Biomedical subjects

J J Liang

Publications and source records attributed to J J Liang.

At least 19 recordsLinked to original sources

PubMed2001

Spectroscopic analysis of lens recombinant betaB2- and gammaC-crystallin.

PURPOSE: To compare the spectroscopic and unfolding properties of human lens [beta]B2- and [gamma]C-crystallin with those of [alpha]A-crystallin. METHODS: Human lens [beta]B2- and [gamma]C-crystallin were cloned and measured spectroscopically. The unfolding curves in response to guanidine HCl (GdnHCl) and heat were also obtained by measuring Trp fluorescence emission intensity or emission maximum wavelength with increasing perturbation. RESULTS: Very similar spectroscopic and unfolding properties were seen with [beta]B2- and [gamma]C-crystallin, but both demonstrated great differences compared with [alpha]A-crystallin. Unlike [alpha]A-crystallin, [beta]B2- and [gamma]C-crystallin showed very little binding to Bis-ANS (4,4'-dianilino-1,1'-binaphthalene-5,5'-disulfonic acid), a hydrophobic fluorescence probe. Both [beta]B2- and [gamma]C-crystallin were more resistant than [alpha]A-crystallin to GdnHCl-induced unfolding, but [alpha]A-crystallin was more resistant than [beta]B2- and [gamma]C-crystallin to heat induced unfolding. CONCLUSIONS: It was observed that [beta]B2- and [gamma]C-crystallin showed more similar spectroscopic and unfolding properties with each other than each of them showed with [alpha]A-crystallin.

Circular Dichroism↗

PubMed2001

Oxidation of human lens recombinant alphaA-crystallin and cysteine-deficient mutants.

Disulfide cross-linking, one of the results of oxidative stress, has been thought to play an important role in cataractogenesis. High molecular mass (HMM) protein aggregation also contributes to cataract development, and a prevailing speculation is that disulfide cross-linking induces HMM aggregation. However, there is no direct evidence to support this speculation. Dimerization is an effect of disulfide cross-linking but cannot explain the size of HMM aggregates observed in the lens. alphaA-crystallin has two cysteine residues (Cys131 and Cys142) and we have prepared three Cys-deficient mutants, two single mutants (C131I and C142I) and one double mutant (C131I/C142I). They were subjected to H202 oxidation in an ascorbate-FeCl(3)-EDTA-H202 system. The effects of oxidation on the mutants, including changes in aggregate size and conformation, were compared with those of the wild-type alphaA-crystallin by FPLC gel filtration, absorption, fluorescence, and circular dichroism measurements. The results indicated that other amino acid residues besides Cys, such as Trp and Tyr, were also oxidized by H202. Disulfide dimerization alone seems to play a less important role in HMM aggregation than does the secondary conformational change resulting from the combined effect of the oxidation of Trp and Tyr as well as Cys.

Amino Acid Substitution↗

PubMed2001

Conformational study of N(epsilon)-(carboxymethyl)lysine adducts of recombinant gammaC-crystallin.

N(epsilon)-(carboxymethyl)lysine, an advanced glycation end product, is present in the human lens. The effects of CML formation on protein conformation and stability were studied using the recombinant gammaC-crystallin as a model. Conformational change was studied by spectroscopic measurements such as fluorescence and circular dichroism. Conformational stability was determined by unfolding with heat. The results indicated that no conformational change was observed due to CML formation, but conformational stability decreased. These observations can be explained in terms of the relatively stable structure of gamma-crystallin, especially when compared with other crystallins. The lens nucleus is rich in gamma-crystallin and its stable conformation can assist gamma-crystallin sustained insults and remain soluble.

Circular Dichroism↗

PubMed2001

Examining multicultural counseling competence and race-related attitudes among white marital and family therapists.

This study investigates the relative contributions of social desirability attitudes, previous number of multicultural counseling courses taken, and racism and White racial identity attitudes together in predicting marital and family therapists' self-reported multicultural counseling competence. Results revealed that, when controlling for social desirability attitudes and the number of multicultural courses taken, racism and White racial identity attitudes in consort accounted for a significant amount of the variance in self-perceived multicultural counseling competence. Implications for marital and family therapy training, practice, and research are discussed.

Attitude of Health Personnel↗

PubMed2000

Interaction between beta-amyloid and lens alphaB-crystallin.

In Alzheimer's disease, beta-amyloid peptides (betaA(1-40) and betaA(1-42)) are deposited on the brain cell surfaces as neurotoxic plaques. Some reports indicate that small heat shock proteins, Hsp27 and alphaB-crystallin, colocalize in the plaques, but their functions are not known. Interaction between betaA and alphaB-crystallin must be determined in order to understand the role of alphaB-crystallin in betaA fibril formation. We used a pyrene (Pyr)-labeled betaA(1-40) in a fluorescence energy transfer experiment. Upon incubation together at 37 degrees C, energy transfer between Trp of alphaB-crystallin and Pyr of Pyr-labeled betaA was observed, indicating that betaA participated in subunit exchange of alphaB-crystallin, which promoted fibril formation.

Alzheimer Disease↗

PubMed2000

Human lens high-molecular-weight alpha-crystallin aggregates.

Alpha-crystallin high-molecular-weight (HMW) aggregates can be formed in vitro by many mechanisms, but the mechanism of in vivo aggregation has not been clearly established. HMW and LMW (low-molecular-weight) alpha-crystallins were isolated from human lenses 50-60 years of age and some spectroscopic measurements were performed. Conformational differences were suggested based on data of increased bis-ANS (4,4'-dianilino-1,1'-binaphthalene-5, 5'-disulfonic acid) and ThT (thioflavin T) fluorescence as well as increased far-UV and decreased near-UV circular dichroism (CD). These results indicated that HMW alpha-crystallin was more hydrophobic than LMW alpha-crystallin, possibly resulting from partial unfolding of alpha-crystallin. On the other hand, the increased ThT fluorescence and far-UV CD intensities indicate that an increased amount of beta-sheet conformation was involved in aggregation. These data, along with little difference in chaperone-like activity between the LMW and HMW alpha-crystallins, strongly suggest that HMW alpha-crystallin aggregates resulted from partial unfolding and disassembling-reassembling of LMW alpha-crystallin caused by posttranslational modification rather than chaperone complex formation.

Chaperonins↗

PubMed2000

RGS7 complex formation and colocalization with the Gbeta5 subunit in the adult rat brain and influence on Gbeta5gamma2-mediated PLCbeta signaling.

This study describes the colocalized distribution and dimeric complex formation between RGS7, a GTPase-activating protein for several heterotrimeric Galpha protein families, and the Gbeta5 subunit in the adult rat brain. Confocal dual immunofluorescence labeling studies indicated a broad regional specificity in the cellular coexpression between RGS7 and Gbeta5 within the cerebral cortical layers I and V-VI, hippocampal formation, caudate-putamen, medial habenula, most thalamic nuclei, and cerebellar molecular and granular layers. In all instances, Gbeta1-beta4 immunoreactivities exhibited no observable colocalization with RGS7, despite their widespread codistribution throughout similar neuronal networks. Coimmunoprecipitation studies confirmed the selective protein-protein interaction between RGS7 and Gbeta5 within brain regions that displayed immunohistochemical colocalization. The influence of RGS7 to modulate Gbeta5gamma2-mediated phosphatidyl inositol (PI) production was examined in COS-7-cotransfected cells. In the presence of Gbeta5gamma2 only, intracellular PI accumulation was increased by 25% above basal levels; addition of RGS7 produced no significant alteration in Gbeta5gamma2-mediated PI accumulation. A similar trend was exhibited when full-length RGS7 was substituted with an RGS7 construct lacking the Gbeta5-interacting region (G protein gamma-like domain; GGL domain) or with RGS4. In conclusion, RGS7/Gbeta5 dimers occurred within most brain regions in which both proteins were cellularly coexpressed. However, an influence of RGS7 on Gbeta5gamma2-mediated PLCbeta signaling activity was not apparent, athough this was in COS-7 cell transfection studies.

Animals↗

PubMed2000

Heat-induced conformational change of human lens recombinant alphaA- and alphaB-crystallins.

PURPOSE: To determine which component of lens alpha-crystallin is responsible for heat-induced transition, conformational change and high molecular weight (HMW) aggregation. METHODS: Recombinant alphaA- and alphaB-crystallins were used. Temperature dependent changes were probed by Trp fluorescence and circular dichroism (CD) measurements. HMW aggregates were induced by heating at 62 degrees C for 1-2 h and then cooling to room temperature. The nature of HMW aggregation was studied with fluorescent probes, 4,4'-dianilino-1, 1'-binaphthalene-5,5'-disulfonic acid (bis-ANS) and thioflavin T (ThT). RESULTS: CD and Trp fluorescence revealed that alphaB-crystallin was more susceptible than alphaA-crystallin to heat-induced conformational change and aggregation. At temperatures greater than 70 degrees C, alphaB-crystallin precipitated but alphaA-crystallin remained soluble. Both bis-ANS and ThT probes displayed increased fluorescence intensity with HMW aggregation, but the increase for bis-ANS was greater with alphaB-crystallin than with alphaA-crystallin, while the reverse was true for ThT. CONCLUSIONS: These results indicate that alphaB-crystallin is more susceptible than alphaA-crystallin to heat-induced conformational change and aggregation and are consistent with the notion that alphaA- and alphaB-crystallins have different biochemical and biophysical properties in spite of their high degree of homology.

Anilino Naphthalenesulfonates↗

PubMed1999

Thermodynamic stability of human lens recombinant alphaA- and alphaB-crystallins.

Lens alpha-crystallin is a 600-800-kDa heterogeneous oligomer protein consisting of two subunits, alphaA and alphaB. The homogeneous oligomers (alphaA- and alphaB-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both alphaA- and alphaB-crystallins. In terms of standard free energy (DeltaG(NU)(H(2)(O))), alphaA-crystallin is slightly more stable than alphaB-crystallin. The significance of the intermediates may be related to the functioning of alpha-crystallins as chaperone-like molecules.

Anilino Naphthalenesulfonates↗

PubMed1999

Site-directed mutations within the core "alpha-crystallin" domain of the small heat-shock protein, human alphaB-crystallin, decrease molecular chaperone functions.

Site-directed mutagenesis was used to evaluate the effects on structure and function of selected substitutions within and N-terminal to the core "alpha-crystallin" domain of the small heat-shock protein (sHsp) and molecular chaperone, human alphaB-crystallin. Five alphaB-crystallin mutants containing single amino acid substitutions within the core alpha-crystallin domain displayed a modest decrease in chaperone activity in aggregation assays in vitro and in protecting cell viability of E. coli at 50 degrees C in vivo. In contrast, seven alphaB-crystallin mutants containing substitutions N-terminal to the core alpha-crystallin domain generally resembled wild-type alphaB-crystallin in chaperone activity in vitro and in vivo. Size-exclusion chromatography, ultraviolet circular dichroism spectroscopy and limited proteolysis were used to evaluate potential structural changes in the 12 alphaB-crystallin mutants. The secondary, tertiary and quaternary structures of mutants within and N-terminal to the core alpha-crystallin domain were similar to wild-type alphaB-crystallin. SDS-PAGE patterns of chymotryptic digestion were also similar in the mutant and wild-type proteins, indicating that the mutations did not introduce structural modifications that altered the exposure of proteolytic cleavage sites in alphaB-crystallin. On the basis of the similarities between the sequences of human alphaB-crystallin and the sHsp Mj HSP16.5, the only sHsp for which there exists high resolution structural information, a three-dimensional model for alphaB-crystallin was constructed. The mutations at sites within the core alpha-crystallin domain of alphaB-crystallin identify regions that may be important for the molecular chaperone functions of sHsps.

Amino Acid Sequence↗

PubMed1999

Expression of dopamine receptors in the subthalamic nucleus of the rat: characterization using reverse transcriptase-polymerase chain reaction and autoradiography.

We analysed the expression of dopamine receptor subtypes in the subthalamic nucleus by means of reverse transcriptase-polymerase chain reaction. We also studied, using autoradiography, all pharmacologically characterized dopamine receptors in four subregions of the subthalamic nucleus. For comparison, dopamine receptor subtypes were also evaluated in brain regions where they are more abundant and well characterized. The radioligands used were: [3H]SCH-23390, [3H]emonapride and [3H]2-dipropylamino-7-hydroxy-1,2,3,4-tetrahydronaphthalene for dopamine D1, D2 and D3 receptors, respectively; and [3H]YM-09151-2 in the presence of raclopride for dopamine D4 receptors. Finally, we also evaluated the effect of unilateral 6-hydroxydopamine injection into the medial forebrain bundle on dopamine receptor levels expressed in the ipsilateral subthalamic nucleus. The lesion was estimated by decrease in the binding of [3H]WIN-35428, a specific dopamine transporter label. D1, D2 and D3 receptor messenger RNAs and binding sites were present in the subthalamic nucleus, but no messenger RNA for D4 receptors was found, although specific binding sites for these receptors were observed. As compared to the intact side, the 6-hydroxydopamine lesion did not change D1 receptors, increased D2 receptors, and decreased D3 receptors and the dopamine transporter. The results suggest that postsynaptic D1, D2 or D3 receptors can mediate the effect of dopamine on subthalamic nucleus neuronal activity. D4 receptors would mediate exclusively presynaptic effects. These results reinforce the idea that dopamine receptors in the subthalamic nucleus may play an important role in the physiology of the basal ganglia and in the pathophysiology of Parkinson's disease.

Animals↗

PubMed1999

Immunohistochemical distribution of RGS7 protein and cellular selectivity in colocalizing with Galphaq proteins in the adult rat brain.

Regulators of G protein signaling (RGS) proteins serve as potent GTPase-activating proteins for the heterotrimeric G proteins alphai/o and aq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Galphaq/11, an important G protein-coupled receptor signal transducer for phospholipase Cbeta-mediated activity. In general, both RGS7 and Galphaq/11 displayed a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity was observed in cortical layers I-VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity was concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1-CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also displayed dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein-specific antibodies indicated a cellular selectivity in the colocalization between RGS7 and Galphaq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Galphaq/11-mediated signaling pathways to modulate dynamically RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13-dibutyrate, a selective protein kinase C activator. A time-dependent increase in levels of mRNA for RGS7, but not RGS4, was observed. Our results provide novel information on the region- and cell-specific pattern of distribution of RGS7 with the transmembrane signal transducer, Galphaq/11. We also describe a possible RGS7-selective neuronal feedback adaptation on Galphaq/11-mediated pathway function, which may play an important role in signaling specificity in the brain.

Age Factors↗

PubMed1999

Conformational study of N(epsilon)-(carboxymethyl)lysine adducts of recombinant alpha-crystallins.

PURPOSE: Lens proteins underwent nonenzymatic glycation, and the advanced glycation end products (AGEs) were detected by immunological assays. One of the major AGE structures is N(epsilon)-(carboxymethyl)lysine (CML). Since the involvement of AGEs in the pathogenesis of diabetic complications is speculated, the effects of CML formation on proteins were studied. METHODS: CML adducts were generated in recombinant alphaA- and alphaB-crystallins by incubation with glyoxylic acid and NaBH3CN. SDS-PAGE and size exclusion chromatography were used to detect subunit degradation and high-molecular-weight (HMW) aggregation. Conformational change was determined by fluorescence and circular dichroism (CD) measurements. The chaperone function was studied by DTT-induced aggregation of insulin. RESULTS: Lysine modification was estimated to be 60-90% depending on the conditions of incubation. No subunit degradation or HMW aggregation was observed. Fluorescence and CD measurements detected a conformational change in CML adducts. Measurements of chaperone-like activity, however, indicated that the formation of CML increased the protein's ability to protect insulin against DTT-induced aggregation. CONCLUSIONS: Although CML adducts of alphaA- and alphaB-crystallins, the major AGE structures formed in vitro, changed protein conformation, no subunit degradation and HMW aggregation were observed. Moreover, the CML adducts increased chaperone-like activity of both alphaA- and alphaB-crystallins. The results suggest that CML formation alone may not play a major role in protein aggregation and lens opacity.

Chromatography, Gel↗

PubMed1999

Spectral contribution of the individual tryptophan of alphaB-crystallin: a study by site-directed mutagenesis.

There are two tryptophan residues in the lens alphaB-crystallin, Trp9 and Trp60. We prepared two Trp --> Phe substituted mutants, W9F and W60F, for use in a spectroscopic study. The two tryptophan residues contribute to Trp fluorescence and near-ultraviolet circular dichroism (UV CD) differently. The major difference in the near-UV CD is the contribution of 1La of Trp: it is positive in W60F but becomes negative in W9F. Further analysis of the near-UV CD shows an increased intensity in the region of 270-280 nm for W60F, suggesting that the Tyr48 is affected by the W60F mutation. It appears that Trp60 is located in a more rigid environment than Trp9, which agrees with a recent structural model in which Trp60 is in a beta-strand.

Amino Acid Substitution↗

PubMed1999

Photooxidized products of recombinant alpha A-crystallin and W9F mutant.

Human lenses contain many photosensitizers that absorb light at wavelengths above 300 nm, most notably UVA light (320-400 nm). Kynurenine (Kyn) and 3-hydroxykynurenine (HK), two of the best-known photosensitizers in the human lens, may play a significant role in photooxidation-related changes in lens proteins, such as conformational change and aggregation. In vitro irradiation experiments with proteins indicate that the Trp residue (with maximal absorption at 295 nm) is more susceptible to photooxidation by UVB light (280-320 nm) than by UVA light, but most UVB light below 300 nm is screened by the cornea and little reaches the lens, especially the nuclear region where nuclear color develops. Therefore, if photooxidation is an important contributor to nuclear color or nuclear cataract, it must arise from a photosensitized reaction. In the present study, we use recombinant alpha A- and its Trp-deficient mutant W9F as models to study the effects of UVA irradiation in the presence of HK or Kyn and of UVB (300 nm) irradiation on alpha-crystallins. alpha A-crystallin showed a large decrease in Trp fluorescence and a large increase in non-Trp (blue) fluorescence after the HK-sensitized or 300 nm photooxidation. For the W9F mutant, a smaller decrease in protein fluorescence (lambda ex at 280 nm) and a smaller increase in blue fluorescence than for the wild-type alpha A-crystallin were observed. A decrease in the near-UV CD was also observed for both photooxidized alpha A and the W9F mutant. The effect of Kyn sensitization is smaller than that of HK sensitization. A study of chaperone-like activity indicated that only 300 nm photooxidized alpha A and the W9F mutant increased the ability to protect insulin from dithiothreitol-induced aggregation. Thus, sensitized photooxidation can occur in amino acids other than Trp by UVA in the presence of HK or Kyn with effects similar to, albeit smaller than, those of direct UVB (300 nm) photooxidation.

Cataract↗

PubMed1998

Subunit exchange of lens alpha-crystallin: a fluorescence energy transfer study with the fluorescent labeled alphaA-crystallin mutant W9F as a probe.

A Trp-free alphaA-crystallin mutant (W9F) was prepared by site-directed mutation. This mutant appears to be identical to the wild-type in terms of conformation (secondary and tertiary structures). W9F was labeled with a sulfhydryl-specific fluorescent probe, 2-(4'-maleimidylanilino) naphthalene-6-sulfonate (MIANS), and used in a subunit exchange between alphaA- and alphaA-crystallins as well as between alphaA- and alphaB-crystallins, studied by measurement of fluorescence resonance energy transfer. Energy transfer was observed between Trp (donor, with emission maximum at 336 nm) of wild-type alphaA- or alphaB-crystallin and MIANS (acceptor, with absorption maximum at 313 nm) of labeled W9F when subunit exchange occurred. Time-dependent decrease of Trp and increase of MIANS fluorescence were recorded. The exchange was faster at 37 degrees C than at 25 degrees C. The energy transfer efficiency was greater between homogeneous subunits (alphaA-alphaA) than between heterogeneous subunits (alphaA-alphaB). A previous exchange study with isoelectric focusing indicated a complete but slow exchange between alphaA and alphaB subunits. The present study showed that the exchange was a fast process, and the different energy transfer efficiencies between alphaA-alphaA and alphaA-alphaB indicated that alphaA- and alphaB-crystallins were not necessarily structurally equivalent.

Anilino Naphthalenesulfonates↗

PubMed1998

Intermolecular interaction of lens crystallins: from rotationally mobile to immobile states at high protein concentrations.

The conformation of lens crystallins in vivo or in a highly concentrated solution is not well established. Most studies were carried out in dilute solutions in which protein-protein interaction is minimal. In order to see whether there is conformational change (tertiary and secondary structures) when crystallin solutions are brought to high concentrations, we have performed the following molecular spectroscopic measurements: circular dichroism (CD) and Fourier transform infrared (FTIR). Near-UV CD measurements showed a more than two-fold increase in CD intensity (molar ellipticity) for the total water-soluble (WS) protein from young calf lens nucleus in a highly concentrated solution (> 300 mg/ml in a 0.01-mm cell), when compared with a dilute solution (1000-fold dilution in a 10-mm cell). The individual crystallins in concentrated solutions also showed an increase in CD intensity, but of different magnitude: alpha-crystallin > beta-crystallin > gamma-crystallin. The increased CD indicates that lens crystallins are in a more compact structure in highly concentrated solutions; they likely undergo a transition from a mobile to an immobile state. Change in near-UV CD usually is caused by restricted mobility of aromatic side groups, particularly Trp. The transition involves not only a change in protein tertiary and/or quaternary structure, but also in protein backbone structure. The change of protein backbone structure was drawn from FTIR measurements. FTIR spectra, sensitive to the secondary structure in the amide I region, could be measured for a highly concentrated solution for which far-UV CD measurement is not feasible. The secondary structure that showed prominent change for alpha-crystallin in a highly concentrated solution was beta-conformation: increase in beta-turn with a concomitant decrease of alpha-helix structure.

Animals↗

PubMed1998

Intermolecular exchange and stabilization of recombinant human alphaA- and alphaB-crystallin.

Lens alpha-crystallin subunits alphaA and alphaB are differentially expressed and have a 3-to-1 ratio in most mammalian lenses by intermolecular exchange. The biological significance of this composition and the mechanism of exchange are not clear. Preparations of human recombinant alphaA- and alphaB-crystallins provide a good system in which to study this phenomenon. Both recombinant alphaA- and alphaB-crystallins are folded and aggregated to the size of the native alpha-crystallin. During incubation together, they undergo an intermolecular exchange as shown by native isoelectric focusing. Circular dichroism measurements indicate that the protein with a 3-to-1 ratio of alphaA- and alphaB-crystallins has the same secondary structure but somewhat different tertiary structures after exchange: the near-UV CD increases after exchange. The resulting hybrid aggregate is more stable than the individual homogeneous aggregates: at 62 degrees C, alphaB-crystallin is more susceptible to aggregation and displays a greater light scattering than alphaA-crystallin. This heat-induced aggregation of alphaB-crystallin, however, was suppressed by intermolecular exchange with alphaA-crystallin. These phenomena are also observed by fast performance liquid chromatography gel filtration patterns. The protein structure of alphaB-crystallin is stabilized by intermolecular exchange with alphaA-crystallin.

Chromatography, Gel↗