Biomedical subjects

K Cai

Publications and source records attributed to K Cai.

34 records · Page 2Linked to original sources

PubMed1999

Structural features and light-dependent changes in the sequence 306-322 extending from helix VII to the palmitoylation sites in rhodopsin: a site-directed spin-labeling study.

Sixteen single-cysteine substitution mutants of rhodopsin were prepared in the sequence 306-321 which begins in transmembrane helix VII and ends at the palmitoylation sites at 322C and 323C. The substituted cysteine residues were modified with a selective reagent to generate a nitroxide side chain, and the electron paramagnetic resonance spectrum of each spin-labeled mutant was analyzed in terms of residue accessibility and mobility. The periodic behavior of these parameters along the sequence indicated that residues 306-314 were in a regular alpha-helical conformation representing the end of helix VII. This helix apparently extends about 1.5 turns above the surface of the membrane, with one face in strong tertiary interaction with the core of the protein. For the segment 315-321, substituted cysteine residues at 317, 318, 320, and 321 had low reactivity with the spin-label reagent. This segment has the most extensive tertiary interactions yet observed in the rhodopsin extra-membrane sequences at the cytoplasmic surface. Previous studies showed the spontaneous formation of a disulfide bond between cysteine residues at 65 and 316. This result indicates that at least some of the tertiary contacts made in the 315-321 segment are with the sequence connecting transmembrane helices I and II. Photoactivation of rhodopsin produces changes in structure detected by spin labels at 306, 313, and 316. The changes at 313 can be accounted for by movements in the adjacent helix VI.

Amino Acid Sequence↗

PubMed1999

Single-cysteine substitution mutants at amino acid positions 55-75, the sequence connecting the cytoplasmic ends of helices I and II in rhodopsin: reactivity of the sulfhydryl groups and their derivatives identifies a tertiary structure that changes upon light-activation.

Cysteines were introduced, one at a time, at amino acid positions 55-75 in the cytoplasmic region connecting helices I and II in rhodopsin. In each of the 21 cysteine mutants, the reactive native cysteine residues (C140 and C316) were replaced by serine. Except for N55C, all mutants formed rhodopsin-like chromophores and had normal photobleaching characteristics. The efficiency of GT activation was reduced only for K66C, K67C, L68C, and P71C. The reactivity of the substituted cysteine in each mutant toward 4, 4'-dithiodipyridine (4-PDS) was investigated in the dark. The mutants F56C to L59C and I75C were unreactive to 4-PDS under the conditions used, suggesting that they are embedded in the micelle or protein interior. The mutants V63C, H65C-T70C, and N73C reacted rapidly, while the remainder of the mutants reacted more slowly, and varied in reactivity with sequence position. For the mutants derivatized with 4-PDS, the rate of release of thiopyridone from the resulting thiopyridinyl-cysteine disulfide bond by dithiothreitol was investigated in the dark and in the light. Marked changes in the rates of thiopyridone release in the light were found at specific sites. Collectively, the data reveal tertiary interactions of the residues in the sequence investigated and demonstrate structural changes due to photoactivation.

Amino Acid Sequence↗

PubMed1999

Relationship between ventral stream for object vision and dorsal stream for spatial vision: an fMRI + ERP study.

Recent imaging studies indicated the existence of two visual pathways in humans: a ventral stream for object and form vision and a dorsal stream for spatial and motion vision. The present study was motivated by a stimulating question: Supposing shape and motion are processed separately in the two pathways, how do the respective cortical areas respond to the stimuli of "forms defined by motion"? fMRI and ERP recordings were combined in order to measure the spatiotemporal activation pattern in the two pathways responding to forms defined by motion, which were produced solely by coherent movement of random dots against a background of dynamic or static random dots. The fMRI data indicated that the stimuli of forms defined by motion indeed activated both dorsal MT/V5 and ventral GTi/GF. Furthermore, the RV curves resulting from fMRI-seeded dipole modeling indicated that each pair of dipoles located at MT/V5 or GTi/GF reached the same best-fit point; a single pair of free dipoles located near the fMRI foci of MT/V5 and GTi/GF could be identified at the corresponding best-fit point; and the source waveforms resulting from fixed dipole modeling also showed simultaneous activation of MT/V5 and GTi/GF dipoles in the time interval around the best-fit point. The present results, therefore, suggest that MT/V5 and GTi/GF appear to be activated in parallel and simultaneously responding to forms defined by motion. Such findings raise interesting issues about the hierarchical organization and the functional specialization in the two pathways.

Adult↗

PubMed1999

[Determination of P in superhigh-P cast iron by EDXRF].

In this paper preparation of standard samples of superhigh-P cast iron has been studied and P in the samples has been determined by EDXRF and powder compacting. In the process the procedure of powder compacting and the software of a multifunctional Lucas-Tooth intenstity correction have been developed. The method is rapid and simple. Analytical results were satisfactory.

English Abstract↗

PubMed1998

Activity and nature of p21(WAF1) complexes during the cell cycle.

Elevated levels of the p21(WAF1) (p21) cyclin-dependent kinase inhibitor induce growth arrest. We have characterized a panel of monoclonal antibodies against human p21 in an effort to understand the dynamic regulatory interactions between this and other cellular proteins during the cell cycle. The use of these reagents has allowed us to address several important, yet unresolved, issues concerning the biological activity of p21, including the potential kinase activity of complexes that associate with this cyclin-dependent kinase inhibitor. We have found that the kinase activity of cyclin A/Cdk2 associated with p21 is significantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled us to begin the study of the molecular architecture of p21 complexes in vivo. In addition, we found that human fibroblasts released from a quiescent state display abundant amounts of p21 devoid of associated proteins ("free" p21), the levels of which decrease as cells approach S phase. Cyclin A levels increase as the amount of monomeric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes that are not bound to, or inactivated by, p21. Our data strengthen the notion that the G1-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21.

Cell Line, Transformed↗

PubMed1998

[The diagnostic value of high field MRI for choroidal melanoma].

OBJECTIVE: To clarify the characteristic magnetic resonance imaging (MRI) features of choroidal melanomas by the high field MRI to improve the accuracy of diagnosis and localization and to differentiate choroidal melanomas from other choroidal lesions. METHODS: 12 patients with choroidal melanomas were examined by GE 1.5T magnet and heat coil, fat suppression technique and GD-DPTA as contrast agent were also used. Ten patients underwent operations and the histopathologic diagnosis was confirmed. RESULTS: The melanomas of the 12 patients had relatively high signals on T(1) weighted image and relatively low signals on T(2) weighted image. This is because of the paramagnetic properties of melanin. The melanomas with melanin can produce stable free radicals, both T(1) and T(2) relaxation times are shortened, with this we can differentiate melanomas from other choroidal lesions. CONCLUSION: Sagittal, coronal, axial and oblique scanning with small feild of view for the orbit by MRI can be made to show the exact position of melanomas and differentiate them from other choroidal lesions.

Adult↗

PubMed1998

[Effects of mitomycin C on haze after photorefractive keratectomy for myopia in rabbits].

OBJECTIVE: To investigate the effects of mitomycin C on haze after photorefractive keratectomy. METHODS: Forty-five rabbits underwent bilateral 193 nm excimer laser photorefractive keratectomy to correct 8 diopters of myopia. All eyes were allocated randomly to be treated with 0.008% mitomycin C during operation, or 0.1% dexamethasone after operation, or no medical treatment as control. Clinical and histopathologic examinations were made with slit-lamp microscope, specular microscope, light microscope and transmission electron microscope. RESULTS: At 4, 8 weeks after operation, the corneal haze was significantly less in mitomycin C group than that in the control group or in dexamethasone group, and the corneal haze was significantly less in dexamethasone group than that in the control group. At 1, 4, 8 weeks, the number of keratocyte in the anterior stroma of ablation area was significantly less in mitomycin C group than in the control group or in dexamethasone group. The difference between dexamethasone group and the control group was insignificant. The changes in time of reepithelialization, thickness of epithelium and keratocyte number of anterior stroma in ablation area were statistically insignificant among all groups. CONCLUSION: Mitomycin C can reduce corneal haze by inhibiting the proliferation of keratocyte and has no toxicity on cornea. Mitomycin C is more effective than dexamethasone on haze.

Animals↗

PubMed1998

[Chitosan pin fixation of rabbit proximal tibia osteotomy].

Degradatable chitosan pin, 4 millimeters in diameter and 30 to 40 millimeters in length, was studied radiographically and histologically (oxytetracycline-labeling) at 3, 6, 12, 24 and 48 weeks after the implantation for fixation of a transverse proximal tibia osteotomy in fourty rabbits. The results showed that new bone formation and oxytetracycline uptake were increased 3-6 weeks after operation and the fracture was completely healed 12 weeks after operation, but in two rabbits the chitosan pin broke, in 3 weeks. Histologically, there was a massive inflammation (non-specific foreign body reaction) 3-12 weeks after implantation, which gradually subsided 24 weeks aften operation. Chitosan pin was slowly degradated in bone and was not replaced by normal tissue 48 weeks after operation. The questions to be addressed in future include how to increase the mechanical strength of chitosan pin and how to improve the characteristics of its surface.

Animals↗

PubMed1997

Structure and function in rhodopsin: topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops.

Cysteine mutagenesis and site-directed spin labeling in the C-terminal region of rhodopsin have been used to probe the local structure and proximity of that region to the cytoplasmic loops. Each of the native amino acids in the sequence T335-T340 was replaced with Cys, one at a time. The sulfhydryl groups of all mutants reacted rapidly with the sulfhydryl reagent 4,4'-dithiodipyridine, which indicated a high degree of solvent accessibility. Furthermore, to probe the proximity relationships, a series of double Cys mutants was constructed. One Cys in all sets was at position 338 and the other was at a position in the sequence S240-V250 in the EF interhelical loop, at position 65 in the AB interhelical loop, or at position 140 in the CD interhelical loop. In the dark state, no significant disulfide formation was observed between C338 and C65 or C140 under the conditions used, whereas a relatively rapid disulfide formation was observed between C338 and C242 or C245. Spin labels in the double Cys mutants showed the strongest magnetic interactions between the nitroxides attached to C338 and C245 or C246. Light activation of the double mutant T242C/S338C resulted in slower disulfide formation, whereas interactions between nitroxides at C338 and C245 or C246 decreased. These results suggest the proximity of the C-terminal residue C338 to residues located on the outer face of a cytoplasmic helical extension of the F helix with an apparent increase of distance upon photoactivation.

Amino Acid Sequence↗

PubMed1996

Structural studies on folding intermediates of serine hydroxymethyltransferase using fluorescence resonance energy transfer.

Previous studies have demonstrated that the in vitro folding pathway of Escherichia coli serine hydroxymethyltransferase has both monomer and dimer intermediates that are stable for periods of minutes to hours at 4 degrees C (Cai K., Schirch, D., and Schirch, V. (1995) J. Biol. Chem. 270, 19294-19299). Single Trp mutant enzymes were constructed and used in combination with other methods to show that on the folding pathway of this enzyme two domains rapidly fold to form a monomer in which the amino-terminal 55 amino acid residues and a segment around the active site region of Lys229 remain in a largely disordered form. This partially folded enzyme can form dimers and slowly undergoes a rate-determining conformational change in which the unstructured segments assume their native state (Cai, K. , and Schirch, V. (1996) J. Biol. Chem. 271, 2987-2994). To further assess the kinetics and structural details of the intermediates during folding, fluorescence energy transfer and fluorescence anisotropy measurements were made of the three Trp residues and pyridoxal 5'-phosphate, attached covalently to the active site by reduction to a secondary amine by sodium cyanoborohydride. These studies confirmed that the basic kinetic folding pathway remained the same in the reduced enzyme as compared to the earlier studies with the apoenzyme. Both equilibrium and kinetic intermediates were identified and their structural characteristics determined. The results show that the active site Lys229-bound pyridoxyl 5'-phosphate remains more than 50 angstroms from any Trp residues until the final rate-determining conformational change when it approaches each Trp residue at the same rate. The environment of each Trp residue and the pyridoxyl phosphate in both an equilibrium folding intermediate and a kinetic folding intermediate are described.

Apoenzymes↗

PubMed1996

Structural studies on folding intermediates of serine hydroxymethyltransferase using single tryptophan mutants.

Previous studies showed that during the in vitro folding of Escherichia coli serine hydroxymethyltransferase at 4 degrees C, both monomer and dimer intermediates accumulated and were stable for periods of minutes to hours (Cai, K., Schirch, D., and Schirch, V.(1995) J. Biol. Chem. 270, 19294-19299). To obtain structural information on these intermediates, two of the three Trp residues in the protein were changed to Phe to generate a set of three single Trp mutant enzymes. These mutant enzymes were purified and characterized and shown to retain essentially all of the properties of the wild-type enzyme. The fluorescence and circular dichroism measurements of each mutant enzyme were studied under unfolding-refolding equilibrium conditions and during refolding. In addition, the sensitivity of the protein to digestion by subtilisin during refolding was investigated. The results of these studies show that the unfolded enzyme has two domains that rapidly fold to form a monomer in which the first 55 amino acids and a segment between residues 225 and 276 remain in a largely disordered form. This partially folded enzyme can form dimers and slowly undergoes a rate determining conformational change in which the unstructured segments assume their native state.

Amino Acid Sequence↗

PubMed1995

The affinity of pyridoxal 5'-phosphate for folding intermediates of Escherichia coli serine hydroxymethyltransferase.

Escherichia coli serine hydroxymethyltransferase is a 94-kDa homodimer. Each subunit contains a covalently attached pyridoxal-P, which is required for catalytic activity. At which step pyridoxal-P binds in the folding pathway of E. coli serine hydroxymethyltransferase is addressed in this study. E. coli serine hydroxymethyl-transferase is rapidly unfolded to an apparent random coil in 8 M urea. Removal of the urea initiates a complete refolding to the native holoenzyme in less than 10 min at 30 degrees C. Several intermediates on the folding pathway have been identified. The most important information was obtained during folding studies at 4 degrees C. At this temperature, the far-UV circular dichroism spectrum and the fluorescence spectrum of the 3 tryptophan residues become characteristic of the native apoenzyme in less than 10 min. Size exclusion chromatography shows that under these conditions the refolding enzyme is a mixture of monomeric and dimeric species. Continued incubation at 4 degrees C for 60 min results in the formation of only a dimeric species. Neither the monomer nor dimer formed at 4 degrees C bind pyridoxal phosphate. Raising the temperature to 30 degrees C results in the formation of a dimeric enzyme which rapidly binds pyridoxal phosphate forming active enzyme. These studies support the interpretation that pyridoxal phosphate binds only at the end of the folding pathway to dimeric apoenzyme and plays no significant role in the folding mechanism.

Circular Dichroism↗

PubMed1995

[Regulatory effect of ventral tegmental area on sleep-wakefulness in rats].

Experiments were performed on 31 male SD rats. The results were as follows: (1) The time of waking was increased during the second and third hours after microinjection of 3.3 and 6.6 nmol bromocriptine into bilateral VTA (P < 0.01), but the 1.33 nmol group was without significant effect. (2) The time of waking was decreased during the second and third hours after microinjection of 2 and 4 nmol SCH23390 into VTA (P < 0.01 and P < 0.05 respectively), while 3.4 nmol sulpiride group was without effect. (3) One week after administration of kainic acid (0.3 microgram), the rats presented less time of waking in the day (P < 0.05), while sleep-wakefulness cycle at night showed no change. The results suggest that bromocriptine could increase wakefulness by activating VTA DA neurons via D1 receptor and that the DA neurons might exert a tonic effect for maintaining the wakefulness at the daytime.

Animals↗

PubMed1994

Photoaffinity-labeling peptide substrates for farnesyl-protein transferase and the intersubunit location of the active site.

--CAAX motif peptides, which are substrates for isoprenylation, were synthetically derivatized with the light-sensitive benzophenone (Bz) group in order to determine their potential use as catalytic site-directed covalent photocross-linking ligands for one of the enzymes catalyzing protein isoprenylation, farnesyl-protein transferase (FPTase). Bz-peptides could be synthesized with [3H]benzophenone and possessed eiter one or two benzophenone groups located at or near the peptide's NH2 terminus (e.g. the mono-Bz probes Bz-ACVIM and Bz-LPCVVM, and the di-Bz derivatized probe Bz-GY-(Bz)PCVVM, referred to as Bz2-GYPCVVM). Each type of derivatized peptide behaved as a substrate for farnesylation in vitro without irradiation, while under 366-nm irradiation each demonstrated covalent cross-linking ability as a catalytic site-directed photoaffinity ligand with tissue-purified or enriched but impure fractions from rat and bovine brain FPTase, as well as with a recombinant human FPTase variant, FPTase (beta alpha t) expressed in Escherichia coli. Without photoactivation, Bz-ACVIM yielded a Kd of 37 nM for the cloned variant of human FPTase. Pseudo first-order photolytic inhibition of FPTase preparations with Bz-peptides, as well as protection from photoinactivation by unmodified -CAAX motif peptides, supported the capacity of these Bz-peptides to serve as co-substrates and their specificity for seeking the catalytic site of the enzyme. SDS-polyacrylamide gel electrophoresis analysis subsequent to photolysis indicated that the mono-Bz-derivatized peptides (e.g. [3H]Bz-LPCVVM or 3H]Bz-ACVIM) became covalently cross-linked preferentially to the approximately 49-kDa beta subunit of the alpha beta dimeric FPTase. The farnesyl-PP cosubstrate bound equally well to unmodified and Bz-ACVIM-labeled enzyme. The di-Bz derivative, [3H]Bz2-GYPCVVM, in contrast, revealed exclusive photocovalent cross-linking with a species of molecular mass approximately 95-97 kDa, indicating that both FPTase subunits were tethered together covalently by the di-Bz probe. Similar differential SDS-polyacrylamide gel electrophoresis cross-linking patterns were obtained with homogeneous FPTases as well as with partially purified rat or bovine brain enzyme preparations. The absence of nonspecific photolabeling of any proteins in the partially purified rat or bovine brain enzyme preparations other than FPTase independently attested to the high efficiency of photocross-linking of the FTPase, and the selective catalytic site-seeking ability of these Bz-derivatized peptide substrates, verifying their potential as structural probes for the active site domain on the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity Labels↗

PubMed1991

[Effects of stimulating periaqueductal gray on the nociceptive neuron discharges of post-thalamic nucleus evoked by stimulation of splanchnic nerve in cats].

The effects of stimulating periaqueductal gray (PAG) on the nociceptive neuron discharges of pronucleus evoked by stimulating the splanchnic nerve in cats were studied. The two kinds of inhibitory action were observed. One was called early phase or prompt inhibition phase which occurred within 30 ms to 150 ms after stimulation of PAG. The other was called slow-pase or sustained inhibition which occurred from a few minutes to more than ten minutes after the stimulation of PAG. The results showed that PAG played an important role in the analgesia of visceral pain. Both kinds of inhibition may be blocked by naloxone, this suggests that endogenous opiate like substances involved in these inhibitory processes caused by stimulating role in the inhibitory effect of PAG.

Animals↗

PubMedDate not supplied

Single UV excitation of Hoechst 33342 and propidium iodide for viability assessment of rhesus monkey spermatozoa using flow cytometry.

Many fluorescent probes excited by visible light have been used to assess sperm quality by flow cytometry. Developing a viability evaluation method using UV excited stains would be useful for multiparameter analysis of sperm function. This investigation was conducted to determine the efficacy of Hoechst 33342 (H342) and propidium iodide (PI) dual staining for evaluating rhesus monkey sperm viability through use of flow cytometry and excited by a single UV laser. The results showed that the live cells stained only with H342 strongly correlated with expected sperm viability, and flow cytometric analyses were highly correlated with fluorescence microscopic observation. Using H342/PI/SYBR-14 triple staining method, it was found that the live/dead sperm distributions were completely concordant in both H342/PI and SYBR-14/PI assays. In addition, this dual staining was extended with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) to simultaneously analyze viability and acrosome integrity of sperm cryopreserved using two different extenders, TTE and TEST, and indicated that TTE offered better preservation of plasma and acrosome integrity than TEST. Therefore, the H342/PI dual staining provides an accurate technique for evaluating viability of rhesus monkey sperm and should be valuable for multiparameter flow cytometric analysis of sperm function.

Animals↗