Phi/psi-chology: Ramachandran revisited.
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Carrier Proteins↗
Biomedical subjects
T A Jones
Publications and source records attributed to T A Jones.
Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase I from Trichoderma reesei.
The roles of the residues in the catalytic trio Glu212-Asp214-Glu217 in cellobiohydrolase I (CBHI) from Trichoderma reesei have been investigated by changing these residues to their isosteric amide counterparts. Three mutants, E212Q, D214N and E217Q, were constructed and expressed in T. reesei. All three point mutations significantly impair the catalytic activity of the enzyme, although all retain some residual activity. On the small chromophoric substrate CNP-Lac, the kcat values were reduced to 1/2000, 1/85 and 1/370 of the wild-type activity, respectively, whereas the KM values remained essentially unchanged. On insoluble crystalline cellulose, BMCC, no significant activity was detected for the E212Q and E217Q mutants, whereas the D214N mutant retained residual activity. The consequences of the individual mutations on the active-site structure were assessed for two of the mutants, E212Q and D214N, by X-ray crystallography at 2.0 A and 2.2 A resolution, respectively. In addition, the structure of E212Q CBHI in complex with the natural product, cellobiose, was determined at 2.0 A resolution. The active-site structure of each mutant is very similar to that of the wild-type enzyme. In the absence of ligand, the active site of the D214N mutant contains a calcium ion firmly bound to Glu212, whereas that of E212Q does not. This supports our hypothesis that Glu212 is the charged species during catalysis. As in the complex of wild-type CBHI with bound o-iodobenzyl-1-thio-beta-D-glucoside, cellobiose is bound to the two product sites in the complex with E212Q. However, the binding of cellobiose differs from that of the glucoside in that the cellobiose is shifted away from the trio of catalytic residues to interact more intimately with a loop that is part of the outer wall of the active site.
Binding Sites↗
Synaptogenesis and dendritic growth in the cortex opposite unilateral sensorimotor cortex damage in adult rats: a quantitative electron microscopic examination.
Unilateral lesions of the forelimb area of the sensorimotor cortex in adult rats resulted in time-dependent increases in the number of synapses per neuron and the volume and membrane surface area of dendritic processes per neuron within layer V of the contralateral motor cortex in comparison to sham-operated rats. Based on previous findings of a behavioral relationship with increased dendritic arborization, these changes may be related to lesion-induced compensatory changes in the use of the non-impaired (ipsilateral to the lesion) forelimb.
Age Factors↗
Storing diffraction data.
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Crystallography, X-Ray↗
Ultrastructural evidence for increased contact between astrocytes and synapses in rats reared in a complex environment.
Rats raised from weaning in a complex environment have an increased number of synapses per neuron in the visual cortex in comparison to animals housed in standard laboratory cages. Previous research has suggested that experience-dependent synaptic changes may be coordinated with changes in astrocytes. The present study used electron microscopy to examine astrocytic processes in the visual cortex of rats raised in a complex environment (EC) or in standard laboratory cages, either individually (IC) or in pairs (social condition, SC). Measurements of the surface density of astrocytic membrane in direct apposition to synaptic elements revealed that astrocytic processes have increased contact with synaptic elements within the visual cortex of EC rats in comparison to SC and IC animals. In contrast, other astrocytic size variables revealed no significant change in astrocytic processes per unit volume of tissue. Previous work has indicated no significant differences in synaptic density in these subjects. The specific increase in the contact between astrocytes and synapses suggests an experience-related enhancement of the astrocytic involvement in synaptic activity.
Animals↗
Effect of temperature on pressure-volume hysteresis of excised lungs.
The objective of this study was to determine if the effect of temperature on excised lung pressure-volume (P-V) hysteresis during various P-V maneuvers would be consistent with predicted effects based on the recruitment-derecruitment (R-D) model of lung P-V hysteresis. Three sets of P-V curves were recorded for excised rat lungs at (1) 24 degrees C, (2) either 42 degrees or 45 degrees C, and (3) 24 degrees C. After full inflation of the lung, deflation-inflation (D-I) cycles were performed between total lung capacity (30 cmH2O) and successively decreasing end-expiratory pressures (EEPs). Normalized hysteresis (K) was plotted vs EEP. K remained relatively constant at EEPs > or = +5 cmH2O at 24 degrees C and 42 degrees C and > +5 cmH2O at 45 degrees C. Large increases in K occurred as the EEP was further reduced, with the relationship of K vs EEP being shifted to the right at 42 degrees C and 45 degrees C relative to 24 degrees C, with the greater shift occurring at 45 degrees C. Previous work has shown that the R-D of lung units contributes to P-V hysteresis and is EEP-dependent, increasing at EEPs < or = +4 +/- 1 cmH2O at room temperature (Cheng et al., 1995). This study suggests that at increased temperatures, R-D of lung units is initiated at higher EEPs and is more extensive than at room temperature.
Animals↗
Rapid laminar-dependent changes in GFAP immunoreactive astrocytes in the visual cortex of rats reared in a complex environment.
Neuronal changes in the visual cortex have previously been found to occur within days of housing weanling rats in a complex environment (EC) compared to rats housed in standard laboratory cages (IC). In contrast, layer IV astrocytes immunostained for glial fibrillary acidic protein (GFAP) have been found to be slow to change. Recent quantitative analysis has shown the surface density of GFAP immunoreactive (GFAP-IR) astrocytes in young rats to be significantly lower in layer IV in comparison to layer II/III. In the present study, the analysis of experience effects on GFAP-IR astrocytes was extended to include layer II/III as well as layer IV of EC and IC rats. The surface density of GFAP-IR processes was found to be significantly increased within layer II/III after 4-10 days of EC rearing in comparison to IC rats. Consistent with previous findings, housing condition did not significantly affect GFAP-IR within layer IV during these early time points. It is possible that GFAP immunocytochemistry is not a sensitive means of detecting experience-induced early changes in astrocytes within layer IV of weanling rats. The rapid astrocytic changes detected in layer II/III are suggestive of a close relationship between astrocytic plasticity and experience-induced synaptic plasticity.
Animals↗
SR8--the establishment and characterisation of a new ovarian carcinoma cell line and xenograft model.
A new cell line, SR8, and xenograft model of ovarian carcinoma has been established in this laboratory over the past 20 months from a patient with advanced ovarian cancer. Electron microscopic examination of SR8 cells demonstrated the presence of desmosomes and tonofilaments; SR8 cells expressed epithelial membrane antigen (EMA) and glandular associated cytokeratin, all of these confirmed the epithelial origin of this cell line. In addition, SR8 cells expressed CA125, as did the original ovarian tumour. EGF-R and TP53 expression was identified by immunocytochemistry (ICC) in this line. Nearly all the SR8 cells (93%) expressed HLA-class I antigen while 13.5% expressed HLA-DR. SR8 cells showed near-diploid and -triploid chromosome populations with several clonal and non-clonal rearrangements. Subcutaneous and intraperitoneal xenografting of SR8 cells resulted in invasive tumour production at both sites in 3/4 and 4/4 female nude mice, respectively. These xenografts exhibited similar morphology as that of original tumour and were found to express EMA, cytokeratin, CA125 and TP53. The potential research applications of this cell line are discussed.
Animals↗
Rats perform better on spatial than brightness delayed matching-to-sample water-escape due to an unlearned bias to use spatial cues.
Rats readily acquire water-escape spatial delayed matching-to-sample (DMTS) tasks and show excellent performance with retention intervals as long as 120 m (17). They also acquire the task more readily with a 5-min retention interval (RI) than with a 1-min RI (16). To determine if these observations are unique to spatial DMTS, or are also true of nonspatial water-escape DMTS, 75-day-old rats were compared on acquisition and subsequent retention of spatial and brightness DMTS. A larger proportion of the rats tested on the spatial problem were able to acquire the task, made fewer acquisition errors, and demonstrated better retention when tested at RIs of 1, 5, 15, 30, 60, and 120 min than did the rats tested on the brightness problem. Acquisition RI did not affect the rate of acquisition on either task. Examination of perserveration errors, the occurrence of intrusions, and position-congruent performance (escape platform in the same physical location on both runs of a trial) revealed that the choices of brightness-trained rats were often more influenced by spatial than brightness cues, suggesting that rats have an unlearned bias to use spatial cues in water-escape DMTS tasks.
Animals↗
The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169.
Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The beta-1,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behavior of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 A resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have a second role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.
Binding Sites↗
Assignment of the human and mouse LIM-kinase genes (LIMK1; Limk1) to chromosome bands 7q11.23 and 5G1, respectively, by in situ hybridization.
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Animals↗
Crystallographic and molecular-modeling studies of lipase B from Candida antarctica reveal a stereospecificity pocket for secondary alcohols.
Many lipases are potent catalysts of stereoselective reactions and are therefore of interest for use in chemical synthesis. The crystal structures of lipases show a large variation in the shapes of their active site environments that may explain the large variation in substrate specificity of these enzymes. We have determined the three-dimensional structure of Candida antarctica lipase B (CALB) cocrystallized with the detergent Tween 80. In another crystal form, the structure of the enzyme in complex with a covalently bound phosphonate inhibitor has been determined. In both structures, the active site is exposed to the external solvent. The potential lid-forming helix alpha 5 in CALB is well-ordered in the Tween 80 structure and disordered in the inhibitor complex. The tetrahedral intermediates of two chiral substrates have been modeled on the basis of available structural and biochemical information. The results of this study provide a structural explanation for the high stereoselectivity of CALB toward many secondary alcohols.
Alcohols↗
Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule.
FAT, a new member of the human cadherin super-family, has been isolated from the T-leukemia cell line J6. The predicted protein closely resembles the Drosophila tumor suppressor fat, which is essential for controlling cell proliferation during Drosophila development. The gene has the potential to encode a large transmembrane protein of nearly 4600 residues with 34 tandem cadherin repeats, five EGF-like repeats, and a laminin A-G domain. The cytoplasmic sequence contains two domains with distant homology to the cadherin catenin-binding region. Northern blotting analysis of J6 mRNA demonstrated full-length, approximately 15-kb, FAT message in addition to several 5'-truncated transcripts. In addition to its presence in J6 cells, in situ hybridization revealed FAT mRNA expression in epithelia and in some mesenchymal compartments. Furthermore, higher levels of expression were observed in fetal, as opposed to adult, tissue, suggesting that its expression may be developmentally regulated in these tissues. FAT shows homologies with a number of proteins important in developmental decisions and cell:cell communication and is the first fat-like protein reported in vertebrates. The gene encoding FAT was located by in situ hybridization on chromosome 4q34-q35. We propose that this family of molecules is likely to be important in mammalian developmental processes and cell communication.
Amino Acid Sequence↗
Structural analysis of human alpha-class glutathione transferase A1-1 in the apo-form and in complexes with ethacrynic acid and its glutathione conjugate.
BACKGROUND: Glutathione transferases (GSTs) constitute a family of isoenzymes that catalyze the conjugation of the tripeptide glutathione with a wide variety of hydrophobic compounds bearing an electrophilic functional group. Recently, a number of X-ray structures have been reported which have defined both the glutathione- and the substrate-binding sites in these enzymes. The structure of the glutathione-free enzyme from a mammalian source has not, however, been reported previously. RESULTS: We have solved structures of a human alpha-class GST, isoenzyme A1-1, both in the unliganded form and in complexes with the inhibitor ethacrynic acid and its glutathione conjugate. These structures have been refined to resolutions of 2.5 A, 2.7 A and 2.0 A respectively. Both forms of the inhibitor are clearly present in the associated electron density. CONCLUSIONS: The major differences among the three structures reported here involve the C-terminal alpha-helix, which is a characteristic of the alpha-class enzyme. This helix forms a lid over the active site when the hydrophobic substrate binding site (H-site) is occupied but it is otherwise disordered. Ethacrynic acid appears to bind in a non-productive mode in the absence of the coenzyme glutathione.
Apoenzymes↗
Where freedom is given, liberties are taken.
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Crystallography, X-Ray↗
Functional significance of arginine 15 in the active site of human class alpha glutathione transferase A1-1.
Arg15 is a conserved active-site residue in class Alpha glutathione transferases. X-ray diffraction studies of human glutathione transferase A1-1 have shown that N epsilon of this amino acid residue is adjacent to the sulfur atom of a glutathione derivative bound to the active site, suggesting the presence of a hydrogen bond. The phenolic hydroxyl group of Tyr9 also forms a hydrogen bond to the sulfur atom of glutathione, and removal of this hydroxyl group causes partial inactivation of the enzyme. The present study demonstrates by use of site-directed mutagenesis the functional significance of Arg15 for catalysis. Mutation of Arg15 into Leu reduced the catalytic activity by 25-fold, whereas substitution by Lys caused only a threefold decrease, indicating the significance of a positively charged residue at position 15. Mutation of Arg15 into Ala or His caused a substantial reduction of the specific activity (200 or 400-fold, respectively), one order of magnitude more pronounced than the effect of the Tyr9-->Phe mutation. Double mutations involving residues 9 and 15 demonstrated that the effects of mutations at the two positions were additive except for the substitution of His for Arg15, which appeared to cause secondary structural effects. The pKa value of the phenolic hydroxyl of Tyr9 was determined by UV absorption difference spectroscopy and was found to be 8.1 in the wild-type enzyme. The corresponding pKa values of mutants R15K, R15H and R15L were 8.5, 8.7 and 8.8, respectively, demonstrating the contribution of the guanidinium group of Arg15 to the electrostatic field in the active site. Addition of glutathione caused an increased pKa value of Tyr9; this effect was not obtained with S-methylglutathione. These results show that Tyr9 is protonated when glutathione is bound to the enzyme at physiological pH values. The involvement of an Arg residue in the binding and activation of glutathione is a feature that distinguishes class Alpha glutathione transferases from members in other glutathione transferase classes.
Arginine↗
Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG.
BACKGROUND: Streptococcal protein G comprises two or three domains that bind to the constant Fc region of most mammalian immunoglobulin Gs (IgGs). Protein G is functionally related to staphylococcal protein A, with which it shares neither sequence nor structural homology. RESULTS: To understand the competitive binding of these two proteins to the Fc region, the crystal structure of a single Ig-binding domain of streptococcal protein G was determined at 3.5 A resolution in complex with the Fc fragment of human IgG and compared with the structures of protein A:Fc and protein G:Fab complexes. Protein G binds to the interface between the second and third heavy chain constant domains of Fc, which is roughly the same binding site used by protein A. Protein G comprises one alpha-helix packed onto a four-stranded beta-sheet. Residues from protein G that are involved in binding are situated within the C-terminal part of the alpha-helix, the N-terminal part of the third beta-strand and the loop region connecting these two structural elements. The identified Fc-binding region of protein G agrees well with both biochemical and NMR spectroscopic data. However, the Fc-binding helices of protein G and protein A are not superimposable. CONCLUSIONS: Protein G and protein A have developed different strategies for binding to Fc. The protein G:Fc complex involves mainly charged and polar contacts, whereas protein A and Fc are held together through non-specific hydrophobic interactions and a few polar interactions. Several residues of Fc are involved in both the protein G:Fc and the protein A:Fc interaction, which explains the competitive binding of the two proteins. The apparent differences in their Fc-binding activities result from additional unique interactions.
Bacterial Proteins↗
Neural tuning characteristics of auditory primary afferents in the chicken embryo.
Primary afferent activity was recorded from the cochlear ganglion in chicken embryos (Gallus domesticus) at 19 days of incubation (E19). The ganglion was accessed via the recessus scala tympani and impaled with glass micropipettes. Frequency tuning curves were obtained using a computerized threshold tracking procedure. Tuning curves were evaluated to determine characteristics frequencies (CFs), CF thresholds, slopes of low and high frequency flanks, and tip sharpness (Q10dB). The majority of tuning curves exhibited the typical 'V' shape described for older birds and, on average, appeared relatively mature based on mean values for CF thresholds (59.6 +/- 20.3 dBSPL) and tip sharpness (Q10dB = 5.2 +/- 3). The mean slopes of low (61.9 +/- 37 dB/octave) and high (64.6 +/- 33 dB/octave) frequency flanks although comparable were somewhat less than those reported for 21-day-old chickens. Approximately 14% of the tuning curves displayed an unusual 'saw-tooth' pattern. CFs ranged from 188 to 1623 Hz. The highest CF was well below those reported for post-hatch birds. In addition, a broader range of Q10dB values (1.2 to 16.9) may related to a greater variability in embryonic tuning curves. Overall, these data suggest that an impressive functional maturity exists in the embryo at E19. The most significant sign of immaturity was the limited expression of high frequencies. It is argued that the limited high CF in part may be due to the developing middle ear transfer function and/or to a functionally immature cochlear base.
Action Potentials↗