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Biomedical subjects

B Li

Publications and source records attributed to B Li.

At least 559 records · Page 31Linked to original sources
PubMed1996

Identification of a unique VP4 serotype that is shared by a human rotavirus (69M strain) and an equine rotavirus (H-2 strain).

A cDNA clone representing the VP4-encoding gene of human rotavirus strain 69M(VP7 serotype 8) was constructed and inserted into a baculovirus expression vector. Baculovirus recombinants that expressed the 69 M VP4 protein in Spodoptera frugiperda (Sf9) cells were screened by immunofluorescence with hyperimmune antiserum to the 69M strain and purified by terminal dilution. The expressed VP4 was detected by Coomassie blue staining of PAGE-separated proteins. The antigenic relationships between the VP4 of the 69M strain and those of various human and other animal rotavirus strains representing ten established VP4 serotypes were examined by plaque reduction neutralization. Hyperimmune antiserum produced in guinea pigs following immunization with a lysate of Sf9 cells infected with a 69M gene 4-baculovirus recombinant neutralized the infectivity of the homologous human rotavirus 69M strain as well as heterologous equine rotavirus H-2 strain to a high titer. The anti-69M VP4 hyperimmune antiserum did not neutralize significantly other rotavirus strains of human, simian, porcine, bovine, or murine origin. It thus appears that the human rotavirus 69M strain has a distinct VP4 serotype (designated as P serotype 4) which is closely related antigenically to equine rotavirus H-2 VP4.

Animals↗
PubMed1996

Gene probe-based detection of type E botulinum neurotoxin binding protein using polymerase chain reaction.

Using primers based on the nucleotide sequence of a neurotoxin binding protein from Type E Clostridium botulinum cultures, an amplified DNA product was obtained through polymerase chain reaction. The 400 base pair amplified DNA fragment was detectable with as low as 0.1 pg template DNA from Type E C. botulinum, and its fidelity was confirmed by Southern blotting using a DNA probe designed to detect the expected amplified DNA fragment. On the other hand, no DNA amplification was observed with as high as 10 ng template DNA from related Types A and B C. botulinum or from C. tetani, indicating the specificity of the probe.

Botulinum Toxins↗
PubMed1996

Inhibition of membrane L-type calcium channel activity and intracellular calcium concentration by 24R, 25-dihydroxyvitamin D3 in vascular smooth muscle.

Pharmacological doses of 24R,25-dihydroxyvitamin D3 (24,25D3) inhibited both phasic and tonic contraction of Sprague-Dawley (SD) rat tail artery helical strips induced by KCl, norepinephrine (NE), and arginine vasopressin (AVP) in organ-bath studies. 24,25D3 also decreased the tension dependent on external calcium influx induced by KCl, AVP, and NE and the tension dependent on internal calcium release from intracellular calcium stores induced by NE. In vascular smooth muscle cells isolated from SD rat tail artery, 24,25D3 reduced membrane L-type calcium channel current and the increment of intracellular calcium concentration induced by KCl. It is suggested that 24,25D3 directly relaxed precontracted SD rat-tail artery by its inhibitory effect on plasma membrane and intracellular organelle calcium channels.

24,25-Dihydroxyvitamin D 3↗
PubMed1996

24,25(OH)2 vitamin D3 modulates the L-type Ca2+ channel current in UMR 106 cells: involvement of protein kinase A and protein kinase C.

In this study, the effect of 24,25(OH)2 vitamin D3 (24,25D3), on the L-type Ca2+ channel current (L-channel current) in UMR 106 cells was investigated using the whole cell version of the patch clamp technique. It was found that 24,25D3 had a dual effect on the L-channel current: a low concentration of 24,25D3 (1 x 10(-8) M) increased the amplitude of the L-channel current by 49 +/- 11%, whereas a high concentration of 24,25D3 (1 x 10(-5) M) reduced the amplitude of the current by 55 +/- 7%. The effect of a low concentration of 24,25D3 was mimicked by 8-bromo-cAMP and inhibited by Rp-cAMPs, indicating the involvement of the cAMP/protein kinase A pathway. In contrast, the effect of a high concentration of 24,25D3 was mimicked by 4 beta-phorbol 12-myristate 13-acetate and inhibited by calphostin C, indicating the involvement of protein kinase C. In comparison, a high concentration of 1,25(OH)2 vitamin D3 (1,25D3) (1 x 10(-6) M) increased the L-channel current in UMR 106 cells. Therefore, 24,25D3 appears to have an action on the L-channel current that is distinct from that of 1,25D3. This demonstration of a non-genomic effect of 24,25D3 on calcium channels suggests that 24,25D3 is an active metabolite of vitamin D3 and may play an important role in regulating the function of bone cells.

24,25-Dihydroxyvitamin D 3↗
PubMed1996

Clinical neuroimmunology.

Clinical research has focused on autoimmune disease (AID) for a couple of decades. More sensitive and specific methods have been developed for neuroimmunological research. Gamma fraction bands (bands separated by electrophoresis and visualized by amino black staining) and IgG fraction bands (bands separated by iso-electric focusing and visualized by immunostaining) are used instead of oligoclonal bands. Myasthenia gravis (MG) mainly involves acetylcholine receptors of the postsynaptic membrane at the neuromuscular junction. Myasthenia gravis has been considered to be a generalized AID, because 7% of patients with myasthenia gravis associate with other AIDs and more than one autoimmune antibody is detected in 52.5% patients with myasthenia gravis. Pyramidal signs in myasthenia gravis patients are described; the possible mechanism may at least be partly due to the acetylcholine receptor antibody. P2 protein and its antibody are studied in patients with acute and chronic inflammatory demyelinating polyneuropathy.

Adolescent↗
PubMed1996

Influence of vehicle, distant topical delivery, and biotransformation on the chemopreventive activity of apigenin, a plant flavonoid, in mouse skin.

PURPOSE: This study was designed to (a) establish a short-term in vivo system to evaluate topical formulations of apigenin, (b) determine whether apigenin should be topically delivered to the local skin tissue or systemic circulation, (c) investigate if biotransformation was involved in apigenin's chemopreventive activity. METHODS: The effect of topical applied apigenin in acetone/DMSO (A/D, 9:1) on the promotion of skin tumorigenesis was studied. The influence of apigenin in DMSO, A/D (4:1), and propylene glycol/DMSO (PG/D, 4:1) on 12-O-tetradecanoylphorbol-13 acetate (TPA) induced ornithine decarboxylase (ODC) activity was compared. Distant topical delivery of apigenin was conducted on abdominal skin and ODC activity was monitored in dorsal skin. Potential glucuronidation/sulfation of apigenin in intact skin was assessed by measuring isolated apigenin before and after enzyme hydrolysis with glucuronidase/sulfatase. The epidermal extracts from apigenin-treated SENCAR mice were analyzed for metabolites by HPLC. RESULTS: Apigenin (5 mumol) in A/D did not significantly reduce skin tumor incidence in contrast to previous data with DMSO. Inhibition of TPA-induced ODC by apigenin in three vehicles was in the order of DMSO > A/D > PG/D. TPA-induced ODC in dorsal skin was not inhibited by apigenin delivered from abdominal skin. The quantity of apigenin recovered from epidermal extract was not different before and after beta-glucuronidase/sulfatase treatment. Metabolites were not observed in the HPLC profiles of epidermal extracts from apigenin treated mice. CONCLUSIONS: (a) The short-term TPA-induced ODC was validated for evaluating topical formulations of apigenin. (b) Topical delivery of apigenin should target the local skin tissue. (c) Glucuronidation/sulfation appeared not to be involved in apigenin's chemopreventive activity.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

In vivo and in vitro percutaneous absorption of cancer preventive flavonoid apigenin in different vehicles in mouse skin.

PURPOSE: In vivo and in vitro percutaneous absorption of apigenin was investigated in three vehicles previously used in cancer prevention studies to determine the drug delivery properties for optimal chemo-preventive activity. METHODS: In vivo percutaneous absorption of apigenin on SENCAR mice was studied with DMSO and acetone/DMSO (A/D, 4:1) vehicle. In vitro percutaneous absorption studies used whole mouse skin, without subcutaneous fat, mounted on Franz diffusion cells with 37 degrees C Dulbecco's phosphate-buffered saline as the receptor fluid. The skin was treated with [G-3H]-apigenin in DMSO, A/D (4:1), or propylene glycol/DMSO (PG/D, 4:1). RESULTS: Apigenin uptake by epidermal cells and distribution in epidermis following in vivo topical treatment in two vehicles was in the order of A/D > DMSO, while apigenin distribution in dermis and subcutaneous fat was not different between DMSO and A/D. Total apigenin absorption in mouse skin in vitro was in the order of A/D > DMSO > PG/D. However, apigenin sub-tissue distribution within epidermis determined by tape-stripping and by determination of apigenin in dermal and epidermal tissue indicated that DMSO delivered more apigenin into viable epidermis than A/D while A/D deposited more apigenin in the stratum corneum. Apigenin absorption in mouse skin with DMSO or A/D showed saturation kinetics while apigenin in PG/D showed very low absorption initially and non-saturated absorption in a period of 6 hr. HPLC-scintillation profiles of in vitro samples showed no evidence of apigenin metabolism in mouse skin. CONCLUSIONS: Delivering apigenin into viable epidermis appears to be a necessary property for an apigenin formulation to be effective in skin cancer prevention.

Acetone↗
PubMed1996

Intragraft TGF-beta 1 mRNA: a correlate of interstitial fibrosis and chronic allograft nephropathy.

Chronic allograft nephropathy is a relentlessly progressive process and a major cause of long-term graft dysfunction and ultimate failure. Interstitial fibrosis, tubular atrophy, and glomerular and vascular lesions characterize this mechanistically unresolved disorder. Given the prominent role of TGF-beta 1 in tissue repair and in fibrosis, we have explored the hypothesis that fibrosis and chronic allograft nephropathy would be distinguished by intragraft TGF-beta 1 mRNA expression. This postulate was tested by mRNA phenotyping of RNA isolated from 127 human renal allograft biopsies. Reverse transcription assisted polymerase chain reaction was used to amplify and identify ingraft gene expression. Our investigation demonstrated a significant correlation between intragraft TGF-beta 1 mRNA display and renal allograft interstitial fibrosis and chronic allograft nephropathy. In contrast, intragraft expression of mRNA encoding immunoregulatory cytokines, IL-2, IFN-gamma, IL-4, IL-10, or cytotoxic attack molecules, granzyme B and perforin was not a correlate of interstitial fibrosis or chronic allograft nephropathy. Our studies identify, for the first time, a significant association between intragraft TGF-beta 1 mRNA expression and renal allograft interstitial fibrosis, and advance a candidate molecular mechanism for chronic allograft nephropathy.

Base Sequence↗
PubMed1996

Characterization of pituitary adenylate cyclase-activating polypeptide 38 (PACAP38)-, PACAP27-, and vasoactive intestinal peptide-stimulated responses in N1E-115 neuroblastoma cells.

In this study, the effects of three related peptides, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, and vasoactive intestinal peptide (VIP), on cyclic AMP (cAMP) accumulation and intracellular Ca2+ concentration ([Ca2+]i) were compared in N1E-115 cells. PACAP38 and PACAP27 stimulated cAMP accumulation up to 60-fold with EC50 values of 0.54 and 0.067 nM, respectively. The effect of VIP on cAMP accumulation was less potent. The binding of 125I-PACAP27 to intact cells was inhibited by PACAP38 and PACAP27 (IC50 values of 0.44 and 0.55 nM, respectively) but not by VIP. In fura-2-loaded cells, both PACAP38 and PACAP27 increased [Ca2+]i with EC50 values around 10 nM. The interactions of these three peptides with ionomycin, a Ca2+ ionophore, and 4 beta-phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were also determined. Ionomycin increased the cAMP accumulation caused by all three peptides. With low concentrations of PACAP38 or PACAP27, the effect of PMA was inhibitory, whereas at higher concentrations of PACAP (> 1 nM), the effect of PMA was stimulatory. Similar to other agents that elevate cAMP, PACAP38 was an effective stimulator of neurite outgrowth. These results show that (a) PACAP27 and PACAP38 stimulate cAMP accumulation and increase [Ca2+]i through the type I PACAP receptors in N1E-115 cells, (b) ionomycin enhances cAMP accumulation by all three peptides, and (c) activation of protein kinase C has a dose-dependent stimulatory or inhibitory effect on the PACAP38- or PACAP27-stimulated cAMP accumulation.

Calcium↗
PubMed1996

The chemopreventive flavonoid apigenin induces G2/M arrest in keratinocytes.

Apigenin is a plant flavonoid which has been shown to significantly inhibit UV-induced mouse skin tumorigenesis when applied topically, and may represent an alternative sunscreen agent in humans. We have investigated the molecular mechanism(s) by which apigenin inhibits skin tumorigenesis. Initial studies examined the effects of apigenin on the cell cycle. DNA flow cytometric analysis indicated that culturing cells for 24 h in medium containing apigenin induced a G2/M arrest in two mouse skin derived cell lines, C50 and 308, as well as in human HL-60 cells. The G2/M arrest was fully reversible after an additional 24 h in medium without apigenin. We investigated the effects of apigenin on cyclin B1 and p34cdc2, since cyclin B1/p34cdc2 complexes regulate G2/M progression. Western blot and immune complex kinase assays using whole cell lysates from 308 and C50 cells treated for 24 h with 0-70 microM doses of apigenin demonstrated that apigenin treatment did not change the steady-state level of p34cdc2 protein, but did inhibit p34cdc2 H1 kinase activity in 308 cells. Western blot analysis showed that apigenin treatment of C50 cells and 308 cells inhibited the accumulation of cyclin B1 protein in a dose-dependent manner. The apigenin levels detected in cultured keratinocytes were relevant to those detected in epidermal cells of Sencar mice treated with tumor inhibitory doses of apigenin. In conclusion, we present evidence that apigenin induces a reversible G2/M arrest in cultured keratinocytes, the mechanism of which is in part due to inhibition of the mitotic kinase activity of p34cd2, and perturbation of cyclin B1 levels.

Animals↗
PubMed1996

PACAP modulates L-type Ca2+ channel currents in vascular smooth muscle cells: involvement of PKC and PKA.

The effect of pituitary adenylate cyclase activating polypeptide (PACAP) on the L-type Ca2+ channel current (L-channel current) was studied in smooth muscle cells prepared from the rat tail artery. PACAP caused an increase in the amplitude of the L-channel current. The maximal increase (56%) occurred at a PACAP concentration of 1 x 10(-8) M; higher concentrations resulted in a smaller increase. Investigation into the intracellular mechanisms of PACAP action revealed that the increase in L-channel currents was blocked by calphostin C and bisindolylmaleimide IV [protein kinase C (PKC) inhibitors] and mimicked by 4 beta-phorbol 12-myristate 13-acetate (PMA), an activator of PKC. PACAP was also found to cause translocation of PKC, suggesting that the increase in the current by PACAP was due to PKC. In contrast, activation of cAMP-dependent protein kinase (PKA) by 8-bromo-cAMP caused an inhibition of the L-channel current. A high concentration of PACAP (1 x 10(-6) M) had no effect on the L-channel current. The null effect of PACAP on the L-channel current could be converted to an increase by Rp-cAMPs, a cAMP antagonist, and a decrease by calphostin C. PACAP also increased cAMP accumulation. These observations indicate the effect of PACAP on the L-channel current represents the integration of two signaling mechanisms that involve the activation of PKA and PKC.

Animals↗
PubMed1996

Promoting effect of unilateral nephrectomy on urinary bladder carcinogenesis in rats.

The effect of unilateral nephrectomy on the growth of bladder tumors was investigated in male F344 rats treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine(BBN) for 6 weeks by means of macroscopical and microscopical examinations. Unilateral nephrectomy 4 weeks after the initiation of BBN administration significantly increased both the number and the volume of visible tumors per rat as compared with the sham-operated group. Histological examination also revealed that the numbers of papillonodular hyperplasia and cancer per basement membrane were significantly increased by unilateral nephrectomy. The results imply a growth-enhancing effect of unilateral nephrectomy on carcinogenesis of the rat bladder.

Animals↗
PubMed1996

IK independent class III actions of MS-551 compared with sematilide and dofetilide during reperfusion in anaesthetized rats.

1. The antiarrhythmic and haemodynamic effects of three class III antiarrhythmic drugs, MS-551, sematilide and dofetilide, were examined in the coronary artery, ligation-reperfusion model of pentobarbitone-anaesthetized rats, a species deficient in functional cardiac IK. MS-551 is a non-selective potassium channel blocker, while both sematilide and dofetilide are selective delayed rectifier potassium (K) channel (IK) blockers. 2. Before coronary ligation, 3 and 10 mg kg-1 MS-551 decreased the heart rate by 6% (P < 0.01) and 12% (P < 0.01), and increased mean arterial pressure (MAP) by 14% (P < 0.05) and 33% (P < 0.01), respectively. Sematilide at 10 and 30 mg kg-1 also decreased the heart rate by 4% (P < 0.01) and 9% (P < 0.01), respectively, and the higher dose of 30 mg kg-1 decreased MAP by 29% (P < 0.01). Dofetilide, 1 mg kg-1, decreased the heart rate (P < 0.01), but had no significant effect on MAP. 3. The QT interval was increased by 10% (P < 0.01) and 31% (P < 0.01), when 3 and 10 mg kg-1 MS-551 were given. Sematilide and dofetilide had no effect on the QT interval. 4. Immediately after reperfusion, lethal ventricular fibrillation (VF) was induced in 80% of the saline group. MS-551 at 3 and 10 mg kg-1, reduced the incidence of lethal VF to 50% and 20% (P < 0.05). Neither dofetilide 1 mg kg-1 nor sematilide (10 and 30 mg kg-1) decreased the incidence of lethal VF (70%, 80% and 50%, respectively). None of the three drugs had any effect on the occurrence of reperfusion-induced VT or the total incidence of VF. However, 10 mg kg-1 MS-551 delayed the onset of reperfusion-induced VF (27 +/- 5 s compared with 12 +/- 2 s of the control group, P < 0.05). 5. In conclusion, in rats which are deficient in cardiac IK MS-551 prolonged the QT interval and reduced the incidence of sustained VF after reperfusion. Blockade of channels other than IK might participate in the defibrillatory effect of MS-551. Sematilide and dofetilide, which are selective IK blockers, did not increase the QT interval nor did they show antiarrhythmic effects Mechanisms other than K channel block may be involved in the different effects of the three drugs on blood pressure.

Animals↗
PubMed1996

Cloning of a cDNA encoding cellobiose dehydrogenase, a hemoflavoenzyme from Phanerochaete chrysosporium.

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by cellulose-degrading cultures of the wood-degrading basidiomycete Phanerochaete chrysosporium. CDH contains one flavin adenine dinucleotide (FAD) and one heme b per molecule, and it oxidizes cellobiose to cellobionolactone. In this report, a 2.4-kb cDNA encoding CDH was isolated by screening an expression library of P. chrysosporium OGC101 with a CDH-specific polyclonal antibody. The cDNA encodes a 755-amino-acid protein with a predicted mass of 80,115 Da. Sequence analysis suggests that the heme domain is located at the N terminus and that the falvin domain is located at the C terminus. The flavin domain shows a beta 1-alpha A-beta 2 motif for FAD binding and has high sequence similarity to several FAD-dependent enzymes. Little sequence similarity to hemoflavoenzymes is found. CDH binds to cellulose similarly to cellulases. However, little sequence similarity is observed with the conserved cellulose-binding sequences of cellulases. This suggests that CDH might possess a specific sequence for cellulose binding which is different from that of cellulases. Northern (RNA) blot analysis of total RNA from cellulose-, glucose-, and cellobiose-grown P. chrysosporium indicated that CDH mRNA is produced only in cellulose-grown cells. This suggests that CDH expression is regulated at the transcriptional level by either cellulose or one of its degradation products. Southern blot analysis suggests the presence of only a single gene for CDH in P. chrysosporium OGC101.

Amino Acid Sequence↗
PubMed1996

Development of type-specific PCR for typing Pneumocystis carinii f. sp. hominis based on nucleotide sequence variations of internal transcribed spacer region of rRNA genes.

The nucleotide sequence variations in the internal transcribed spacer region 1 (ITS1) and region 2 (ITS2) of rRNA genes were found to be useful for typing Pneumocystis carinii isolates that infect humans. Two types of ITS1 (A and B) and three types of ITS2 (a, b, and c) sequences have been found, and P. carinii isolates are classified based on sequence types of ITS1 and ITS2 as Ax or Bx (where x may be a, b, or c). Type determination has been achieved by sequencing the ITS regions or by reacting the ITS regions amplified by PCR with type-specific oligonucleotide (TSO) probes. However, TSO typing alone does not work on a specimen from an individual who is infected by more than one strain of P. carinii where different ITS1 types are present in the same specimen. In this study, type-specific PCR assays were developed to supplement TSO typing. Type-specific PCR primers were made so that they differ at their 3' ends by the two nucleotides which distinguish type A from type B of ITS1 plus an additional "A" residue at the extreme 3' ends of the primers. These two primers were paired separately with a general primer which anneals to a region downstream from ITS2 to specifically amplify Ax or Bx. The amplified products were then reacted separately with ITS2-specific probes 2-a, 2-b, and 2-c to identify their types.

Base Sequence↗
PubMed1996

Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract.

Analyses of Drosophila cells have revealed that RNA polymerase II is paused in a region 20 to 40 nucleotides downstream from the transcription start site of the hsp70 heat shock gene when the gene is not transcriptionally active. We have developed a cell-free system that reconstitutes this promoter-proximal pausing. The paused polymerase has been detected by monitoring the hyperreactivity of thymines in the transcription bubble toward potassium permanganate. The pattern of permanganate reactivity for the hsp70 promoter in the reconstituted system matches the pattern found on the promoter after it has been introduced back into files by P-element-mediated transposition. Matching patterns of permanganate reactivity are also observed for a non-heat shock promoter, the histone H3 promoter. Further analysis of the hsp70 promoter in the reconstituted system reveals that pausing does not depend on sequence-specific interactions located immediately downstream from the pause site. Sequences upstream from the TATA box influence the recruitment of polymerase rather than the efficiency of pausing. Kinetic analysis indicates that the polymerase rapidly enters the paused state and remains stably in this state for at least 25 min. Further analysis shows that the paused polymerase will initially resume elongation when Sarkosyl is added but loses this capacity within minutes of pausing. Using an alpha-amanitin-resistant polymerase, we provide evidence that promoter-proximal pausing does not require the carboxy-terminal domain of the polymerase.

Amanitins↗
PubMed1996

Essential and nonessential histone H2A variants in Tetrahymena thermophila.

Although variants have been identified for every class of histone, their functions remain unknown. We have been studying the histone H2A variant hv1 in the ciliated protozoan Tetrahymena thermophila. Sequence analysis indicates that hv1 belongs to the H2A.F/Z type of histone variants. On the basis of the high degree of evolutionary conservation of this class of histones, they are proposed to have one or more distinct and essential functions that cannot be performed by their major H2A counterparts. Considerable evidence supports the hypothesis that the hv1 protein in T. thermophila and hv1-like proteins in other eukaryotes are associated with active chromatin. In T. thermophila, simple mass transformation and gene replacement techniques have recently become available. In this report, we demonstrate that either the HTA1 gene or the HTA2 gene, encoding the major H2As, can be completely replaced by disrupted genes in the polyploid, transcriptionally active macronucleus, indicating that neither of the two genes is essential. However, only some of the HTA3 genes encoding hv1 can be replaced by disrupted genes, indicating that the H2A.F/Z type variants have an essential function that cannot be performed by the major H2A genes. Thus, an essential gene in T. thermophila can be defined by the fact that it can be partially, but not completely, eliminated from the polyploid macronucleus. To our knowledge, this study represents the first use of gene disruption technology to study core histone gene function in any organism other than yeast and the first demonstration of an essential gene in T. thermophila using these methods. When a rescuing plasmid carrying a wild-type HTA3 gene was introduced into the T. thermophila cells, the endogenous chromosomal HTA3 could be completely replaced, defining a gene replacement strategy that can be used to analyze the function of essential genes.

Animals↗