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J J Liang

Publications and source records attributed to J J Liang.

35 records · Page 2Linked to original sources
PubMed1998

Conformational change of human lens insoluble alpha-crystallin.

Human lens alpha-crystallin becomes progressively insoluble with age and is the major crystallin component in the water-insoluble (WI) fraction. The mechanism that causes the originally water-soluble (WS) alpha-crystallin to become insoluble is unknown. A conformational change by chemical modification may be the cause, but the nature of insolubility renders it impossible to study protein conformation in the WI fraction by most spectroscopic measurements. In the present study, alpha-crystallin in the WI fraction was extracted by urea and reconstituted to a folded protein by dialysis. The refolded urea-soluble (US) alpha-crystallin was compared with WS alpha-crystallin. The US alpha-crystallin has a greater amount of polymeric species, but fewer degraded subunits than the WS alpha-crystallin as shown by SDS-PAGE and Western blot. Circular dichroism (CD) measurements indicate that they have the same secondary structure but a different tertiary structure, possibly a partial unfolding in the US alpha-crystallin. This is supported by fluorescence measurements: Trp residues are more exposed and protein has a more-hydrophobic surface in the US than in the WS alpha-crystallin. Blue fluorescence further indicates that the US alpha-crystallin has a greater amount of pigment than the WS alpha-crystallin. Together, these results indicate that the US alpha-crystallin is a chemically and conformationally modified protein.

Aged↗
PubMed1998

Thermodynamic and kinetic characterization of calf lens gammaF-crystallin.

Gamma-crystallin is reported to be conformationally stable because of its internal structural symmetry, and gammaF (gammaIVa) is the most stable among the various gamma-crystallin gene products. However, there is no detailed report on its thermodynamic and kinetic stability. In the present study, detailed unfolding of gammaF-crystallin was investigated by equilibrium and kinetics methods with fluorescence and far-UV CD spectroscopic measurements. The GdnHCl-induced unfolding curves probed by Trp emission maximum and intensity showed a sharp single-step transition. Upon widening the unfolding transition with the use of urea in 1.5 M GdnHCl, a more proper fit for thermodynamic analysis was obtained. GammaF-Crystallin underwent a straightforward two-state process (N <==> U) without showing any measurable amount of intermediate. The conformational stability, as measured by deltaG(D)H2O (approximately 9 kcal/mol), indicates that gammaF-crystallin is a very stable protein. The high activation energy deltaG++H2O (approximately 24 kcal/mol), calculated from unfolding kinetics monitored by far-UV CD at 218 nm, also indicates that the native and unfolded states are separated by a high activation energy barrier.

Animals↗
PubMed1998

Immunohistochemical localization of G protein beta1, beta2, beta3, beta4, beta5, and gamma3 subunits in the adult rat brain.

The regional distributions of the G protein beta subunits (Gbeta1-beta5) and of the Ggamma3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gbeta and Ggamma3 subunits were widely distributed throughout the brain, with most regions containing several Gbeta subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gbeta immunostaining. Negative immunostaining was observed in cortical layer I for Gbeta1 and layer IV for Gbeta4. The hippocampal dentate granular and CA1-CA3 pyramidal cells displayed little or no positive immunostaining for Gbeta2 or Gbeta4. No anti-Gbeta4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gbeta1 was absent from the cerebellar molecular layer, and Gbeta2 was not detected in the Purkinje cells. No positive Ggama3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Ggamma3 antibody and individual anti-Gbeta1-beta5 antibodies displayed regional selectivity with Gbeta1 (cortical layers V-VI) and Gbeta2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gbeta1-beta5 with Ggamma3, specific dimeric associations in situ were observed within discrete brain regions.

Age Factors↗
PubMed1998

Fluorescence and immunochemical studies of advanced glycation-related lens pigments.

PURPOSE: To establish whether advanced glycation is the major mechanism for yellowing of lens proteins. METHODS: Synchronous fluorescence (SF) and immunochemical assays were used to study glycation in vitro and in vivo. In the in vitro study, advanced glycation end products (AGEs) were prepared and used as antigens to induce antibodies to AGEs. The in vitro AGEs and classified nuclear cataracts were analyzed by SF and immunochemical assays. RESULTS: In vitro AGEs generated from various glycating agents and carrier proteins displayed strong SF above 350 nm; the spectra were well resolved with major bands at 380 nm and 420 nm. Samples from human lenses manifested a band at 395 nm in addition to the two bands shown by in vitro AGEs. SF intensity is greater for the water-insoluble (WI) than water-soluble (WS) fraction, but both increased with increasing nuclear color. The immunoreactivity data also showed that the WI fraction contained more AGEs than the WS fraction and that the amount of AGEs increased with increasing nuclear color. CONCLUSIONS: Fluorescence and immunoassays indicated that pigmented AGEs contributed to yellowing of the crystalline lens nucleus.

Animals↗
PubMed1997

Detection and characterization of alpha-crystallin intermediate with maximal chaperone-like activity.

Lens alpha-crystallin has been reported to act like a chaperone molecule, with the chaperone-like activity enhanced by partial unfolding. The nature of the partial unfolding, however, is not fully understood. In this project, the unfolding and refolding process of alpha-crystallin was studied with guanidine hydrochloride (GdnHCl). Trp fluorescence (tertiary structure) and far-ultraviolet circular dichroism (UVCD) (secondary structure) demonstrated the presence of an intermediate in the unfolding pathway. ANS (1-anilino-8-naphthalenesulfonate) fluorescence clearly indicated a two-step transition in the unfolding-refolding process and showed that maximum hydrophobicity of the alpha-crystallin occurred at 0.8-1.0 M GdnHCl. This alpha-crystallin intermediate appears to be in a molten globule state; conformational study by near- and far-UVCD measurements indicated that alpha-crystallin intermediate exhibited tertiary structure which was significantly altered from that of the native protein, but had nearly the same secondary structure. Quaternary structure (size of aggregate) of the intermediate also remained unchanged from that of the native protein, as shown by FPLC size exclusion chromatography. The maximal hydrophobicity of the alpha-crystallin intermediate in the unfolding-refolding pathway was accompanied by maximal protection of betaH-crystallin from aggregation. However, an adverse effect of partial unfolding is that the alpha-crystallin intermediate aggregates at high concentrations. Together, these results clearly demonstrated the biological significance of the alpha-crystallin intermediate: it is a more effective chaperone than native alpha-crystallin.

Animals↗
PubMed1997

Conformational and functional differences between recombinant human lens alphaA- and alphaB-crystallin.

Human and other mammalian lens proteins are composed of three major crystallins: alpha-, beta-, and gamma-crystallin. alpha-Crystallin plays a prominent role in the supramolecular assembly required to maintain lens transparency. With age, the crystallins, especially alpha-crystallin, undergo posttranslational modifications that may disrupt the supramolecular assembly, and the lens becomes susceptible to other stresses resulting in cataract formation. Because these modifications occur even at a relatively young age, it is difficult to obtain pure, unmodified crystallins for in vitro experiments. alpha-Crystallin is composed of two subunits, alphaA and alphaB. Before the application of recombinant DNA technology, these two alpha-crystallin subunits were separated from calf lens in the denatured state and reconstituted by the removal of the denaturant, but they were not refolded properly. In the present studies, we applied the recombinant DNA technology to prepare native, unmodified alphaA- and alphaB-crystallins for conformational and functional studies. The expressed proteins from Escherichia coli are in the native state and can be studied directly. First, alphaA and alphaB cDNAs were isolated from a human lens epithelial cell cDNA library. The cDNAs were cloned into a pAED4 expression vector and then expressed in E. coli strain BL21(DE3). Pure recombinant alphaA- and alphaB-crystallins were obtained after purification by gel filtration and DEAE liquid chromatography. They were subjected to conformational studies involving various spectroscopic measurements and an assessment of chaperone-like activity. alphaA- and alphaB-crystallins have not only different secondary structure, but also tertiary structure. 1-Anilino-8-naphthalene sulfonate fluorescence indicates that alphaB-crystallin is more hydrophobic than alphaA-crystallin. The chaperone-like activity, as measured by the ability to protect insulin aggregation, is about 4 times greater for alphaB- than for alphaA-crystallin. The resulting data provide a base line for further studies of human lens alpha-crystallin.

Blotting, Western↗
PubMed1997

Cloning and in situ hybridization analysis of the expression of polysialyltransferase mRNA in the developing and adult rat brain.

Polysialyltransferase (PST) is an enzyme that catalyzes the addition of polysialic acid (PSA), a homopolymer of alpha-2,8-linked sialic acid residues, onto neural cell adhesion molecule (NCAM). The expression of PSA-NCAM in the brain is developmentally regulated and is of critical importance; however, the temporal and spatial developmental expression of brain PST, a potential key player in the control of PSA-NCAM levels, remains unclear. In the present study, we have cloned the coding region of rat PST cDNA by reverse transcription-polymerase chain reaction, using primers based on the hamster PST-1 cDNA sequence. A 39-mer oligonucleotide complementary to rat PST cDNA was synthesized to investigate the distribution of its mRNA in the developing and adult rat brain by Northern blot and in situ hybridization. In the embryonic rat brain, PST mRNA was detected abundantly throughout the neuroepithelia of most brain regions. At post-natal days 1 and 14, PST was detected throughout the neocortex, in the pyramidal cells (PC) of the hippocampus proper, the granule cell layer (GCL) of the dentate gyrus, the anterior ventral nucleus of the thalamus (AVNT) and the GCL and external germinal layer of the cerebellum. Finally, from PD21 until adulthood, expression of PST mRNA was restricted to the PC layer of the hippocampus proper, the GCL of the dentate gyrus, the AVNT, the GCL of the cerebellum and the dorsal and lateral nucleus of the anterior olfactory bulb. The developmental profile of PST mRNA is paralleled in some structures by that of the PSA-NCAM, there are, however, notable exceptions. Therefore, our results demonstrate that expression of rat PST mRNA is developmentally regulated, is present in the adult rat brain in restricted areas and may be involved in regulating temporal and spatial expression of PSA-NCAM.

Aging↗
PubMed1997

Identification and localization of dopamine receptor subtypes in rat olfactory mucosa and bulb: a combined in situ hybridization and ligand binding radioautographic approach.

Olfactory bulb (OB) of mammals contains a large population of dopaminergic interneurons within the glomerular layer. Dopamine has been shown in vivo to modulate several aspects of olfactory information processing. The dopamine receptors of olfactory bulb and mucosa are assessed here at the levels of mRNAs and radioligand binding sites with presently available tools. D1A mRNA was found in OB glomerular-, plexiform-, mitral-cell and granular layers, but not in olfactory mucosa. D1B mRNA was absent in olfactory bulb and mucosa. D1-like binding sites were detected with two distinct radioligands, in glomerular-, plexiform-, mitral cell- and granular layers of OB but not in olfactory mucosa. We thus demonstrate the previously doubtful presence of D1-like receptors in OB. D2 mRNAs were localized in the glomerular and granular layers of OB and in olfactory mucosa; lesser amounts of D3 mRNAs were found in OB glomerular and granular layer, but not in olfactory mucosa. No D4 mRNA was detected in either structure. High densities of D2-like, [125I]Iodosulpride-labelled binding sites, were revealed within lamina propria of olfactory mucosa, and confirmed in the olfactory nerve- and glomerular layers of OB. A faint but significant density of [3H]7-hydroxy-dipropyl-aminotetralin (OH-DPAT) labelled, D3 binding sites was detected in olfactory nerve- and glomerular layers of OB, but not in olfactory mucosa. Competition of [125I]Iodosulpride specific binding by three D2/D3 selective drugs yielded kinetics typical of the D2 receptor subtype in olfactory bulb and mucosa. Olfactory nerve- and glomerular layers of OB are proved thus to contain a predominant contingent of D2 receptors and a minor population of D3 receptors, while olfactory mucosa expresses only D2 receptors.

Animals↗
PubMed1997

Heat-induced conformational change and increased chaperone activity of lens alpha-crystallin.

PURPOSE: Alpha-crystallin is the major structural protein of the eye lens known to have chaperone-like activity. Our objective is to elucidate the nature of the thermal transition that alpha-crystallin undergoes at 60 degrees C and the effect of this transition on the chaperone activity. METHODS: FPLC size exclusion chromatography, far- and near-ultraviolet circular dichroism, and tryptophan (Trp) and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence were used to study conformational change. Turbidity of dithiothreitol (DTT)-reduced insulin was used to study chaperone activity. RESULTS: The thermal transition was identified as a conformational change in mainly tertiary (partial unfolding) and quaternary high-molecular-weight (HMW) aggregation structures, along with a loss of 10 percentage points of secondary structure (beta-sheet). Initial partial perturbation in tertiary structure increased chaperone activity, but the increase was less in the HMW aggregate. Similar results were observed in in vivo-formed HMW alpha-crystallin. CONCLUSIONS: The conformational change and HMW aggregation of alpha-crystallin observed at 60 degrees C, as well as in vivo-formed HMW aggregates, increased chaperone activity.

Anilino Naphthalenesulfonates↗
PubMed1996

Enhanced amphetamine sensitivity and increased expression of dopamine D2 receptors in postpubertal rats after neonatal excitotoxic lesions of the medial prefrontal cortex.

Functional and structural abnormalities in the medial prefrontal cortex (MPFC) and overactive dopamine (DA) neurotransmission are thought to be the key pathologies in schizophrenia. To understand the role of MPFC in the pre- and postpubertal development of the subcortical DA system, the effects of neonatal [postnatal day 7 (PD7)] MPFC excitotoxic lesions on locomotor behaviors and the expression of DA receptor subtypes and DA transporter were investigated in Sprague Dawley rats at PD35 and PD56, respectively. No significant differences in the novelty of d-amphetamine-induced locomotion were observed between sham-operated and ibotenic acid-lesioned rats at PD35. Postpubertally (at PD56), however, the locomotor activity of lesioned rats in the novel environment and after d-amphetamine administration was enhanced significantly compared with controls. The expressions of DA D1, D2, D3, and D4 receptors and DA transporter were then estimated in MPFC-lesioned and sham-operated rats at PD59 and PD60. The levels of DA D2 receptors, measured using [3H]-YM-09151-2 binding, and its mRNA by in situ hybridization, were observed to be significantly increased at PD60 in striatal and limbic areas of lesioned rats. Levels of other DA receptor subtypes were not significantly affected at any time points. Lesioned rats at PD39 show a small increase in DA transporter level in the shell of nucleus accumbens; however, this effect seems to wear off at PD60. The data suggest that neonatal MPFC lesions may alter the functional development and maturation of mesolimbic/nigrostriatal DA systems in that neonatally lesioned rats grow into a behavioral/neurochemical deficit.

Amphetamine↗
PubMed1995

Decreased expression of the embryonic form of the neural cell adhesion molecule in schizophrenic brains.

The regulated expression of neural cell adhesion molecule (NCAM) isoforms in the brain is critical for many neurodevelopmental processes including neurulation, axonal outgrowth, and the establishment of neuronal connectivity. We have investigated the expression of the major adult isoforms of NCAM (NCAM-180, NCAM-140, and NCAM-120) and its embryonic highly polysialylated isoform (PSA-NCAM) in the hippocampal region of postmortem brains from 10 schizophrenic and 11 control individuals. Immunohistochemical analysis with a monoclonal antibody recognizing the PSA-NCAM revealed immunoreactivity primarily in the dentate gyrus and in a subset of cells in the hilus region. We have observed a 20-95% reduction in the number of hilar PSA-NCAM-immunoreactive cells in the great majority of schizophrenic brains. The change in PSA-NCAM immunoreactivity is not obvious in other hippocampal subfields. Western blots of tissues from the hippocampal region (as well as from the frontal cortex) probed with a polyclonal antibody recognizing all NCAM isoforms did not reveal significant changes in the overall expression of NCAM, suggesting that the decrease in PSA-NCAM-immunoreactive cells may be related to post-translational processing of the molecule. The expression of this embryonic form of NCAM has been proposed to be related to synaptic rearrangement and plasticity. Therefore, the decrease in PSA-NCAM immunoreactivity in schizophrenic hippocampi may suggest an altered plasticity of this structure in a large proportion of schizophrenic brains. These findings may bear significance to the "neurodevelopmental hypothesis" of schizophrenia.

Adult↗
PubMed1993

Role of the cellular stress response in the biogenesis of cysteamine-induced astrocytic inclusions in primary culture.

Cysteamine (CSH; 2-mercaptoethylamine) stimulates the accumulation of peroxidase-positive inclusions in cultured astroglia akin to those observed in the aging periventricular brain. Because CSH induces the synthesis of a stress protein (heme oxygenase) in rat liver, we hypothesized that aspects of the cellular stress response may play a role in the biogenesis of CSH-induced astrocyte granules. In the present study, we performed indirect immunofluorescent staining and immunoblotting for various stress proteins in rat neuroglial cultures. Exposure of astrocyte cultures to CSH enhanced immunostaining for heme oxygenase-1 (HO-1) and heat-shock proteins 27, 72, and 90, but not glucose-regulated protein 94, relative to untreated cultures. CSH-pretreated astrocytes exhibited enhanced tolerance to H2O2 toxicity relative to untreated cells, providing physiological evidence of an antecedent stress response in the former. In addition, exposure for 12 days to H2O2, a known inducer of the stress response, elicited astrocyte granulation similar to that observed with CSH. Chronic induction of HO-1 and other stress proteins may participate in the biogenesis of metalloporphyrin-rich inclusions in CSH-treated astroglial cultures and in astrocytes of the aging periventricular brain.

Animals↗
PubMed1993

Quiescent and cycling cell compartments in the senescent and Alzheimer-diseased human brain.

Statin is a 57-kd nuclear protein expressed exclusively in nonproliferating cells. In the present study, immunohistochemical localization of statin in normal, senescent human brain revealed that virtually all neurons, ependymal cells, vascular smooth muscle cells, and endothelial cells are statin-positive and, hence, postmitotic. As we previously demonstrated in rodents, an unexpectedly large fraction of neuroglial cells throughout the aging human brain is statin-negative (range, 41 to 45%), consistent with the substantial retention of neuroglial proliferative capacity well into the senium. In Alzheimer's disease, there is a significant increase in the proportion of statin-negative neuroglia (range, 51 to 58%). In all regions except cerebellum, loss of statin in Alzheimer neuroglia could be accounted for by changes involving the astrocyte subpopulation. These results provide evidence that reactive gliosis in this neurodegenerative disorder encompasses some degree of astrocyte hyperplasia in addition to astrocyte hypertrophy. Maintenance of normal compartments of cycling and quiescent neuroglia in the senescent human brain may serve to define neurologic well-being during the aging process. Conversely, deviations in neuroglial cytokinetics may indicate the presence and extent of intervening neuropathologic processes.

Aged↗
PubMed1992

Expression of the non-proliferation-specific protein, statin, in grey matter neuroglia of the aging rat brain.

The monoclonal antibody, S-44, identifies statin, a 57 kDa nuclear protein which appears to be expressed exclusively in non-proliferating cells. We previously demonstrated that in the aging rat corpus callosum approximately one third of neuroglia are statin-negative, suggesting the existence of an unexpectedly large cycling glial compartment. In the present study, double-labeling of individual cultured astroglia with [3H]thymidine and the S-44 antibody provided direct evidence for the non-proliferative status of statin-positive cells. The S-44 antibody was used to immuno-localize statin and thereby determine growth fractions for neuroglia in various grey matter regions of 3-, 18-, and 33-month-old rats. The proportion of statin-negative (cycling) cells for the three ages combined ranged from about 24% in the molecular layer of the dentate gyrus to 38% in the molecular layer of the parietal cortex. In most regions surveyed total glial counts and proportions of statin-positive and -negative cells did not vary significantly as a function of advancing age. These results suggest that (i) as in corpus callosum, pools of cycling neuroglia in various grey matter regions are far in excess of those previously predicted by S-phase labeling with [3H]thymidine or BUdR, and (ii) ratios of proliferating-to-quiescent neuroglia are tightly regulated over much of the animal's adult life span. These conserved ratios may be used as markers of normal CNS senescence, and deviations thereof may indicate the presence and extent of intervening neuropathologic processes.

Aging↗
PubMed1992

Spectroscopic studies on the interaction of calf lens membranes with crystallins.

The interaction of crystallins with lens membranes and liposomes was studied by fluorescence and circular dichroism (CD) measurements. Two extrinsic fluorescence probes ANS (1-anilino-naphthalene-8-sulfonic acid) and DPH (1,6-diphenyl, 1,3,5-hexatriene) were used to detect the binding and to explore the binding site. The ANS fluorescence intensity is greater in membranes than in liposomes, but is reverse for DPH. Among alpha, beta and gamma-crystallins, only alpha c-crystallin decreased the ANS fluorescence intensity in membranes, indicating a binding between membranes and alpha c-crystallin. The binding site appears to be at the polar-apolar interface in membrane protein (MIP26) and alpha c-crystallin. Fluorescence polarization measurements show that lipid bilayer becomes less mobile with alpha c-crystallin binding. The change in the near UV CD due to the binding also indicates a decreased freedom of rotation of aromatic amino acid residues either in MIP26 or in alpha-crystallin.

Anilino Naphthalenesulfonates↗
PubMed1991

Spectral characteristics of electroretinogram in X-linked dichromats--a preliminary study.

Spectral characteristics of X-linked Dichromats (13 protanopes, 20 deuteranopes) were studied with spectral ERG. The results are as follows: The maximal spectral response of the b-wave in protanopes tended to shift toward the short wavelength side and the sensitivity to long wavelengths decreased obviously. The ratio value of the amplitude in 500 nm and in 620 nm (500/620) was greater in the protanope than that in the normal subject. Like the normals, the maximal response of the b-wave in deuteranopes appeared at 550-570 nm. Though the differences between the normals and deuteranopes in the ratio values (600/620 and b/a at 520 nm) were found by means of analysing a, b wave responses at each wavelength, most of the ratio values (600/620 or b/a at 520 nm) overlapped between the normals and deuteranopes. We established the discriminant function or score made up by the combination of two ratios basing on Fisher's rule, which can raise the diagnostic rate for deuteranopes.

Adolescent↗