Gonyaulax luciferase: gene structure, protein expression, and purification from recombinant sources.
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Animals↗
Biomedical subjects
L Li
Publications and source records attributed to L Li.
Single-cell MALDI: a new tool for direct peptide profiling.
Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry (MS) is a rapid and sensitive analytical approach that is well suited for obtaining molecular weights of peptides and proteins from complex samples. MALDI-MS can profile the peptides and proteins from single-cell and small tissue samples without the need for extensive sample preparation, except for the cell isolation and matrix application. Strategies for peptide identification and characterization of post-translational modifications are presented. Furthermore, several recent enhancements in MALDI-MS technology, including in situ peptide sequencing as well as the direct spatial mapping of peptides in cells and tissues are discussed.
Animals↗
Downregulation of TrkA expression in primary sensory neurons after unilateral lumbar spinal nerve transection and some rescuing effects of nerve growth factor infusion.
Peripheral nerve injury results in sprouting of sympathetic and sensory nerve terminals around large diameter neurons in the dorsal root ganglia (DRG), but the underlying mechanism is not clear. Current study sought to examine changes of the nerve growth factor (NGF) receptor TrkA in DRG and spinal cord after a spinal nerve transection by an immunohistochemical technique and to investigate effects of NGF on the expression of TrkA protein in the same animal model. In the control rat, TrkA immunoreactivity was localized to about 55 +/ -1% of total neurons in DRG and to laminae I and II of the spinal cord. The percentage of TrkA immunoreactive neurons in DRG and TrkA staining intensity of spinal cord were reduced 1 week after the nerve lesion. The changes became maximal 2 weeks, but recovered partially 4 weeks after the lesion. The size of TrkA immunoreactive neurons dramatically shifted to smaller sizes, becoming more remarkable 4 weeks after the lesion. In the contralateral DRG, the percentage of TrkA immunoreactive neurons also decreased significantly. Exogenous NGF delivered to DRG for 2 weeks partially reversed the reduction of TrkA expression as well as atrophy of TrkA immunoreactive neurons. No TrkA immunoreactive basket was found around neuronal somata. Our data show that unilateral peripheral nerve injury results in dynamic downregulation of TrkA in sensory neurons in bilateral DRG and spinal cord, and that TrkA expression in sensory neurons is partially regulated by target-derived NGF.
Animals↗
The French neurotropic vaccine strain of yellow fever virus accumulates mutations slowly during passage in cell culture.
This study of the yellow fever French neurotropic vaccine strain from the Institut Pasteur (FNV-IP) demonstrates that this viral genome is not as stable as that of the 17D-204 vaccine virus. FNV-IP was plaque-purified three times and then passaged eight times in Vero cells. Viral populations from the second and eighth passage post purification were sequenced and compared to the published sequences of FNV-IP. The passage-2 viral population had 31 nucleotide and nine amino acid changes compared to the parental virus while the passage-8 virus had six additional nucleotide changes encoding a single amino acid substitution. The plaque-purified virus also had two sequence deletions in the 3'-noncoding region. The plaque purification resulted in selection of a passage-2 virus that had a mouse LD(50) of 20 pfu/ml, 67-fold greater than parental FNV-IP which had an LD(50) of 0.3 pfu/ml. Subsequent passage in Vero cells resulted in a passage-8 virus which had increased neurovirulence with an LD(50) of 3.2 pfu/ml. The only amino acid difference between the passage-2 and passage-8 viruses was at amino acid 638 of NS5 which lies within domain V of the RNA-dependent-RNA polymerase. Overall, these data indicate that FNV-IP virus has an inherently less stable genome than 17D vaccine virus and a variable viral population.
Amino Acid Sequence↗
Apoptotic death of hair cells in mammalian vestibular sensory epithelia.
Hair cell death was examined in cultured explants of vestibular organs from mature guinea pigs and gerbils. The effects of gentamicin were compared with those of staurosporine, a membrane-permeable kinase inhibitor that induces programmed cell death in almost all cell types. Under the conditions used staurosporine killed hair cells but supporting cells appeared unaffected, and a topographic pattern of differential sensitivity to staurosporine amongst hair cells, similar to that described for aminoglycoside antibiotics, was revealed. This suggests such differential sensitivity is an inherent property of the hair cell population. Thin sectioning, and examination of whole mount preparations after application of the TUNEL procedure or after double fluorescent labelling with phalloidin and with propidium iodide, which labels nuclei, revealed that hair cells after exposure to gentamicin show features identical to those of apoptotic cells after exposure to staurosporine. Furthermore, cells showing features of apoptosis constitute a major proportion of the hair cells that are ultimately lost following exposure to gentamicin. Incubation of cultures with gentamicin in the presence of broad-spectrum inhibitors of caspases, proteases involved specifically in the cell death pathway, prevented almost all of the hair cell deaths normally triggered by gentamicin. This confirms that apoptosis is the predominant mode of hair cell death after gentamicin exposure. Hair cells exposed to gentamicin in the presence of caspase inhibitors appeared to be preserved intact. This, and the thin section observations, suggests that apoptotic death is the fate of the majority of hair cells affected by that drug and that any sub-lethal damage to hair cells exposed to gentamicin does not result in significant morphological alterations. Hair cell death was also prevented by deferoxamine which has been shown to protect cochlear hair cells in vivo from the effects of gentamicin. Explant cultures of mature vestibular organs may be, therefore, a useful model system for examining putative hair cell protecting agents.
Animals↗
Association and linkage analysis of the alpha-adducin gene and blood pressure.
In Milan hypertensive rats, a variant in the alpha-adducin gene has been shown to account for approximately 50% of the interindividual variation in blood pressure levels between these animals and their normotensive counterparts. Additional studies have suggested that a polymorphism within exon 10 of the human alpha-adducin gene (Gly-460-Trp) may be associated with hypertension and salt sensitivity. On the basis of these observations, we investigated variation within or near the human alpha-adducin gene for linkage and association with a locus influencing blood pressure levels in 281 nuclear families (774 siblings aged 5 to 37 years; 380 parents aged 26 to 57 years), selected from the white population of Rochester, Minnesota, without regard to health. Sib pair linkage analyses (n = 852 sibling pairs) using a dinucleotide repeat marker (D4S43) that maps approximately 660 kb from the alpha-adducin gene provided no evidence of linkage between this marker locus and a locus influencing systolic, diastolic, or mean blood pressure levels. Allele frequencies for the Gly-460-Trp polymorphism were similar to those reported in other white populations (Gly = 0.812, Trp = 0.188); however, this polymorphism was not associated with any measure of blood pressure level in either parents or siblings. Therefore, variation within the alpha-adducin gene does not appear to have a major influence on measures of blood pressure in white families from Rochester, Minnesota.
Adolescent↗
Total synthesis and antifungal evaluation of cyclic aminohexapeptides.
The need for new therapies to treat systemic fungal infections continues to rise. Naturally occurring hexapeptide echinocandin B (1) has shown potent antifungal activity via its inhibition of the synthesis of beta-1,3 glucan, a key fungal cell wall component. Although this series of agents has been limited thus far based on their physicochemical characteristics, we have found that the synthesis of analogues bearing an aminoproline residue in the 'northwest' position imparts greatly improved water solubility (> 5 mg/mL). The synthesis and structure-activity relationships (SAR) based on whole cell and upon in vivo activity of the series of compounds are reported.
Acute Disease↗
Discerning matrix-cluster peaks in matrix-assisted laser desorption/ionization time-of-flight mass spectra of dilute peptide mixtures.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is widely used for the analysis of peptide mixtures such as those resulting from protein digestion. Among several useful peptide matrices, alpha-cyano-4-hydroxycinnamic acid (4-HCCA) appears to be the most popular. This matrix does not generally give matrix-cluster peaks at the mass region covered by enzyme-digested peptides (i.e., m/z above approximately 500). However, when an analyte mixture is very dilute and/or the sample contains a large amount of salts, ion peaks from matrix clusters can be quite intense, compared to peptide peaks. This matrix-cluster interference becomes more pronounced as the amount of analyte decreases. In this paper, a simple scheme for matrix-cluster identification is reported. It is shown that matrix-cluster formation follows a systematic pattern, although the relative intensities of these cluster ions cannot be predicted. Discerning the matrix-cluster peaks from the peptide peaks is found to be critical in analyzing dilute peptide mixtures with both conventional and microspot MALDI-TOF techniques.
Hydrolysis↗
Using intracranial electrical stimulation to study the timing of prepulse inhibition of the startle reflex.
Due to the short latency and briefness of the startle reflex, event-related inhibition of startle has high temporal resolution and is useful for studying the hierarchical organization of sensorimotor gating and motive-motor gating. In this article, we describe methods for measuring the inhibitory effects of electrically stimulating each of the following four brain structures on startle in awake rats: the inferior colliculus (IC), the deeper layers of the superior colliculus (SC), the pedunculopontine tegmental nucleus (PPTg), and the ventral pallidum (VP). These four brain structures have been reported to be important in mediating sensorimotor or motive-motor gating. Startle responses are elicited by either intense noise bursts or electrical stimulation of the principal trigeminal nucleus. The time course of the IC-inhibited startle reflex is used as a standard for estimating timing of the neural transfer of startle-inhibitory information to motor outputs. We also discuss how these methods can be used in combination with neuropharmacology.
Animals↗
Study on using I- as heavy atom perturber in cyclodextrin-induced room temperature phosphorimetry.
A cyclodextrin induced room temperature phosphorimetry (CD-RTP) for determine beta-NOA, which using I- as a heavy atom perturber (HAP) and sodium sulfite as a deoxygenator, was developed. The phosphorescence peak wavelength maxima lambda(ex)/lambda(em) = 287/496,521 nm. The analytical curve of beta-NOA gives a linear dynamic range of 2.0 x 10(-7)-6.0 x 10(-6) mol/l and a detection limit of 4 x 10(-8) mol/l. The relative standard deviation (RSD; n = 7) was 3.2% for the 4.0 x 10(-6) mol/l beta-NOA in spiked apple samples. The influence of I- concentration on RTP lifetime of beta-NOA was studied in detail, the static Stern-Volmer equation for phosphorescence was derived and the luminescence kinetic parameters were calculated. It is found that the relation between I- concentration (x) and RTP lifetime (tau) can be expressed as tau = 1.047 e(-0.354x) and the rate constants of phosphorescence emission k(p) and non-radiation process k(i) from T1 --> S0 were 0.9551 s(-1) and 0.4276 s(-1) l(-1) mol, respectively.
Cyclodextrins↗
The inhibited xanthophyll cycle is responsible for the increase in sensitivity to low temperature photoinhibition in rice leaves fed with glutathione.
Exposure of intact rice leaves to an irradiance of 1000 mumol m(-2) s(-1) at 6 degrees C for 2 h caused severe photoinhibition of Photosystem II. The rate and extent of photoinhbition were greatly exacerbated in leaves fed with 10 mM reduced glutathione (GSH) or 10 mM cysteine. Analyses of antioxidant enzyme activities as well as the application of protein synthesis inhibitors revealed that the increased sensitivity to photoinhibition following GSH feeding was not related to its effect on cellular antioxidant systems. On the other hand, feeding with GSH markedly suppressed the formation of zeaxanthin and antheraxanthin via the xanthophyll cycle and its associated nonradiative energy dissipation in leaves chilled in high light, suggesting that the stimulating effect of exogenous GSH on photoinhibition may be attributable to its action on the xanthophyll cycle. In vitro experiments using isolated thylakoids indicated that GSH is a weak inhibitor of violaxanthin deepoxidation. The possible implications of these results are discussed.
Journal Article↗
Estimation of distribution functions using data from different environments.
Suppose that when a unit operates in a certain environment, its lifetime has distribution G, and when the unit operates in another environment, its lifetime has a different distribution, say F. Moreover, suppose the unit is operated for a certain period of time in the first environment and is then transferred to the second environment. Thus we observe a censored lifetime in the first environment and a failure time of a "used" unit in the second environment. We propose an EM algorithm approach for obtaining a self-consistent estimator of F using observations from both environments. The case where failure times are subject to right censoring is considered as well. We also establish the maximum likelihood estimator of F when the unit is repairable. Application and simulation studies are presented to illustrate the methods derived.
Algorithms↗
Morphology of liver repair following cholestatic liver injury: resolution of ductal hyperplasia, matrix deposition and regression of myofibroblasts.
BACKGROUND/AIMS: Myofibroblasts are the primary cells responsible for increased matrix deposition in hepatic fibrosis. Activation of hepatic stellate cells and portal fibroblasts to myofibroblasts during cholestatic liver injury is accompanied by increased expression of the activation marker, alpha-smooth muscle actin (SMA), and collagen genes. In contrast to our understanding of injury, the cellular mechanisms of liver repair are not well defined. This study was designed to examine the morphological relationship between bile duct hyperplasia, matrix deposition and myofibroblast phenotype in a model of chronic cholestatic liver injury and repair. METHODS: Reversible extrahepatic obstruction was accomplished in rats using a soft vessel loop suspended from the anterior abdominal wall: duct manipulation alone was performed in sham-operated controls. After 7 days, rats were either sacrificed or decompressed by release of the loop and subsequently sacrificed 2-10 days after reversal. Liver sections were obtained for in situ hybridization for procollagen alpha1(I) mRNA, immunohistochemical staining for SMA and cytokeratin 19, and histochemical staining for reticulin. RESULTS: Cholestatic livers demonstrated bile duct hyperplasia, which reversed to normal within 10 days after decompression. Fibrosis was also substantially reduced during this period. SMA-positive myofibroblasts were abundant and localized to regions adjacent to proliferating ducts and excess matrix in the obstructed animals. Decompressed livers showed a dramatic time-dependent reduction in the number of SMA-positive cells and in the expression of procollagen I mRNA. CONCLUSIONS: Our results show that the disappearance of bile duct hyperplasia after biliary decompression is accompanied by a similarly rapid loss of SMA-positive myofibroblasts. Both cellular events may abrogate enhanced matrix synthesis and allow repair to occur.
Actins↗
A novel approach to gene therapy of albino hair in histoculture with a retroviral streptomyces tyrosinase gene.
In order to induce melanin production in mammalian cells with pigment disorders such as albino hair, a recombinant retrovirus containing the mel locus of Streptomyces antibioticus was constructed. The S. antibioticus mel locus, which consists of the open reading frame (ORF)-438 and the tyrosinase gene, was specifically derived by polymerase chain reaction (PCR) from Streptomyces plasmid pIJ702. The ORF-438 is required for the transfer of copper to apotyrosinase, which is essential for tyrosinase enzymatic activity. The tyrosinase gene was inserted into the XhoI/BamHI cloning site of the pLXSN retroviral vector to obtain pLtyrSN. An internal ribosome entry site (IRES) suitable for mammalian cell expression was obtained from the pLXIN retroviral vector by PCR. The ORF-438 and IRES DNA fragments were inserted into the pLtyrSN vector to obtain the tyrosinase-expression retroviral vector pLmelSN. The expression vector was amplified in murine PT67 packaging cells, where the ORF-438 and tyrosinase genes were also co-expressed as determined by reverse transcription-PCR. In order to evaluate the vector's ability to restore pigment production in cells with a pigment disorder, albino-mouse skins were histocultured and then infected with the pLmelSN retrovirus. Six days after infection, melanin granules were observed in approximately 60% of albino-mouse hair follicles in the histocultured skin. These results demonstrated that the S. antibioticus mel operon could express an active tyrosinase and produce melanin in the albino-mouse hair follicles. This novel gene therapy approach, using a small and simple tyrosinase operon in a high-expression vector, has a potentially wide application for therapy of pigment disorders in hair follicles.
Albinism↗
Cardiac defects and renal failure in mice with targeted mutations in Pkd2.
PKD2, mutations in which cause autosomal dominant polycystic kidney disease (ADPKD), encodes an integral membrane glycoprotein with similarity to calcium channel subunits. We induced two mutations in the mouse homologue Pkd2 (ref.4): an unstable allele (WS25; hereafter denoted Pkd2WS25) that can undergo homologous-recombination-based somatic rearrangement to form a null allele; and a true null mutation (WS183; hereafter denoted Pkd2-). We examined these mutations to understand the function of polycystin-2, the protein product of Pkd2, and to provide evidence that kidney and liver cyst formation associated with Pkd2 deficiency occurs by a two-hit mechanism. Pkd2-/- mice die in utero between embryonic day (E) 13.5 and parturition. They have structural defects in cardiac septation and cyst formation in maturing nephrons and pancreatic ducts. Pancreatic ductal cysts also occur in adult Pkd2WS25/- mice, suggesting that this clinical manifestation of ADPKD also occurs by a two-hit mechanism. As in human ADPKD, formation of kidney cysts in adult Pkd2WS25/- mice is associated with renal failure and early death (median survival, 65 weeks versus 94 weeks for controls). Adult Pkd2+/- mice have intermediate survival in the absence of cystic disease or renal failure, providing the first indication of a deleterious effect of haploinsufficiency at Pkd2on long-term survival. Our studies advance our understanding of the function of polycystin-2 in development and our mouse models recapitulate the complex human ADPKD phenotype.
Animals↗
Kinetics, thermodynamics and evolution of non-native interactions in a protein folding nucleus.
A lattice model with side chains was used to investigate protein folding with computer simulations. In this model, we rigorously demonstrate the existence of a specific folding nucleus. This nucleus contains specific interactions not present in the native state that, when weakened, slow folding but do not change protein stability. Such a decoupling of folding kinetics from thermodynamics has been observed experimentally for real proteins. From our results, we conclude that specific non-native interactions in the transition state would give rise to straight phi-values that are negative or larger than unity. Furthermore, we demonstrate that residue Ile 34 in src SH3, which has been shown to be kinetically, but not thermodynamically, important, is universally conserved in proteins with the SH3 fold. This is a clear example of evolution optimizing the folding rate of a protein independent of its stability and function.
Amino Acid Sequence↗
Structural basis of the recognition of the dishevelled DEP domain in the Wnt signaling pathway.
The DEP domain of Dishevelled (Dvl) proteins transduces signals to effector proteins downstream of Dvl in the Wnt pathway. Here we report that DEP-containing mutants inhibit Wnt-induced, but not Dvl-induced, activation of the transcription factor Lef-1. This inhibitory effect is weakened by a K434M mutation. Nuclear magnetic resonance spectroscopy revealed that the DEP domain of mouse Dvl1 comprises a three-helix bundle, a beta-hairpin 'arm' and two short beta-strands at the C-terminal region. Lys 434 is located at the tip of the beta-hairpin 'arm'. Based on our findings, we conclude that DEP interacts with regulators upstream of Dvl via a strong electric dipole on the molecule's surface created by Lys 434, Asp 445 and Asp 448; the electric dipole and the putative membrane binding site are at two different locations.
3T3 Cells↗
The collecting duct, dopamine and vasopressin-dependent hypertension.
AVP not only increases osmotic water permeability (Pf) in the rat cortical collecting duct (CCD), but also acts synergistically with aldosterone to augment sodium reabsorption (JNa). These effects are inhibited by catecholamines via alpha2 adrenergic receptors, and by dopamine. We review here studies designed to determine the mechanism and receptor involved in dopamine action. The inhibitory effect of dopamine on Na+ and water transport was found to be reversible, and was not produced by agonists specific to D1A and D1B receptors. D2-type (D2, D3 or D4) receptors and activation of the GTP-binding protein Gi were implicated by the observation that dopamine had no inhibitory effect when JNa and Pf were stimulated by a cyclic AMP analogue plus isobutylmethylxanthine. The only dopaminergic antagonist that reversed the inhibitory effect of dopamine was clozapine, which is relatively D4-specific. We also found that dopamine or D1-specific agonists by themselves had no effect on cAMP production. However, dopamine inhibited the high rate of AVP-dependent cAMP production, and this effect of dopamine was reversed by clozapine but not other antagonists or by inhibitors of protein kinase C. The D4 receptor was observed in western blots of renal cortical proteins, and it was localized to the collecting duct by RT-PCR and immuno-histochemistry using a D4-specific antibody. These results show that at least a portion of the natriuretic effect of dopamine can be attributed to inhibition of AVP-dependent Na+ reabsorption by the CCD, and they introduce another signalling system as a candidate in the aetiology of low-renin, salt-dependent hypertension.
Animals↗