scholarly journals Experimental and Theoretical Bases of Specific Affinity, a Cytoarchitecture-Based Formulation of Nutrient Collection Proposed To Supercede the Michaelis-Menten Paradigm of Microbial Kinetics

2004 ◽  
Vol 70 (9) ◽  
pp. 5511-5521 ◽  
Author(s):  
D. K. Button ◽  
Betsy Robertson ◽  
Elizabeth Gustafson ◽  
Xiaoming Zhao
Keyword(s):  
Kinetic Curve ◽  
Enzyme Concentration ◽  
Whole Cells ◽  
Cytoplasmic Enzyme ◽  
Multiple Substrates ◽  
Microbial Kinetics ◽  
Solute Uptake ◽  
Sufficient Energy ◽  
Specific Affinity

ABSTRACT A theory for solute uptake by whole cells was derived with a focus on the ability of oligobacteria to sequester nutrients. It provided a general relationship that was used to obtain the kinetic constants for in situ marine populations in the presence of naturally occurring substrates. In situ affinities of 0.9 to 400 liters g of cells−1 h−1 found were up to 103 times smaller than those from a “Marinobacter arcticus ” isolate, but springtime values were greatly increased by warming. Affinities of the isolate for usual polar substrates but not for hydrocarbons were diminished by ionophores. A kinetic curve or Monod plot was constructed from the best available data for cytoarchitectural components of the isolate by using the theory together with concepts and calculations from first principles. The order of effect of these components on specific affinity was membrane potential > cytoplasmic enzyme concentration > cytoplasmic enzyme affinity > permease concentration > area of the permease site > translation coefficient > porin concentration. Component balance was influential as well; a small increase in cytoplasmic enzyme concentration gave a large increase in the effect of permease concentration. The effect of permease concentration on specific affinity was large, while the effect on Km was small. These results are in contrast to the Michaelis-Menten theory as applied by Monod that has uptake kinetics dependent on the quality of the permease molecules, with Km as an independent measure of affinity. Calculations demonstrated that most oligobacteria in the environment must use multiple substrates simultaneously to attain sufficient energy and material for growth, a requirement consistent with communities largely comprising few species.

High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level

1995 ◽  
Vol 53 ◽  
pp. 752-753
Author(s):  
B.A. Hamkalo ◽  
S. Narayanswami ◽  
A.P. Kausch
Keyword(s):  
Nucleic Acid ◽  
Interphase Nucleus ◽  
Genome Mapping ◽  
Precise Location ◽  
Electron Microscope Level ◽  
Rna Sequences ◽  
Fundamental Interest ◽  
Whole Cells ◽  
Dna And Rna

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.

Bioencapsulation in Sol-Gel Glasses

1998 ◽  
Vol 519 ◽  
Author(s):  
L. Bergogne ◽  
S. Fennouh ◽  
J. Livage ◽  
C. Roux
Keyword(s):  
Sol Gel ◽  
Porous Silica ◽  
Silica Matrix ◽  
Whole Cells ◽  
The Past ◽  
Wide Range ◽  
Activity Of Enzymes ◽  
Gel Glasses ◽  
Micro Organisms

AbstractBioencapsulation in sol-gel materials has been widely studied during the past decade. Trapped species appear to retain their bioactivity in the porous silica matrix. Small analytes can diffuse through the pores allowing bioreactions to be performed in-situ, inside the sol-gel glass. A wide range of biomolecules and micro-organisms have been encapsulated. The catalytic activity of enzymes is used for the realization of biosensors or bioreactors. Antibody-antigen recognition has been shown to be feasible within sol-gel matrices. Trapped antibodies bind specifically the corresponding haptens and can be used for the detection of traces of chemicals. Even whole cells are now encapsulated without any alteration of their cellular organization. They can be used for the production of chemicals or as antigens for immunoassays.

scholarly journals Chromatin fibers observed in situ in frozen hydrated sections. Native fiber diameter is not correlated with nucleosome repeat length.

1994 ◽  
Vol 125 (1) ◽  
pp. 11-19 ◽  
Author(s):  
C L Woodcock
Keyword(s):  
Cell Types ◽  
Repeat Length ◽  
Strong Positive Correlation ◽  
Whole Cells ◽  
Chromosome Architecture ◽  
Chromatin Fibers ◽  
The Moment ◽  
Chicken Erythrocytes ◽  
Patiria Miniata

Chromatin fibers have been observed and measured in frozen hydrated sections of three types of cell (chicken erythrocytes and sperm of Patiria miniata and Thyone briareus) representing an approximately 20-bp range of nucleosomal repeat lengths. For sperm of the starfish P. miniata, it was possible to obtain images of chromatin fibers from cells that were swimming in seawater up to the moment of cryo-immobilization, thus providing a record of the native morphology of the chromatin of these cells. Glutaraldehyde fixation produced no significant changes in the ultrastructure or diameter of chromatin fibers, and fiber diameters observed in cryosections were similar to those recorded after low temperature embedding in Lowicryl K11M. Chromatin fiber diameters measured from cryosections of the three types of nuclei were similar, a striking contrast to the situation for chromatin isolated from these cell types, where a strong positive correlation between diameter and nucleosomal repeat length has been established. The demonstration of chromatin fibers in unfixed whole cells establishes an unequivocal baseline for the study of native chromatin and chromosome architecture. The significant differences between chromatin fibers in nucleo and after isolation supports a previous observation (P. J. Giannasca, R. A. Horowitz, and C. L. Woodcock. 1993. J. Cell Sci. 105:551-561), and suggests that structural studies on isolated material should be interpreted with caution until the changes that accompany chromatin isolation are understood.

Regulation of transmembrane electrical potential gradient in rat hepatocytes in situ

1987 ◽  
Vol 252 (1) ◽  
pp. G56-G64 ◽  
Author(s):  
J. G. Fitz ◽  
B. F. Scharschmidt
Keyword(s):  
Electrical Potential ◽  
Potential Gradient ◽  
Rat Hepatocytes ◽  
Solute Uptake ◽  
Tightly Coupled ◽  
Transmembrane Electrical Potential ◽  
Cell Variation ◽  
Over Time ◽  
Electrical Potential Gradient

The transmembrane electrical potential gradient (Em) has been measured in hepatocytes from intact anesthetized rats using conventional intracellular microelectrodes under a variety of conditions. Em measurements in control animals were normally distributed around a mean of -35.5 +/- 4.6 mV (SD) with a coefficient of variation (CV) of 13.1% and a range of -26 to -54 mV. In individual livers, however, measurements of Em at a given point in time exhibited little cell-to-cell variation (cv of 4.5%). The Em was noted to fluctuate spontaneously over time and to change consistently in response to a variety of physiological stimuli including fasting (depolarization to -28.5 +/- 3.8 mV) and infusion of glucagon in physiological amounts (hyperpolarization to -45.0 +/- 1.8 mV). Hepatocyte Em abruptly depolarized (2-5 mV) after an intravenous bolus of taurocholate (3 mumol) or alanine (45 mumol), suggesting that both solutes exhibit electrogenic uptake. The Em returned to or below preinfusion values within 5 min. Continued infusion of alanine (10.8 mumol/min), but not taurocholate (810 nmol/min), caused a sustained and unexpected hyperpolarization of Em of 8.2 +/- 3.1 mV that lasted at least 60 min. In separate studies, alanine administration did not alter the biliary excretion of a taurocholate load. Taken together, these observations demonstrate that rat hepatocytes in situ are tightly coupled electrically and that physiological stimuli, including fasting, glucagon, and sodium-coupled solute uptake can change Em considerably over time. The late hyperpolarization of Em caused by alanine appears to offset the rise in intracellular Na+ associated with alanine uptake and preserve the Na+ electrochemical gradient such that Na+-coupled taurocholate transport is maintained.

scholarly journals Machine Learning to Quantify In Situ Humoral Selection in Human Lupus Tubulointerstitial Inflammation

2020 ◽  
Vol 11 ◽  
Author(s):  
Andrew J. Kinloch ◽  
Yuta Asano ◽  
Azam Mohsin ◽  
Carole Henry ◽  
Rebecca Abraham ◽  
...  
Keyword(s):  
Machine Learning ◽  
Somatic Hypermutation ◽  
Specific Binding ◽  
Renal Tissue ◽  
Affinity Maturation ◽  
Variable Regions ◽  
Whole Cells ◽  
Antibody Staining ◽  
Tubulointerstitial Inflammation

In human lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ expansion of B cells expressing anti-vimentin antibodies (AVAs). The mechanism by which AVAs are selected is unclear. Herein, we demonstrate that AVA somatic hypermutation (SHM) and selection increase affinity for vimentin. Indeed, germline reversion of several antibodies demonstrated that higher affinity AVAs can be selected from both low affinity B cell germline clones and even those that are strongly reactive with other autoantigens. While we demonstrated affinity maturation, enzyme-linked immunosorbent assays (ELISAs) suggested that affinity maturation might be a consequence of increasing polyreactivity or even non-specific binding. Therefore, it was unclear if there was also selection for increased specificity. Subsequent multi-color confocal microscopy studies indicated that while TII AVAs often appeared polyreactive by ELISA, they bound selectively to vimentin fibrils in whole cells or inflamed renal tissue. Using a novel machine learning pipeline (CytoSkaler) to quantify the cellular distribution of antibody staining, we demonstrated that TII AVAs were selected for both enhanced binding and specificity in situ. Furthermore, reversion of single predicted amino acids in antibody variable regions indicated that we could use CytoSkaler to capture both negative and positive selection events. More broadly, our data suggest a new approach to assess and define antibody polyreactivity based on quantifying the distribution of binding to native and contextually relevant antigens.

In-Lake Bioreactors for the Treatment of Acid Mine Water in Pit Lakes

2007 ◽  
Vol 20-21 ◽  
pp. 271-274 ◽  
Author(s):  
Volker Preuss ◽  
Martin Horn ◽  
Matthias Koschorreck ◽  
Günter Luther ◽  
Katrin Wendt-Potthoff ◽  
...  
Keyword(s):  
Mine Water ◽  
Treatment Process ◽  
Mining District ◽  
Pit Lake ◽  
Acid Mine Water ◽  
Acid Mine ◽  
Results Of Treatment ◽  
Sufficient Energy ◽  
Mode Of Operation

For the treatment of acid mine water an in-situ pilot plant with a self-sufficient energy supply and remote data transmission was tested in acidic pit lake 111, a small lake in the Lusatian mining district in Germany. In this paper the design of the enclosure-bioreactor in-lake system, the mode of operation of a three-stage treatment process by the use of anaerobic fixed film reactors and the results of treatment are shown.

scholarly journals Evaluation of a mathematical model of rumen digestion and an in vitro simulation of rumen proteolysis to estimate the rumen-undegraded nitrogen content of feedstuffs

1983 ◽  
Vol 50 (3) ◽  
pp. 555-568 ◽  
Author(s):  
U. Krishnamoorthy ◽  
C. J. Sniffen ◽  
M. D. Stern ◽  
P. J. Van Soest
Keyword(s):  
Mathematical Model ◽  
Rumen Fluid ◽  
Full Range ◽  
Enzyme Concentration ◽  
Total N ◽  
Protease Enzyme ◽  
Residual N

1. Twelve grain mixtures, one lucerne (Medicago sativa) hay and one maize silage which had been used in mixed diets for which dietary nitrogen undegraded in the rumen (UDN) had been estimated with duodenally-cannulated cows, were studied. Total N in the feeds was fractionated into pool A (N soluble in borate–phosphate buffer), pool B (total N–(pool A + pool C)) and pool C (acid-detergent-insoluble N or residual N after 24 h incubation in protease solution).2. N solubilization in protease solution containing 6·6 units/ml (substrate-saturating enzyme concentration) indicated the presence of subfractions in pool B, with different rates of solubilization. Such subfractions were not detectable from in situ, Dacron bag, estimates of N solubilization.3. UDN was estimated using a dynamic mathematical model and rate-constants obtained from N solubilization in protease solution or in situ.For three grain mixtures tested using the protease technique the model predicted UDN values of 7, 10 and 12% compared with values of 47, 66 and 59% estimated in vivo. The full range of experimental feeds was tested using the in situtechnique and UDN values predicted by the model were used to derive UDN values for twelve mixed diets. The latter values were significantly but not closely correlated with those determined in vivo (r2 0·41, P < 0·05).4. An attempt was made to simulate rumen proteolysis in vitro by choosing a protease enzyme concentration (0·066 units/ml) providing a proteolytic activity similar to that of whole rumen fluid. The experimental samples of feed were subjected to simulated rumen proteolysis for 18 or 48 h to resemble the mean retention times in the rumen for grain mixtures and roughages respectively. The residual N at the end of incubation was considered as an estimate of UDN. The UDN values estimated from simulated rumen proteolysis and those determined in vivo for twelve mixed diets were in close agreement (r2 0·61, P < 0·01).5. Simulated rumen proteolysis can serve as a simple, rapid and sensitive method to estimate UDN in a variety of feedstuffs.

Abundant nuclear ribonucleoprotein form of CAD RNA

1985 ◽  
Vol 5 (3) ◽  
pp. 569-575
Author(s):  
R Sperling ◽  
J Sperling ◽  
A D Levine ◽  
P Spann ◽  
G R Stark ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Short Length ◽  
Wild Type ◽  
Whole Cells ◽  
Physiological Conditions ◽  
Mature Mrna ◽  
Nuclear Rna ◽  
Cad Gene ◽  
Nuclear Ribonucleoprotein

Transcripts of the CAD gene in Syrian hamster cells are as abundant in the nucleus as in the cytoplasm. This was shown by in situ hybridization of whole cells and by solution and blot hybridization of subcellular fractions. Similar results were obtained both for wild-type cells and for a mutant containing amplified CAD genes in which the level of CAD RNA is 150-fold greater. CAD nuclear RNA is indistinguishable from mature mRNA by gel electrophoresis and blot hybridization. Discrete higher-molecular-weight precursors are undetectable, although the persistence of a short length of intervening sequence in the otherwise fully processed RNA is not excluded. CAD RNA is released from nuclei by sonication in physiological conditions in a ribonucleoprotein form that sediments as a broad peak at about 200S in a sucrose gradient. CAD sequences extracted from nuclei by treatment with EDTA and RNase are found in the 30S particles previously described.

scholarly journals Simultaneous in situ detection of beta-interferon mRNA and DNA in the same cell.

1989 ◽  
Vol 37 (5) ◽  
pp. 697-701 ◽  
Author(s):  
F J Tang ◽  
P O Ts'o ◽  
S A Lesko
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
In Situ Hybridization ◽  
Single Cells ◽  
Specific Gene ◽  
Whole Cells ◽  
Beta Interferon ◽  
Ht1080 Cells ◽  
In Situ Detection

We report a quantitative method that combines in situ mRNA hybridization with microfluorometric analysis of DNA content to detect gene expression in single cells of a heteroploid cell population. The model was a human fibrosarcoma HT1080 cell line which consisted of diploid and tetraploid cells that were induced with polyI:polyC for production of beta-interferon. The level of beta-interferon mRNA detected by in situ hybridization was found to be two to three times higher in tetraploid compared to diploid HT1080 cells, and correlated with beta-interferon activity in that a subclone of tetraploid HT1080 cells secreted two- to fivefold more beta-interferon than a subclone of diploid HT1080 cells. Interestingly, beta-interferon-related transcripts were detected during S-phase in uninduced tetraploid HT1080 cells. In addition, beta-interferon induced by polyI:polyC was expressed in all phases of the cell cycle as demonstrated with a human diploid fibroblast, HF926. The unique features offered by the combination of microfluorometry and in situ hybridization provide a valuable tool to investigate specific gene expression related to ploidy or cell-cycle stage in the same individual cell of an unsynchronized population. Since the method allows direct observation of morphology, one can be assured that all quantitative measurements were made on whole cells with intact nuclei.

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