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Biomedical subjects

R A Warren

Publications and source records attributed to R A Warren.

At least 19 recordsLinked to original sources
PubMed1997

Saturation of the endocytic pathway for the transferrin receptor does not affect the endocytosis of the epidermal growth factor receptor.

Cell-surface receptors that undergo clathrin-mediated endocytosis contain short amino acid sequences in their cytoplasmic domain that serve as internalization signals. Interactions between these sequences and components of the endocytic machinery should become limiting upon overexpression of the constitutively recycling transferrin receptor (TfR). A tetracycline-responsive system was used to induce overexpression of the TfR up to 20-fold in HeLa cells. Internalization assays indicate the rate of 125I-transferrin uptake per surface TfR is reduced by a factor of 4 in induced cells. Consistent with endocytosis being the rate-limiting step, TfRs shift from an endosomal to more of a plasma membrane distribution with TfR overexpression. The clathrin-associated protein AP-2 has been proposed to interact directly with the cytoplasmic domain of many receptors, yet no changes in the amount or distribution of AP-2 were detected in induced cells. The internalization rate for the epidermal growth factor receptor was also measured, with or without induction of TfR expression. Even though endocytosis of the TfR is saturated in induced cells, 125I-labeled epidermal growth factor continues to be internalized at a rate identical to that seen in uninduced cells. We propose that there are different limiting steps for the endocytosis of these two receptors.

Adaptor Protein Complex alpha Subunits↗
PubMed1997

Maturation of neuronal form and function in a mouse thalamo-cortical circuit.

Postnatal development of physiological properties underlying slow intrathalamic oscillations was studied by whole-cell recording from synaptically coupled neurons of the reticular nucleus (RTN) and ventral posterior nucleus (VPN) of mouse brain slices in vitro and compared with the morphological development of dye-injected cells. Between postnatal days 3 and 11 (P3-P11), progressive changes in RTN and VPN neurons included shortening of the membrane time constant, decreasing input resistance, and lowering of the resting membrane potential (RMP). Low-threshold Ca2+ spikes (LTS) were present from P3, but their capacity to sustain multispike bursts was limited before P11. Synaptic responses were evoked in RTN and VPN neurons by electrical stimulation of the internal capsule from P3. Younger RTN neurons responded with a single spike, but their capacity to fire bursts gradually improved as the RMP reached levels below the LTS activation potential. Concomitantly, as the reversal potential of the inhibitory postsynaptic potential in VPN neurons became more negative, its capacity to deinactivate the LTS increased, and rebound bursts that could maintain oscillations were produced; sustained oscillations became the typical response to internal capsule stimulation at P12. The functional maturation of the intrathalamic circuitry, particularly between P10 and P14, occurs in parallel with the morphological maturation (size, dendritic growth, and dendritic field structure) of individual RTN and VPN neurons, as studied by confocal microscopy. Maturation of RTN cells led that of VPN cells by 2-3 d. The appearance of intrathalamic oscillations is probably correlated with the appearance of slow-wave sleep in postnatal animals.

Aging↗
PubMed1997

GABA(B)-receptor-mediated inhibition in developing mouse ventral posterior thalamic nucleus.

Inhibitory postsynaptic potentials (IPSPs) generated by activation of the thalamic reticular nucleus (RTN) were recorded in neurons of the ventral posterior nucleus (VP) in vitro in slices from mice aged postnatal day (P)1-P17. An early IPSP peaking 41 +/- 2.5 (SE) ms after electrical stimulation of the internal capsule or RTN was found in 96% of VP neurons. This early IPSP was blocked by bicuculline, showing its dependence on gamma-aminobutyric acid-A (GABA(A)) receptors. A late IPSP peaking 357 +/- 27 ms after the stimulus was observed in 22% of VP neurons in control medium but was uncovered in 38% of neurons when bicuculline was added. The late IPSP was blocked by addition of a GABA(B) antagonist, 2-hydroxysaclofen, to the medium (n = 7); it had a reversal potential of -98 +/- 1.3 mV, 14 mV negative to the early component. In contrast to the early IPSP, whose reversal potential became more negative during postnatal development, the reversal potential of the late IPSP remained constant throughout the postnatal period studied. The most significant change in the late IPSP was shortening in duration, with reduction in latency-to-peak by >400 ms, between P1 and P10. No changes of comparable magnitude were observed in the duration of the earlier GABA(A) response. These results show that both GABA(A) and GABA(B) IPSPs are present very early in the postnatal thalamus and that their characteristics evolve along independent paths during postnatal development.

Animals↗
PubMed1996

Mechanistic consequences of mutation of active site carboxylates in a retaining beta-1,4-glycanase from Cellulomonas fimi.

The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate. The roles of three conserved active site carboxylic acids in this enzyme have been probed by detailed kinetic analysis of mutants modified at these three positions. Elimination of the catalytic nucleophile (E233A) results in an essentially inactive enzyme, consistent with the important role of this residue. However addition of small anions such as azide or formate restores activity, but as an inverting enzyme since the product formed under these conditions is the alpha-glycosyl azide. Shortening of the catalytic nucleophile (E233D) reduces the rates of both formation and hydrolysis of the glycosyl-enzyme intermediate some 3000-4000-fold. Elimination of the acid/base catalyst (E127A) yields a mutant for which the deglycosylation step is slowed some 200-300-fold as a consequence of removal of general base catalysis, but with little effect on the transition state structure at the anomeric center. Effects on the glycosylation step due to removal of the acid catalyst depend on the aglycon leaving group ability, with minimal effects on substrates requiring no general acid catalysis but large (> 10(5)-fold) effects on substrates with poor leaving groups. The Brønsted beta 1g value for hydrolysis of aryl cellobiosides was much larger (beta 1g approximately -1) for the mutant than for the wild-type enzyme (beta 1g = -0.3), consistent with removal of protonic assistance. The pH-dependence was also significantly perturbed. Mutation of a third conserved active site carboxylic acid (E123A) resulted in rate reductions of up to 1500-fold on poorer substrates, which could be largely restored by addition of azide, but without the formation of glycosyl azide products. These results suggest a simple strategy for the identification of the key active site nucleophile and acid/base catalyst residues in glycosidases without resort to active site labeling.

Binding Sites↗
PubMed1996

Substrate specificity of endoglucanase A from Cellulomonas fimi: fundamental differences between endoglucanases and exoglucanases from family 6.

Values of kcat. and Km for the hydrolysis of cellotetraose, cellotriose, beta-cellobiosyl fluoride and various beta-aryl cellobiosides by endoglucanase A (CenA) from Cellulomonas fimi indicate that specific binding interactions between the reducing-end glucose residues of cellotetraose and cellotriose and the enzyme at the transition state provide enormous stabilization, endowing glucose with the "effective leaving group ability' of 2,4-dinitrophenol. As has been seen with several other inverting glycosidases, CenA hydrolyses the "wrong' anomer of its glycosyl fluoride substrate, alpha-cellobiosyl fluoride, according to non-Michaelian kinetics. This indicates that CenA carries out this hydrolysis by a mechanism involving binding of two substrate molecules in the active site (Hehre, Brewer and Genghof (1979) J. Biol. Chem. 254, 5942-5950] in contrast with that reported for cellobiohydrolase II, another family-6 enzyme [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 833-838]. The pH profiles for wild-type CenA indicate that kcat. for CenA depends on the presence of both a protonated group and a deprotonated group for full activity, consistent with the presence of an acid and a base catalyst at the active site. By contrast, the profile for the Asp252Ala mutant of CenA shows a dependence only on a base-catalytic group, thereby confirming the role of Asp-252 as an acid catalyst. These results show that hydrolysis by CenA occurs by a typical inverting mechanism involving both acid and base catalysis, as first proposed by Koshland. It also suggests that endoglucanases from family 6 may function by fundamentally different mechanisms for exoglucanases in this family.

Actinomycetales↗
PubMed1996

Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification.

The cellulose-binding domain (CBDCex) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the beta-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604-607], each of the exposed tryptophans interacts reversibly with cellulose.

Binding Sites↗
PubMed1996

Characterization of CenC, an enzyme from Cellulomonas fimi with both endo- and exoglucanase activities.

The cenC gene, encoding beta-1,4-glucanase C (CenC) from Cellulomonas fimi, was overexpressed in Escherichia coli with a tac-based expression vector. The resulting polypeptide, with an apparent molecular mass of 130 kDa, was purified from the cell extracts by affinity chromatography on cellulose followed by anion-exchange chromatography. N-terminal sequence analysis showed the enzyme to be properly processed. Mature CenC was optimally active at pH 5.0 and 45 degrees C. The enzyme was extremely active on soluble, fluorophoric, and chromophoric glycosides (4-methylumbelliferyl beta-glycosides, 2'-chloro-4'-nitrophenyl-beta-D-cellobioside, and 2'-chloro-4'-nitrophenyl-lactoside) and efficiently hydrolyzed carboxymethyl cellulose, barley beta-glucan, lichenan, and, to a lesser extent, glucomannan. CenC also hydrolyzed acid-swollen cellulose, Avicel, and bacterial microcrystalline cellulose. However, degradation of the latter was slow compared with its degradation by CenB, another C. fimi cellulose belonging to the same enzyme family. CenC acted with inversion of configuration at the anomeric carbon, in accordance with its classification as a family 9 member. The enzyme released mainly cellobiose from soluble cellodextrins and insoluble cellulose. Attack appeared to be from the reducing chain ends. Analysis of carboxymethyl cellulose hydrolysis suggests that CenC is semiprocessive enzyme with both endo- and exoglucanase activities.

Base Sequence↗
PubMed1996

Microbial hydrolysis of polysaccharides.

Microorganisms are efficient degraders of starch, chitin, and the polysaccharides in plant cell walls. Attempts to purify hydrolases led to the realization that a microorganism may produce a multiplicity of enzymes, referred to as a system, for the efficient utilization of a polysaccharide. In order to fully characterize a particular enzyme, it must be obtained free of the other components of a system. Quite often, this proves to be very difficult because of the complexity of a system. This realization led to the cloning of the genes encoding them as an approach to eliminating other components. More than 400 such genes have been cloned and sequenced, and the enzymes they encode have been grouped into more than 50 families of related amino acid sequences. The enzyme systems revealed in this manner are complex on two quite different levels. First, many of the individual enzymes are complex, as they are modular proteins comprising one or more catalytic domains linked to ancillary domains that often include one or more substrate-binding domains. Second, the systems are complex, comprising from a few to 20 or more enzymes, all of which hydrolyze a particular substrate. Systems for the hydrolysis of plant cell walls usually contain more components than systems for the hydrolysis of starch and chitin because the cell walls contain several polysaccharides. In general, the systems produced by different microorganisms for the hydrolysis of a particular polysaccharide comprise similar enzymes from the same families.

Bacteria↗
PubMed1996

Transient and long-lasting effects of iontophoretically administered norepinephrine on somatosensory cortical neurons in halothane-anesthetized cats.

We examined the modulatory effects of iontophoretically administered norepinephrine (NE) on the excitability of 117 neurons in cat somatosensory cortex. NE was released in the vicinity of neurons with receptive fields (31/117) while they were excited by somatic stimuli and near neurons without receptive fields (86/117) while they were excited by glutamate. In 54% of the neurons (n = 63) the effects were inhibitory, decreasing both the spontaneous and the driven activity. Most of these cells were found in the middle layers of cortex. In 36% of the neurons (n = 42), mostly located in either the upper or lower layers, the effects were excitatory, enhancing either or both driven and spontaneous activity, but 52% (n = 22) of this group displayed a transient phase of inhibition. NE usually had a proportionately greater effect on the spontaneous activity than on the evoked activity. Effects of specific NE agonists and antagonists indicated that alpha 2- and beta-receptors mediated the inhibition, but that alpha 1-receptors mediated the excitation. We hypothesize that when NE is released in cat somatosensory cortex, it modulates neuronal responses to afferent activity by generally reducing the excitability of cells in the middle layers and by increasing their signal-to-noise ratios. However, in the upper and lower layers NE will enhance neuronal activity, encouraging exchanges with other cortical areas. In addition, we tested and confirmed the hypothesis that short treatments with NE can modify the excitability of neurons in adult cat somatosensory cortex for long periods of time. Of 69 cells, 59% showed effects of NE lasting for more than 5 min. Response enhancement lasting for the duration of the recording session (5 to 36 min) occurred in 82% (18/22) of silent cells without a receptive field and in 63% (17/27) of spontaneously active cells without a receptive field, but only in 14% (2/14) of cells with a receptive field, suggesting an inverse relationship between cell excitability and this effect of NE. The magnitude of the enhancement followed the same relationship, being greatest for silent cells and least for cells with receptive fields (125, 58, and 49%, respectively). The long-lasting enhancement was blocked by an alpha 1-receptor antagonist in 6 of 9 neurons tested, while the administration of alpha 2- and beta-receptor agonists never produced a long-lasting effect, suggesting that the effect is mediated by alpha 1-adrenoceptors.

Animals↗
PubMed1995

Identification of the acid/base catalyst in Agrobacterium faecalis beta-glucosidase by kinetic analysis of mutants.

The catalytic mechanism of the retaining beta-glucosidase (Abg) from Agrobacterium faecalis involves a double-displacement process in which an alpha-glucosyl-enzyme intermediate is formed with general acid catalytic assistance and then hydrolyzed with general base assistance. Glu170 was identified as an important residue, possibly the acid/base catalyst, on the basis of sequence alignments. This glutamate is conserved in almost all enzymes in family 1 of glycoside hydrolases. Detailed pre-steady-state and steady-state kinetic analyses of the mutant E170G suggested very strongly that Glu170 is the acid/base catalyst. First, kcat values were invariant with pH over the range of 5.0-9.0. Secondly, rates of formation of the glycosyl-enzyme, calculated from kcat/Km and k2, were similar to those of wild-type enzyme for substrates not requiring protonic assistance but dramatically reduced for those needing acid catalysis. Thirdly, addition of azide as a competitive nucleophile increased kcat values 100-300-fold for substrates whose rate-limiting step is deglycosylation, yielding beta-glucosyl azide, but had no effect on the wild-type enzyme. Other anionic nucleophiles had similar, but less dramatic effects. Previous results [Gebler, J.C., et al. (1995) 34, 14547-14553] had indicated that Tyr298F is important for catalysis. The kinetic consequences of the mutations in the double mutant E170G-Y298F are additive, resulting in a 10(6)-fold reduction in kcat values and allowing the accumulation of a stable (t1/2 > 7 h) glucosyl-enzyme intermediate. Thus, Glu170 and Tyr298 function independently, and a possible role for Tyr298 in modulating the pKa of the catalytic nucleophile is proposed.

Amino Acid Sequence↗
PubMed1995

Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic bacterium Cellulomonas fimi.

The gene cbhB from the cellulolytic bacterium Cellulomonas fimi encodes a polypeptide of 1090 amino acids. Cellobiohydrolase B (CbhB) is 1037 amino acids long, with a calculated molecular mass of 109765 Da. The enzyme comprises five domains: an N-terminal catalytic domain of 643 amino acids, three fibronectin type III repeats of 97 amino acids each, and a C-terminal cellulose-binding domain of 104 amino acids. The catalytic domain belongs to family 48 of glycosyl hydrolases. CbhB has a very low activity on CM-cellulose. Viscometric analysis of CM-cellulose hydrolysis indicates that the enzyme is an exoglucanase. Cellobiose is the major product of hydrolysis of cellulose. In common with two other exoglycanases from C. fimi, CbhB has low but detectable endoglucanase activity. CbhB is the second exo-cellobiohydrolase found in C. fimi. Therefore, the cellulase system of C. fimi resembles those of fungi in comprising multiple endoglucanases and cellobiohydrolases.

Actinomycetales↗
PubMed1995

The pTugA and pTugAS vectors for high-level expression of cloned genes in Escherichia coli.

Plasmids pTugA and pTugAS, designed for expression of cloned genes in Escherichia coli, possess the features of high-level inducible transcription, enhanced RNA translation, portability, high copy number, stability and versatility. In addition, pTugAS can be used to produce fusion proteins comprising a target protein and a cellulose-binding domain. Such fusion proteins can be purified in a single step by affinity chromatography on cellulose. Expression of two model gene fusions using the pTug plasmids resulted in yields of 500 mg of intracellular and 250 mg of extracellular recombinant protein per liter.

Alkaline Phosphatase↗
PubMed1995

Enhancement of the endo-beta-1,4-glucanase activity of an exocellobiohydrolase by deletion of a surface loop.

In the commonly accepted mechanism for enzymatic hydrolysis of cellulose, endo-beta-1,4-glucanases randomly cleave glucosidic bonds within glucan polymers, providing sites for attack by exo-cellobiohydrolases (EC 3.2.1.91). It has been proposed that hydrolysis by Trichoderma reesei cellobiohydrolase II is restricted to the ends of cellulose polymers because two surface loops cover its active site to form a tunnel. In a closely related endoglucanase, E2 from Thermomonospora fusca, access to the substrate appears to be relatively unhindered because the carboxyl-proximal loop is shortened, and the amino-proximal loop is displaced. The hypothesis was examined by deletion of a region in Cellulomonas fimi cellobiohydrolase A corresponding to part of the carboxyl-proximal loop of T. reesei cellobiohydrolase II. The mutation enhanced the endoglucanase activity of the enzyme on soluble O-(carboxymethyl)cellulose and altered its activities on 2',4'-dinitrophenyl-beta-D-cellobioside, insoluble cellulose, and cellotetraose.

Amino Acid Sequence↗
PubMed1995

Site-directed mutation of the putative catalytic residues of endoglucanase CenA from Cellulomonas fimi.

The catalytic domains of beta-1,4-glucanases can be grouped into families of related amino acid sequences. The endoglucanase CenA from Cellulomonas fimi is a member of family B. All enzymes from this family are believed to hydrolyze beta-1,4-glucosidic bonds using a general acid-base catalytic mechanism resulting in inversion of anomeric configuration at the scissile bond. Three-dimensional structures for two cellulases from family B have been determined by X-ray crystallographic analysis. These structures show that there are four Asp residues which are in a position to function as acid catalyst, base catalyst, and/or transition state stabilizers. These aspartates are conserved in all members of family B. The roles of Asp216, Asp252, Asp287, and Asp392, the corresponding amino acids in CenA, were determined. These aspartates have been systematically replaced with alanine and glutamate via site-directed mutagenesis, and the resulting effect on activity, substrate specificity, and overall structure has been determined. Changes in overall structure were monitored using circular dichroism spectroscopy, and no significant differences between the wild-type and mutant proteins were found. Active site structure was also found to be intact as all proteins bound to a cellobiose affinity column. The Michaelis-Menten parameters of the enzyme were determined on 2,4-dinitrophenyl cellobioside as well as (carboxymethyl)-cellulose and phosphoric acid-swollen cellulose. Initial characterization of mutant proteins indicates that Asp252 and Asp392 are the acid and base catalysts, respectively, in CenA. Residue Asp287 appears to aid Asp252 in acid catalysis, and Asp216 is not absolutely required for catalysis.

Aspartic Acid↗
PubMed1995

Synaptic distribution of afferents from reticular nucleus in ventroposterior nucleus of cat thalamus.

This study was aimed at determining the synaptic circuitry that contributes to the alterations in thalamic function that accompany changes in behavioral states. The somatosensory sector of the thalamic reticular nucleus (RTN) was identified by microelectrode recording in cats and injected with Phaseolus vulgaris-leucoagglutinin (PHA-L). The axons of labeled RTN cells gave rise to collaterals within the RTN and continued into the dorsal thalamus where they terminated predominately in the ventral posterior lateral nucleus (VPL). After small injections in the upper limb representation of RTN, most labeled terminations in VPL were confined to its medial part, suggesting the presence of a topographic organization in the projection. Terminations were concentrated in localized, focal aggregations of boutons. Combined electron microscopic immunocytochemistry, using immunogold labeling for gamma-aminobutyric acid (GABA), showed that the PHA-L labeled boutons were GABA-positive terminals that ended in symmetrical synapses. Eighty-two percent of these synapses were on dendrites of relay neurons, 8.5% on dendrites of interneurons, and 9.3% on somata. The terminals of RTN axons form the majority of axon terminals ending in symmetrical synapses in VPL. Their concentration on relay neurons probably underlies the capacity of the RTN projection to reduce background activity of VPL relay neurons in the awake state and to maintain oscillatory behavior of these neurons in drowsiness and early phases of sleep.

Animals↗