Levbid
Tropane alkaloid production and shoot regeneration in hairy and adventitious root cultures of Duboisia myoporoides-D. leichhardtii hybrid.

Yoshimatsu K, Sudo H, Kamada H, Kiuchi F, Kikuchi Y, Sawada J, Shimomura K.

Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences, Ibaraki, Japan. yoshimat nihs.go.jp

Co-culture conditions for Duboisia myoporoides-D. leichhardtii hybrid hairy root induction were investigated using leaf explants and Agrobacterium rhizogenes ATCC 15834. The bacteria density and duration of co-culture greatly affected the induction rate; the highest rate of 50% was obtained when the leaf explants were co-cultured for 2 d with 10(6) bacteria. One hairy root clone that showed the fastest root growth was selected and used for comparison study with adventitious roots cultured with 0.5 mg/l indole-3-acetic acid (IAA). The hairy roots cultured in Murashige and Skoog (MS) liquid medium grew well and yielded much more tropane alkaloids (35 mg/l scopolamine and 17 mg/l hyoscyamine) than adventitious roots cultured in 0.5 mg/l IAA after 6 weeks of culture at 25 degrees C in the dark. The hairy and adventitious roots (2.5 cm) grown in liquid media were divided into 5 parts (each 0.5 cm) along the root axis. Distribution of scopolamine and IAA was then determined by enzyme-linked immunosorbent assay (ELISA). Inverse relationship between contents of scopolamine and IAA was observed in the hairy roots; increase of scopolamine and decrease of IAA were proportional to the distance from the root meristem. In contrast, the contents of scopolamine and IAA were relatively constant in the adventitious roots. In shoot regeneration experiments, the hairy and adventitious root segments (1 cm) were placed onto 1/2 MS solid medium containing various concentrations of IAA and BA cultured at 25 degrees C under 16 h light. In adventitious roots, the shoots regenerated on media containing 6-benzyladenine (BA) (0.5 to 5 mg/l), and 100% regeneration was observed in medium with 0.1 mg/l IAA and 2 mg/l BA. On the other hand, shoot regeneration was only observed in 33% of hairy roots cultured on medium containing 5 mg/l BA.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15305033&dopt=Abstract hyoscyamine Levbid SL



Levbid
Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don.

Vazquez-Flota F, De Carolis E, Alarco AM, De Luca V.

Departement de Sciences Biologiques, Universite de Montreal, Quebec, Canada.

A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of desacetoxyvindoline was purified to homogeneity. Three oligopeptides isolated from a tryptic digest of the purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6 beta-hydroxylase from Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence of all three peptides. Primer extension analysis suggested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5' end of the clone and sequencing of the genomic clone, gD4H-8, confirmed these results. Southern blot analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the significant differences in the sequence of the 3'-UTR between cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydroxylase. The identity of the clone was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4H clone encoded a putative protein of 401 amino acids with a calculated molecular mass of 45.5 kDa and the amino acid sequence showed a high degree of similarity with those of a growing family of 2-oxoglutarate-dependent dioxygenases of plant and fungal origin. The similarity was not restricted to the dioxygenase protein sequences but was also extended to the gene structure and organization since the 205 and 1720 bp introns of d4h were inserted around the same highly conserved amino acid consensus sequences as those for e8 protein, hyoscyamine-6 beta-hydroxylase and ethylene-forming enzyme. These results provide further support that a common ancestral gene is responsible for the appearance of this family of dioxygenases. Hydroxylase assays and RNA blot hybridization studies showed that enzyme activity followed closely the levels of d4h transcripts, occurring predominantly in young leaves and in much lower levels in stems and fruits. In contrast, etiolated seedlings which contained considerable levels of d4h transcripts had almost undetectable hydroxylase activity, whereas exposure of seedlings to light resulted in a rapid increase of enzyme activity without a significant further increase in d4h transcripts over those detected in dark-grown seedlings. These results suggest that the activating effect of light may occur at a point downstream of transcription which remains to be elucidated.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9290645&dopt=Abstract hyoscyamine Levbid SL



Levbid
Use of antimuscarinic toxins to facilitate studies of striatal m4 muscarinic receptors.

Purkerson SL, Potter LT.

Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101, USA.

Striatal m4 muscarinic receptors are important because their blockade controls movement, and they are preferentially located on striatal neurons that project to the internal globus pallidus. The following studies were performed in vitro to provide a basis for using antimuscarinic toxins to study the effects of selective m4 blockade on movement in vivo. Because m4-toxin has limited selectivity alone (102-fold higher affinity for m4 than m1 receptors), m1-toxin was used first to occlude m1 receptors selectively, fully and irreversibly. It blocked 42% of the sites for 1.0 nM 3H-N-methylscopolamine in rat striatal membranes and 43% in sections of cat striatum. m4-Toxin (>500-fold higher affinity for m4 than m2, m3 or m5 receptors) blocked 88% of the residual, non-m1 sites in membranes, showing 64 pmol m4 receptors/g tissue. In comparison, AFDX-116, biperiden, clozapine, gallamine, hexahydrodifenidol, himbacine, R(+)hyoscyamine, methoctramine, pirenzepine, silahexocyclium, trihexyphenidyl and tripitramine did not distinguish m4 from other non-m1 receptors. 3H-Pirenzepine dissociated twice as rapidly from non-m1 as m1 receptors. Autoradiography was used to test the idea that m4 receptors are localized preferentially in the striosomes of the cat striatum. Non-m1 receptors were distributed equally in striosomes and matrix, indicating that striatal neurons with m4 receptors are in both compartments. Thus m1-toxin facilitates studies of m4 receptors by occluding m1 receptors, and m4-toxin is a selective antagonist for residual m4 receptors.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9454818&dopt=Abstract hyoscyamine Levbid SL



Levbid
Post-marketing surveillance using pharmacy-based cohorts: results of a pilot study.

Louik C, Mitchell AA.

Slone Epidemiology Center, Boston University, Boston, Massachusetts, USA.

PURPOSE: To assess the feasibility of recruiting subjects for follow-up studies of drug exposures using pharmacy records but without involving dispensing pharmacists. METHODS: Working with Eckerd Corporation, a large chain pharmacy, we attempted to enroll subjects taking either hyoscyamine (Levsin and others) or dicyclomine (Bentyl and others). Adults who filled prescriptions during the recruitment period were randomly assigned to one of four enrollment approaches that used a script and materials we provided: (1) an introductory phone call from an Eckerd pharmacy technician with an offer of a $5 payment; (2) an introductory phone call with no payment; (3) a questionnaire mailed from Eckerd with introductory letters enclosed and an offer of a $5 payment and (4) the same mailed packet but with no payment offered. Willing subjects responded directly to us; they received a follow-up questionnaire approximately 6 weeks following enrollment. This method also provided limited information about subjects who chose not to enroll, permitting us to assess the representativeness of the study population. RESULTS: The enrollment rates for the four groups were 35, 22, 21 and 17% respectively. Rates of completion of the follow-up questionnaire were 86, 83, 83 and 79% respectively. Participants appeared to be representative of the target population. The differential cost per enrolled subject in each group was $39.25, $50.45, $41.95 and $48.26 respectively. CONCLUSIONS: This method provides an efficient way to create cohorts of users of specific prescription medications, with enrollment and retention rates that compare favorably with other approaches, allows a limited evaluation of representativeness, and is logistically feasible. Copyright 2004 John Wiley & Sons, Ltd.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15372672&dopt=Abstract hyoscyamine Levbid SL



Levbid
Combination therapy in the treatment of persistent nocturnal enuresis.

Cendron M, Klauber G.

Department of Surgery, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA.

OBJECTIVE: To evaluate, in a retrospective study, the response rate of older children to combination therapy using a sustained-release anticholinergic agent, hyoscyamine, and a synthetic analogue of antidiuretic hormone, desmopressin acetate. PATIENTS AND METHOD: Twenty-eight patients (20 males and eight females, aged 9-18 years) diagnosed with nocturnal enuresis were evaluated using a questionnaire, history and physical examination. None had success with single-agent pharmacological therapy. All were begun on 0.375 mg of hyoscyamine and 20 microg of desmopressin intranasally at bedtime. The response rate was monitored at 2 and 4 weeks, and then every 3 months by recording dry nights on a calendar. To improve efficacy, the dosage of medication was adjusted up to 0.750 mg of hyoscyamine and 60 microg of desmopressin. Upon achieving dryness and spontaneous awakening to void, medication doses were tapered. RESULTS: Within 6 months 16 (57%) patients were completely dry and six (21%) were dry at least 80% of nights. Nine patients relapsed during dose tapering and therapy was reinstituted. Presently, 17 (60%) patients are off medication (after a mean of 8 months of medication). Eight patients are still on medication and are dry at least 80% of nights. Combination therapy failed in three patients and they have transferred to a different regimen. None experienced untoward side-effects from the medications. CONCLUSION: Most older children with nocturnal enuresis responded to combination therapy. These children require long-term follow-up and may need medication for up to 6 months because the relapse rate is fairly high. Combination therapy appears safe and reliable in treating nocturnal enuresis in older children who have had no success with other treatment modalities.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9634015&dopt=Abstract hyoscyamine Levbid SL