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PubMed1988

Soluble and polymerized tubulin levels in the anterior pituitary lobe of the lactating rat during suckling.

A [3H]colchicine-binding assay was employed for estimating the relative amounts of soluble and polymerized tubulin present in an individual anterior pituitary lobe. Colchicine binding to these two tubulin pools was time dependent and dose responsive. Scatchard analysis of the binding revealed that [3H]colchicine bound to both fractions with similar affinity. After establishing the optimal conditions for the binding assay, the effect of suckling on the relative levels of the two tubulin pools was studied. Groups of primiparous rats on days 12-14 postpartum were isolated from their pups for 4-5 h, then suckled for 30, 60, and 90 min, and killed, and the anterior pituitary lobes were dissected out. Each pituitary lobe was processed to obtain the two tubulin pools, viz. soluble and polymerized tubulin fractions. Suckling for 30 min resulted in an increase in soluble tubulin levels and a concomitant decrease in the polymerized pools. At 60 and 90 min, the soluble tubulin levels gradually decreased to presuckled levels, whereas the polymerized pools increased from the 30-min levels to those observed at 0 min. In another experiment, suckling for 10 min resulted in a decrease in the soluble tubulin levels and a corresponding increase in the polymerized tubulin pool. Treatment of nonsuckled rats with domperidone for 10 min also resulted in a shift of the equilibrium between the two tubulin pools similar to that observed at 10 min of suckling. Administration of bromocriptine 30 min beforehand to nonsuckled rats and then suckling them for 10 min blocked the suckling-induced rise in the polymerized tubulin levels. These results suggest a regulatory role for dopamine in the suckling-induced shift in the equilibrium between the soluble and polymerized tubulin pools in the anterior pituitary lobe.

Animals↗
PubMed1976

Adsorption of albumin on rabbit sperm membranes.

When mammalian sperm cells are exposed to solutions of albumin there are changes in the membranes of some species that resemble those that normally occur in the uterus prior to fertilization. We have shown that albumin molecules absorb on to the membranes of ejaculated rabbit sperm cells, and that the equilibrium binding constant, K, (1) varies inversely with the albumin concentration, (2) is independent of the sperm cell concentration in the range of 10(6)--10(7) per ml, (3) is independent of the time of exposure of the sperm cells to the albumin solution, and (4) decreases in the presence of Ca++ and Mg++ ions. An unusual aspect of the adsorption is that if the albumin concentration is given the symbol [A], K[A] is a constant in our measurements. This means that for virtually the entire range of [A] studied, the sperm cells bind albumin so that half of the available surface is coated and half remains uncoated. This situation is rather remarkable and it suggests a role that adsorption could play in the physical processes preceding fertilization. In purely physical systems, the optimum for the bridging and flocculation of particles that are coated with adsorbed macromolecular films occurs when half of the available surface is covered. The sperm cell appears to provide the optimal situation for interacting with itself or with another surface.

Adsorption↗
PubMed2005

Evolutionary dynamics and highly optimized tolerance.

We develop a numerical model of a lattice community based on Highly Optimized Tolerance (HOT), which relates the evolution of complexity to robustness tradeoffs in an uncertain environment. With the model, we explore scenarios for evolution and extinction which are abstractions of processes which are commonly discussed in biological and ecological case studies. These include the effects of different habitats on the phenotypic traits of the organisms, the effects of different mutation rates on adaptation, fitness, and diversity, and competition between generalists and specialists. The model exhibits a wide variety of microevolutionary and macroevolutionary phenomena which can arise in organisms which are subject to random mutation, and selection based on fitness evaluated in a specific environment. Generalists arise in uniform habitats, where different disturbances occur with equal frequency, while specialists arise when the relative frequency of different disturbances is skewed. Fast mutators are seen to play a primary role in adaptation, while slow mutators preserve well-adapted configurations. When uniform and skewed habitats are coupled through migration of the organisms, we observe a primitive form of punctuated equilibrium. Rare events in the skewed habitat lead to extinction of the specialists, whereupon generalists invade from the uniform habitat, adapt to their new surroundings, ultimately leading their progeny to become vulnerable to extinction in a subsequent rare disturbance.

Adaptation, Physiological↗
PubMed2004

Body temperature regulation during hemodialysis in long-term patients: is it time to change dialysate temperature prescription?

During hemodialysis procedures, changes in the dialysate temperature can raise or lower body temperature because the blood is returned to the patient in thermal equilibrium with the dialysate. Even a dialysate temperature equal to the patient's body temperature as measured from the tympanic membrane, oral cavity, or axilla can result in an increase in the patient's body temperature, leading to cutaneous vasodilation and the potential for cardiovascular instability and hypotension. This deleterious cycle of events can be prevented by suitably adjusting the dialysate temperature. Lowering the dialysate temperature from 37 degrees C to 34-35.5 degrees C has improved the cardiovascular stability of many hemodialysis patients. Continuous monitoring of blood temperature allows the practitioner to make preemptive changes in dialysate temperature because a small change in body temperature can have enormous cardiovascular implications. For example, only 0.3 degrees C to 0.8 degrees C separates the thresholds for skin vasodilation from that for shivering. A suggested improvement in the hemodialysis procedure is to use devices that allow continuous monitoring of arterial and venous blood temperatures and adjust the dialysate temperature automatically, keeping the patient, not the dialysate, isothermic. Less optimal solutions appear to be (1) to monitor arterial and venous temperatures while manually adjusting the dialysate temperature to maintain arterial (and hence body) temperature stability; (2) to monitor peripheral temperatures (oral, tympanic) at regular intervals and adjust dialysate temperature to maintain the body temperature constant; (3) routinely use a dialysate temperature <37.0 degrees C in all patients unless contraindicated.

Adult↗
PubMed1976

A contribution to the pharmacokinetics of bencyclane (Fludilat) in man.

The quantitative determination of bencyclane from the biological material was carried out with the aid of a combined microchemical method (thin-layer chromatography and measurement of fluorescence) using NBD chloride. The original method [J. Reisch, Z. Analyt. Chemie, 247 (1969) 56; J. Monforte, Clinical Chemistry 18 (1972) 1329; R.S. Fager, Anal. Biochemistry, 53 (1973) 290, etc.] was so modified as to enable attainment of optimal results in respect of sensitivity and accuracy in the determination of bencyclane. The sensitivity of this modified method is 0.1 mug/ml plasma. Volunteer subjects and patients received under standard conditions 2 coated tablets Fludilat (i.e. 200 mg bencyclane hydrogen fumarate) orally as a single dose or repeated 3 times daily over 5 days, or 4 ampoules (= 200 mg) in a single intravenous injection. After a single oral administration, maximum plasma concentrations of approximately 2 mug/ml were attained in about 2 hours. The elimination half-life was about 360-480 min. The appearance of a second peak after about 6-7 hours indicates involvement of several compartments. On intravenous administration, maximum plasma concentrations of above 2 mug/ml were attained. A second peak in the late phase of the elimination was also detected here. The repeated oral administration led to maximum plasma concentrations of above 3 mug/ml without there being any indication of accumulation. Protein binding of about 30% was determined with the aid of the equilibrium dialysis method. A parallel "in vitro" study with 14C-bencyclane (U.R. Kleeberg, 1973, unpublished) showed an approx. 40% protein binding, an approx. 30% erythrocyte binding, and an approx. 10% thrombocyte binding. About 20% bencyclane remain free.

Administration, Oral↗
PubMed2000

Rectal cancer in pregnancy: a new management based on blended anesthesia and monitoring of fetal well being.

Colorectal carcinoma presenting during pregnancy is an extremely rare condition associated with a poor prognosis. In this report we studied a patient referred to our hospital at 26 weeks of gestation with the diagnosis of rectal adenocarcinoma. Tumor resection with a colostomy was planned in the attempt to preserve pregnancy until fetal viability could be reached. Blended anesthesia (general and epidural) was chosen to avoid surgical and anesthesiological risks; in fact this technique allows either an optimal block of neurohormonal response or a good control of surgical stress to be obtained. In order to monitor fetal well being during surgery, Doppler evaluations of fetal heart rate and umbilical artery flow velocity waveforms were performed. The patient was dismissed in good health and then rehospitalized at 32 weeks of gestation in order to perform an elective cesarean section. In conclusion we suggest that, with the choice of a good anesthesiological technique and monitoring of fetal well being, surgical treatment in case of rectal cancer could be performed without affecting the course of pregnancy.

Acid-Base Equilibrium↗
PubMed2000

Mutations in the N-terminal cooperativity domain of gene 32 protein alter properties of the T4 DNA replication and recombination systems.

The gene 32 protein (gp32) of bacteriophage T4 is the essential single-stranded DNA (ssDNA)-binding protein required for phage DNA replication and recombination. gp32 binds ssDNA with high affinity and cooperativity, forming contiguous clusters that optimally configure the ssDNA for recognition by DNA polymerase or recombination enzymes. The precise roles of gp32 affinity and cooperativity in promoting replication and recombination have yet to be defined, however. Previous work established that the N-terminal "B-domain" of gp32 is essential for cooperativity and that point mutations at Arg(4) and Lys(3) positions have varying and dramatic effects on gp32-ssDNA interactions. Therefore, we examined the effects of six different gp32 B-domain mutants on T4 in vitro systems for DNA synthesis and homologous pairing. We find that the B-domain is essential for gp32's stimulation of these reactions. The stimulatory efficacy of gp32 B-domain mutants generally correlates with the hierarchy of relative ssDNA binding affinities, i.e. wild-type gp32 approximately R4K > K3A approximately R4Q > R4T > R4G gp32-B. However, the functional defect of a particular mutant is often greater than can be explained simply by its ability to saturate the ssDNA at equilibrium, suggesting additional defects in the proper assembly and activity of DNA polymerase and recombinase complexes on ssDNA, which may derive from a decreased lifetime of gp32-ssDNA clusters.

Bacteriophage T4↗
PubMed2003

Equilibrium and balanced growth of a vegetative crop.

MODEL: A previously developed dynamic model, NICOLET, designed to predict growth and nitrate content of a lettuce crop, is subjected to (virtual) constant environmental conditions. For every combination of shoot and root environment, the cell sap, here assumed to reside in the "vacuole" compartment, equilibrates at a certain nitrate concentration level. This, in turn, defines the composition of the crop in terms of carbon and nitrogen content in each of the three compartments of the model. Growth under constant environmental conditions is defined as "equilibrium" growth (EG). If, in addition, the source strengths of carbon and nitrogen balance each other, as well as the sink strength of the growing crop, the growth is said to be "balanced" (BG). RESULTS: It is shown that the range of BG approximately coincides with the range of "mild" nitrogen stress, where reduction in nitrogen availability results in a mild reduction of relative growth rate (RGR). Beyond a certain low nitrate concentration in the cell sap, the N-stress becomes "severe" and the loss of growth increases considerably. CONCLUSIONS: The model is able to mimic the five central observations of many constant-environment growth-chamber experiments, namely (1) the initial exponential growth and later decline of the RGR, (2) the constant chemical composition, (3) the equality of the RGR and the relative nutrient supply rate (RNR), (4) the proportionality between the N : C ratio and the RNR, and (5) the proportionality between the water content and the reduced N content. Guidelines for the optimal combination of the shoot and root environments are suggested.

Crops, Agricultural↗
PubMed1993

The storage of energy as a cause of malignant transformation: a 7-phase model of carcinogenesis.

Although many theories for the development of cancer exist, a new hypothesis for carcinogenesis is suggested and a new 7-phase model of malignant transformation described. Both the hypothesis and the model are based on the principle of a critical point in local energy (entropy?) storage, at a certain level of structural organisation within the cells. This principle has been previously formulated by the author from the rules of non-equilibrium thermodynamics. The model introduces a new terminology and explores the concepts of both high work value of energy and bifurcation. The ability (A) of cells is suggested to be the most important cellular feature in respect to cell survival. This ability implies that the cells follow the requirement Kir > 1, even in dangerous situations and under harmful environmental influence. But, when the cells have lost their ability (A) and all levels of the cell defence machine have been exhausted, then the local energy storage may provoke a cascade of harmful events within the cells. The S-stage is the most unstable state of the cell cycle. If such harmful events take place during DNA synthesis within the 'premalignant' cell, in the phase 'promotion' at the 4th bifurcation, they lead ultimately to carcinogenesis (i.e. malignant transformation). The 7-phase model of carcinogenesis is consistent and offers many advantages in comparison to the previous hypotheses. This model could help the design of experiments, development of new drugs and optimization of medical treatment. The new hypothesis could contribute to a better understanding of the processes of carcinogenesis and the uncontrolled division, growth and lability of tumour cells.

Animals↗
PubMed1998

Immunocytochemistry of formalin-fixed human brain tissues: microwave irradiation of free-floating sections.

Formalin fixation, the chemical process in which formaldehyde binds to cells and tissues, is widely used to preserve human brain specimens from autolytic decomposition. Ultrastructure of cellular and mitochondrial membranes is markedly altered by vesiculation, but this does not interfere with diagnostic evaluation of neurohistology by light microscopy. Serious difficulties are encountered, however, when immunocytochemical staining is attempted. Antigens that are immunoreactive in unfixed frozen sections and protein extracts appear to be concealed or destroyed in formalin-fixed tissues. In dilute aqueous solution, formaldehyde is in equilibrium with methylene glycol and its polymeric hydrates, the balance by far in favor of methylene glyco. Carbonylic formaldehyde is a reactive electrophilic species well known for crosslinking functional groups in tissue proteins, nucleic acids, and polysaccharides. Some of its methylene crosslinks are readily hydrolyzed. Others are stable and irreversible. During immunostaining reactions, intra- and inter-molecular links between macromolecules limit antibody permeation of tissue sections, alter protein secondary structure, and reduce accessibility of antigenic determinants . Accordingly, immunoreactivity is diminished for many antigens. Tissues are rapidly penetrated by methylene glycol, but formaldehyde binding to cellular constituents is relatively slow, increasing progressively until equilibrium is reached. In addition, prolonged storage in formalin may result in acidification of human brain specimens. Low pH favors dissociation of methylene glycol into formaldehyde, further reducing both classical staining and antigen detectability. Various procedures have been devised to counter the antigen masking effects of formaldehyde. Examples include pretreatment of tissue sections with proteases, formic acid, or ultrasound. Recently, heating of mounted sections in ionic salt solution by microwave energy was found to restore many antigens. Theory and practice of microwave antigen retrieval are covered extensively in the handbook Microwave Cookbook for Microscopists. A concise overview of microwave methods in the neurosciences has been published, and clinical applications have been reviewed. In this context, it should be noted that fresh tissues may be stabilized for immunocytochemistry by reversible, non-chemical binding processes such as cryosectioning after microwave treatment and freeze-drying. Thus, it may be possible to enhance immunostaining for some antigens by microwave irradiation of unfixed as well as fixed specimens. Parameters to be optimized for microwave retrieval of specific antigens include temperature, irradiation time, tissue buffer composition, salt concentration, and pH. Temperature, irradiation time, and pH are key variables. With this in mind, an optimal method was developed for retrieval of a wide variety of antigens in human brain tissues. Typical microwave protocols employ elevated temperatures that may reach 100 degrees C, where denaturation causes irreversible uncoiling and disruption of protein secondary and tertiary structures. Under these conditions, stable covalent bonds securing methylene crosslinks between polypeptides remain intact, but more reactive links formed by Schiff bases may be hydrolyzed. Resultant conformational changes presumably expose buried loops of continuous amino acids and protruding regions, increasing accessibility of their epitopes. Protein denaturation seems to be a reasonable explanation for the effects of microwaves on antigen retrieval. This idea is supported by the observation that denaturing solutions such as 6 M urea increase immunoreactivity of some antigens. Still, the molecular basis of these effects remains unresolved, in part due to the complex chemistry of formaldehyde reactions with tissue constituents. Indeed, some methylene bridges between similar groups such as NH2 and NH may be hydrolyzed by washing fixed tissues in distilled wa

Benzothiazoles↗
PubMed2004

Fractionation effects in phase equilibria of polydisperse hard-sphere colloids.

The equilibrium phase behavior of hard spheres with size polydispersity is studied theoretically. We solve numerically the exact phase equilibrium equations that result from accurate free energy expressions for the fluid and solid phases, while accounting fully for size fractionation between coexisting phases. Fluids up to the largest polydispersities that we can study (around 14%) can phase separate by splitting off a solid with a much narrower size distribution. This shows that experimentally observed terminal polydispersities above which phase separation no longer occurs must be due to nonequilibrium effects. We find no evidence of reentrant melting; instead, sufficiently compressed solids phase separate into two or more solid phases. Under appropriate conditions, coexistence of multiple solids with a fluid phase is also predicted. The solids have smaller polydispersities than the parent phase as expected, while the reverse is true for the fluid phase, which contains predominantly smaller particles but also residual amounts of the larger ones. The properties of the coexisting phases are studied in detail; mean diameter, polydispersity, and volume fraction of the phases all reveal marked fractionation. We also propose a method for constructing quantities that optimally distinguish between the coexisting phases, using principal component analysis in the space of density distributions. We conclude by comparing our predictions to Monte Carlo simulations at imposed chemical potential distribution, and find excellent agreement.

Journal Article↗
PubMed1991

Characterization of epidermal growth factor binding to hepatic plasma membranes of rainbow trout (Oncorhynchus mykiss).

Epidermal growth factor (EGF) is a potent mitogen which exerts its effects through a transmembrane receptor located on target cells. Since little is known about EGF in nonmammalian animals, experiments were conducted to characterize the EGF receptor in rainbow trout hepatic cell membranes. The binding of mouse EGF with rainbow trout receptors was peptide-specific, saturable, reversible, and of high affinity. Optimal binding was observed at pH 7.2. Both monovalent and divalent cations augmented the specific binding of EGF, by a factor of two- to threefold over control values, but were not needed for binding to occur. Scatchard plot of the saturation data was curvilinear. Analysis of the data by kinetic methods indicated the curvilinear nature of the Scatchard plot was due to multiple receptor sites of differing affinity and not to site-to-site interactions. Rate constants for association (K11) were 9.38 X 10(8) M-1 * min-1 and 2.28 X 10(7) M-1 * min-1 for the high and low affinity sites, respectively. Rate constants for dissociation (K-1) were 2.03 X 10(-3) min-1 and 2.07 X 10(-1) min-1 for high and low affinity sites, respectively. The apparent dissociation constants determined from the rate constants for the high affinity (KD = 2.3 X 10(-11) M) and low affinity (KD = 9.1 X 10(-9) M) were in agreement with constants estimated by equilibrium methods. Maximum binding capacities were 13 fmol EGF bound/mg protein of protein and 270 fmol EGF bound/mg of protein for the high and low affinity receptor sites, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Biosorption of cadmium by various types of dried sludge: an equilibrium study and investigation of mechanisms.

Batch equilibrium sorption experiments were used for screening for cost-effective four types of sludge, which were DWS (drinking water treatment plant sludge), LLS (landfill leachate sludge), ADSS (anaerobically digested sewage sludge), and SS (sewage sludge). SS removed cadmium most efficiently from aqueous solution (0.38 mmol/g), and showed the highest desorption efficiency (26.3%). Only the SS can be fitted by Langmuir isotherm model (r2 = 0.996). The FT-IR spectra of SS and cadmium loaded SS indicated that carboxyl groups were major binding sites of cadmium binding sites. In kinetic experiment, it was found that the uptake of the metal by the SS was accompanied with proton release, indicating that the metal binding occurs via an ion exchange as well as by electrostatic interaction between carboxylate groups and cadmium ions. This sorbent may have a potential for use as high-value biosorbent of heavy metals and it deserves further investigations into the details of practical application, for example on the development of desorption methods and on sorption process optimization.

Adsorption↗
PubMed1975

Bacterial bioluminescence: equilibrium association measurements, quantum yields, reaction kinetics, and overall reaction scheme.

The characteristics of the bioluminescence reactions with bacterial luciferase from two different cell types, Photobacterium fischeri and Beneckea harveyi, are reported. The reduced flavine mononucleotide (FMNH2)-luciferase association constant, directly measured by equilibrium dialysis and gel filtration is the same for both luciferases, 3 times 10(-4) Mminus1 at romm temperature, and is significantly different from the kinetic reciprocal Michaelis-Menten constant. The luciferase bioluminescence quantum yield for the highest activity preparations is the same as for the aldehyde. Rapid stopped-flow observations show that the oxidation of FMNH2 in the presence of sufficient luciferase to outcompete autoxidation, is bimodal. A long-lived intermediate, formed before reaction with aldehyde, has an activation energy for decay of 35 kcal mol-1, much greater than for the light reaction, 14 kcal mol-1. The ratio of bioluminescence quantum yields with respect to aldehyde and FMNH2 is independent of temperature, however, and also of aldehyde chain lenght longer than octanal, pH (6.5-8), and type of luciferase and its specific activity. Even when the aldehyde concentration limits the rate of the light reaction, the quantum yield of the long-lived intermediate is unchanged, and together these data mean that, under the optimal conditions chosen for quantum yield measurements, no dark side reactions effectively compete with the main reaction leading to light emission. A series of reactions involving one-electron steps and the sequential oxidation of two FMNH2 molecules is postulated for the formation of the long-lived intermediate.

Aldehydes↗
PubMed1994

Multiple-site binding interactions in metal-affinity chromatography. I. Equilibrium binding of engineered histidine-containing cytochromes c.

Mechanisms of protein retention in immobilized metal-affinity chromatography (IMAC) have been probed using a set of Saccharomyces cerevisiae iso-1-cytochrome c histidine variants constructed by site-directed mutagenesis. Proteins containing a single accessible histidine exhibit Langmuir-type isotherms with maximum protein binding capacities between 5 and 10% of the maximum copper loading and the capacity of the support to bind imidazole. A simple model that assumes that the copper sites are densely packed and can be blocked by protein adsorption yields binding constants for single-histidine proteins that are similar to the binding constant for free imidazole. Proteins containing multiple accessible histidines do not exhibit simple Langmuir-type behavior; they appear to interact with the support by simultaneous coordination to more than one metal ion, the result of which is to increase the apparent binding affinity by as much as a factor of 1000. The protein binding constant depends on the availability of copper sites: binding is significantly weaker at low surface concentrations of copper that presumably cannot support multiple-site interactions. The protein binding capacity drops to zero at copper loadings less than one-half the maximum, indicating that immobilized iminodiacetic acid ligands are sufficiently close together that two can coordinate a single copper ion, which precludes its interaction with a protein. Protein adsorption via multiple-site coordination has important consequences for the optimization of IMAC separations and the design of new IMAC supports.

Animals↗
PubMed2004

Commercial application of marker- and gene-assisted selection in livestock: strategies and lessons.

During the past few decades, advances in molecular genetics have led to the identification of multiple genes or genetic markers associated with genes that affect traits of interest in livestock, including genes for single-gene traits and QTL or genomic regions that affect quantitative traits. This has provided opportunities to enhance response to selection, in particular for traits that are difficult to improve by conventional selection (low heritability or traits for which measurement of phenotype is difficult, expensive, only possible late in life, or not possible on selection candidates). Examples of genetic tests that are available to or used in industry programs are documented and classified into causative mutations (direct markers), linked markers in population-wide linkage disequilibrium with the QTL (LD markers), and linked markers in population-wide equilibrium with the QTL (LE markers). In general, although molecular genetic information has been used in industry programs for several decades and is growing, the extent of use has not lived up to initial expectations. Most applications to date have been integrated in existing programs on an ad hoc basis. Direct markers are preferred for effective implementation of marker-assisted selection, followed by LD and LE markers, the latter requiring within-family analysis and selection. Ease of application and potential for extra-genetic gain is greatest for direct markers, followed by LD markers, but is antagonistic to ease of detection, which is greatest for LE markers. Although the success of these applications is difficult to assess, several have been hampered by logistical requirements, which are substantial, in particular for LE markers. Opportunities for the use of molecular information exist, but their successful implementation requires a comprehensive integrated strategy that is closely aligned with business goals. The current attitude toward marker-assisted selection is therefore one of cautious optimism.

Animals↗
PubMed1986

Orientation selectivity of synaptic potentials in neurons of cat primary visual cortex.

Neurons of the visual cortex of the cat were penetrated with intracellular electrodes and postsynaptic potentials evoked by visual stimuli recorded. By alternately polarizing the cell with steady current injected through the recording electrode, IPSPs and EPSPs could be recorded and analyzed independently. Hyperpolarizing current suppressed IPSPs and enhanced EPSPs by moving the membrane potential toward the IPSP equilibrium potential. Depolarizing the cell toward the EPSP equilibrium potential enhanced IPSP. The responses to electrical stimulation of the LGN, where EPSPs and IPSPs could be distinguished easily by virtue of their characteristic latencies and shapes, were used to set the current injection to the appropriate level to view the two types of synaptic potential. EPSPs were found to be well oriented in that maximal depolarizing responses could be evoked at only one stimulus orientation; rotating the stimulus orientation in either direction produced a fall in the EPSP response. IPSPs were also well tuned to orientation, and invariably the preferred orientations of EPSPs and IPSPs in any one cell were identical. In addition, no systematic difference in the width of tuning of the two types of potential was seen. This result has been obtained from penetrations of over 30 cortical cells, including those with simple and complex receptive fields. It is concluded that orientation of cortical receptive fields is neither created nor sharpened by inhibition between neurons with different orientation preference. The function of inhibition evoked simultaneously with excitation by optimally oriented stimuli has yet to be determined, though it is likely to be the mechanism underlying other cortical receptive field properties, such as direction selectivity and end-stopping.

Animals↗
PubMed1995

Effects of altered phosphorylation sites on the properties of CTP:phosphocholine cytidylyltransferase.

To investigate the role of phosphorylation and dephosphorylation in modulating the activity and location of CTP:phosphocholine cytidylyltransferase, we used site-directed mutagenesis to construct four mutant forms of cytidylyltransferase. These forms were 5SP-->AP, in which five of the seven Ser-Pro sequences were converted to Ala-Pro; 7SP-->AP, in which all of the seven Ser-Pro sequences converted to Ala-Pro; 16S-->A, in which all sixteen Ser residues that can be phosphorylated in wild type cytidylyltransferase were converted to Ala; and 16S-->E, in which all sixteen Ser residues were converted to Glu. The mutant enzymes were expressed in the strain 58 Chinese hamster ovary cell line, which is temperature-sensitive for growth and cytidylyltransferase activity. All mutant enzyme forms were enzymatically as active as the wild type when assayed under optimal conditions. In untreated cells, more of the Ser-->Ala mutants were membrane-associated than in cells expressing wild type enzyme, consistent with the phosphorylation state of the enzyme affecting its affinity for membranes. About half of the 16S-->A mutant remained soluble, however, indicating that dephosphorylation alone does not trigger membrane association. Although the amount of membrane-associated enzyme in the 16S-->A mutant was about 10-fold greater than that of wild type, phosphatidylcholine synthesis was increased by only about 75%, suggesting that membrane association does not necessarily cause full activation. All mutant forms, including the 16S-->E mutant, translocated to the particulate fraction upon oleate treatment, indicating that a high negative charge in the phosphorylation region does not preclude association of cytidylyltransferase with membranes. All mutant enzymes were able to support growth of strain 58 at 40 degrees C, and the rate of phosphatidylcholine synthesis was not greatly altered in the cell lines expressing mutant cytidylyltransferase forms. These results are consistent with a role for phosphorylation in the equilibrium distribution of cytidylyltransferase but suggest that changes in enzyme activity and location are not triggered exclusively by changes in the phosphorylation state.

Amino Acid Sequence↗