracellular entities to facillitate the above mentioned functionalities.     

Effect of flushing of blastocysts on days 10-13 on the life-span of the corpora lutea in the pig

Blastocysts were flushed out of both uterine horns of gilts on Days 10, 11, 12 or 13. In mated non-pregnant gilts flushing had no effect on progesterone profile or cycle length (20.8 +/- 0.4 versus 20.6 +/- 0.6 days in the preflush cycle, N = 6, mean +/- s.e.m.). Flushing the blastocysts out of the uterine horns on Day 10 resulted in a cycle with a normal progesterone profile and a normal length (21.2 +/- 0.4 days, N = 5). Flushing on Days 11, 12 or 13 resulted in a normal cycle or in maintenance of the CL for 3-13 days as indicated by elevated progesterone concentrations and an increased interoestrous interval of, respectively, 22.0 +/- 1.2 versus 19.8 +/- 0.6 days (Day 11; N = 6), 24.8 +/- 1.4 versus 21.0 +/- 0.6 days (Day 12; N = 5; P less than 0.05) and 26.3 +/- 2.3 versus 20.5 +/- 0.4 days (Day 13; N = 6; P less than 0.05). There was a positive relationship between the change in interoestrous interval and the interval between the first observed standing oestrus and flushing of the blastocysts (rs = 0.350; n = 22; P less than 0.1). There was a large variation in the diameter of the blastocysts flushed on the same day. Only in those gilts in which the blastocysts were greater than or equal to 8 mm or filamentous were the CL maintained for 3 or more days. These results indicate that a first signal for maternal recognition of pregnancy is generated on Day 12 and that blastocysts greater than or equal to 8 mm are required for prolongation of CL function for 3 or more days. Since CL function is only extended for a maximum of 13 days (mean 7.4 +/- 1.0), a second signal seems necessary to maintain the CL for the whole period of pregnancy.     

A general method for highly selective cross-linking of unprotected polypeptides via pH-controlled modification of N-terminal alpha-amino groups

A method is described for the highly selective modification of the alpha-amino groups at the N-termini of unprotected peptides to form stable, modified peptide intermediates which can be covalently coupled to other molecules or to a solid support. Acylation with iodoacetic anhydride at pH 6.0 occurs with 90-98% selectivity for the alpha-amino group, depending on the N-terminal residue (as shown with a series of model hexapeptides containing a competing Lys residue). Although Cys residues must be protected (reversibly or irreversibly) before the anhydride reaction, there are no detectable side reactions of the alpha-amino moiety--of the reagent or of modified peptide--with the side chains of His, Met, or Lys. The reaction works well in denaturants, so that inhibitory effects of noncovalent structure can be minimized. In a second step the iodoacetyl-peptide can be reacted with a thiol group on a protein, on a solid chromatography matrix, on a spectroscopic probe, etc. This is illustrated by reaction of a series of N alpha-iodoacetyl-peptides with murine interferon-gamma, which contains a C-terminal Cys residue. Data are presented which suggest that this iodoacetic anhydride scheme is superior in selectivity for alpha-amino groups to conventional chemical approaches to cross-linking such as use of 2-iminothiolane or N-hydroxysuccinimide-activated carboxylic acid esters. The reaction is ideally suited for modifying peptide fragments, as pure species or as mixtures, derived from proteolytic or chemical fragmentation of proteins. Furthermore, polypeptides synthesized biosynthetically, for example via recombinant DNA techniques, can be cross-linked in this way.(ABSTRACT TRUNCATED AT 250 WORDS)     

Freeze-Sectioning of Plant Tissues

Techniques are described for freeze-sectioning a wide range of both fresh and fixed plant tissues. Gelatin-antifreeze media are used to support but not infiltrate the tissue during sectioning. At cryostat temperatures of -10 to -15 C, 15% gelatin (w/v) containing 0.8% dimethyl sulfoxide (DMSO), or 1.5% ethanediol (ethylene glycol), or 2% glycerol is used. Lower concentrations of gelatin and higher concentrations of antifreezes are required for sectioning at -24 C. Petri plates of media are stored at 2 C, and used by simply melting a hole in the medium. Fresh tissues can be placed directly in the hole, or prefrozen at temperature of liquid nitrogen, or equilibrated in antifreeze solution, before freeze-sectioning in the gelatin antifreeze medium. Many plant tissues have highly vacuolated cells and need equilibration in antifreeze solutions prior to freeze-sectioning. Fixed tissues are rehydrated and washed in water or buffer for 15-24 hr before equilibrating in a 10% solution of either DMSO, ethanediol or glycerol (named in order of rapidity of equilibration). Pretreatment in 10% DMSO is usually for 1-6 hr at 2 C for histochemical studies; or in 10% ethanediol or glycerol for 15-24 hr at either room temperature or 37 C for morphological studies. These methods permit serial cryostat sections free from freezing and thawing artifacts to be cut as thin as 2 μ.     

酵母糖化酶基因高效表达的剂量效应研究

通过原生质体融合和秋水仙素加倍技术,将糖化酵母的３个等效异位基因分别构建成交配型位点等痊基因纯合和糖化酶基因各自纯各加倍的纯合二倍体（ＳＴＡ１／ＳＴＡ１、ＳＴＡ２／ＳＴＡ２和ＳＴＡ３ＳＴＡ３）以及糖化酶基因相互组织加倍的组合二倍体（ＳＴＡ１／ＳＴＡ２、ＳＴＡ２／ＳＴＡ３和ＳＴＡ１／ＳＴＡ３）。研究了这３个糖化酶基因表达的剂量效及其相互关系。糖化酶酶活测定的结果表明,在交配型位点等位基因纯合状态下,     

Preparing Single or Serial Sections of Calcified Bones and Teeth

An apparatus for cutting single or serial sections of calcified bone and teeth consists of a motor-driven shaft on which is mounted one saw (for single section cutting) or a gang (for serial sectioning at one cutting operation). The plastic-embedded specimen is attached to a cylindrical plastic holder which is in turn mounted on the machine and fed into the saw. Prior to cutting the specimen may be oriented in two planes, as well as rotated, with respect to the cutting edge. Single or serial sections made by means of repeated cuts with a single saw, may be 0.3 mm or more thick as determined by the setting of a micrometer screw. For serially sectioning a tooth or bone specimen at one cutting operation, the thickness of the separators between adjacent saws (0.5 mm or more) determines the section thickness. After sectioning, specimens may be ground and polished, with or without reimbedding in fresh plastic.     

Effects of lipids on the activity of ferrochelatase.

Removal of lipids from submitochondrial particles or detergent-solubilized mitochondrial preparations of rat liver resulted in a 90% loss of ferrochelatase (protochemeferro-lyase, EC 4.99.1.1) activity. The addition of either a fatty acid or phospholipid restored enzyme activity; the extent of reactivation being correlated with the degree of unsaturation of the fatty acid or acyl chain and independent of the polar head group of the phospholipid, Arrhenius plots of the ferrochelatase activities of submitochondrial particles and detergent-solubilized mitochondrial preparations showed transition temperatures of 37 and 28.5 degrees C, respectively. Ferrochelatase of submitochondrial particles or detergent-solubilized preparations had an absolute requirement for Ca2+. The ferrous salt of oxalic acid, a Ca2+ chelator, was a very poor substrate for these preparations. In contrast, ferrochelatase activities of fatty acid- or lipid-supplemented acetone extracts of these preparations were not dependent on the presence of Ca2+ and ferrous oxalate served as substrate for these extracts.20