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M J Palazzolo

Publications and source records attributed to M J Palazzolo.

16 recordsLinked to original sources
PubMed1997

Toxicokinetic evaluation of a selegiline transdermal system in the dog.

The toxicology and toxicokinetics of a selegiline transdermal system (STS) were evaluated in a 3 month dog study of daily 24 h applications of placebo 4, 8, or 12 STSs in 32 male and 32 female beagle dogs. Each STS delivered approximately 5 mg selegiline over 24 h. No drug-related signs of toxicity were noted in any group with respect to clinical observations, dermal effects, body weight, food consumption, hematology, urinalysis data, or ophthalmoscopic or electrocardiographic examinations. Clinical chemistry data revealed no consistent adverse effects except for an increase in alanine aminotransferase in dogs receiving 8 and 12 STSs. Histological evaluation of tissues revealed the presence of pigment in the Kupffer cells of dogs treated with 8 and 12 STSs. There were no pathology findings suggestive of hemolysis or cholestasis. The no-effect level (NOEL) was 4 STSs (2.9 mg kg-1 d-1). There were no degenerative or life-threatening toxic effects up to 12 STSs (8.5 mg kg-1 d-1). Gender-related differences in steady-state plasma levels were not observed. Steady-state plasma concentrations were similar to maximum plasma concentrations obtained in single-dose studies, suggesting that drug accumulation was not evident. Simulation of systemic exposure following oral administration of 16.8 mg kg-1 d-1 from previous toxicology studies indicated that selegiline exposure following 12 STSs is sixfold greater while N-desmethylselegiline, L-amphetamine, and L-methamphetamine exposure is 0.5, 0.15, and 0.14 times the exposure in the oral study. The threefold difference in NOEL between oral and transdermal studies in the dog (0.8 versus 2.9 mg kg-1 d-1) is probably related to greater L-amphetamine and L-methamphetamine exposure following oral administration. The reduction in metabolite formation, relative exposure of selegiline in the dog at the NOEL compared to oral toxicology studies, and margin of safety provided, given that the expected clinical dose is less than the dosage of oral Eldepryl (0.15 mg kg-1 d-1), documents the safety of the selegiline drug substance and indicates that additional toxicologic findings with the STS may not be expected.

Administration, Cutaneous↗
PubMed1997

Computational and biological analysis of 680 kb of DNA sequence from the human 5q31 cytokine gene cluster region.

With the human genome project advancing into what will be a 7- to 10-year DNA sequencing phase, we are presented with the challenge of developing strategies to convert genomic sequence data, as they become available, into biologically meaningful information. We have analyzed 680 kb of noncontiguous DNA sequence from a 1-Mb region of human chromosome 5q31, coupling computational analysis with gene expression studies of tissues isolated from humans as well as from mice containing human YAC transgenes. This genomic interval has been noted previously for containing the cytokine gene cluster and a quantitative trait locus associated with inflammatory diseases. Our analysis identified and verified expression of 16 new genes, as well as 7 previously known genes. Of the total of 23 genes in this region, 78% had similarity matches to sequences in protein databases and 83% had exact expressed sequence tag (EST) database matches. Comparative mapping studies of eight of the new human genes discovered in the 5q31 region revealed that all are located in the syntenic region of mouse chromosome 11q. Our analysis demonstrates an approach for examining human sequence as it is made available from large sequencing programs and has resulted in the discovery of several biomedically important genes, including a cyclin, a transcription factor that is homologous to an oncogene, a protein involved in DNA repair, and several new members of a family of transporter proteins.

Animals↗
PubMed1995

Extent and character of circadian gene expression in Drosophila melanogaster: identification of twenty oscillating mRNAs in the fly head.

BACKGROUND: Although mRNAs expressed with a circadian rhythm have been isolated from many species, the extent and character of circadianly regulated gene expression is unknown for any animal. In Drosophila melanogaster, only the period (per) gene, an essential component of the circadian pacemaker, is known to show rhythmic mRNA expression. Recent work suggests that the encoded Per protein controls its own transcription by an autoregulatory feedback loop. Per might also control the rhythmic expression of other genes to generate circadian behavior and physiology. The goals of this work were to evaluate the extent and character of circadian control of gene expression in Drosophila, and to identify genes dependent on per for circadian expression. RESULTS: A large collection of anonymous, independent cDNA clones was used to screen for transcripts that are rhythmically expressed in the fly head. 20 of the 261 clones tested detected mRNAs with a greater than two-fold daily change in abundance. Three mRNAs were maximally expressed in the morning, whereas 17 mRNAs were most abundant in the evening--when per mRNA is also maximally expressed (but when the flies are inactive). Further analysis of the three 'morning' cDNAs showed that each has a unique dependence on the presence of a light-dark cycle, on timed feeding, and on the function of the per gene for its oscillation. These dependencies were different from those determined for per and for a novel 'evening' gene. Sequence analysis indicated that all but one of the 20 cDNAs identified previously uncloned genes. CONCLUSIONS: Diurnal control of gene expression is a significant but limited phenomenon in the fly head, which involves many uncharacterized genes. Diurnal control is mediated by multiple endogenous and exogenous mechanisms, even at the level of individual genes. A subset of circadianly expressed genes are predominantly or exclusively dependent on per for their rhythmic expression. The per gene can therefore influence the expression of genes other than itself, but for many rhythmically expressed genes, per functions in conjunction with external inputs to control their daily expression patterns.

Amino Acid Sequence↗
PubMed1995

Complete sequence of the bithorax complex of Drosophila.

The bithorax complex (BX-C) of Drosophila, one of two complexes that act as master regulators of the body plan of the fly, is included within a sequence of 338,234 bp (SEQ89E). This paper presents the strategy used in sequencing SEQ89E and an analysis of its open reading frames. The BX-C sequence (BXCALL) contains 314,895 bp obtained by deletion of putative genes that are located at each end of SEQ89E and appear to be functionally unrelated to the BX-C. Only 1.4% of BXCALL codes for the three homeodomain-containing proteins of the complex. Principal findings include a putative ABD-A protein (ABD-AII) larger than a previously known ABD-A protein and a putative glucose transporter-like gene (1521 bp) located at or near the bithoraxoid (bxd), infra-abdominal-2 (iab-2) boundary on the opposite strand relative to that of the homeobox-containing genes.

Animals↗
PubMed1994

Direct sequencing of terminal regions of genomic P1 clones. A general strategy for the design of sequence-tagged site markers.

A method for the preparation of P1 DNA is presented, which allows the direct sequencing of ends of inserts in genomic P1 clones using the Applied Biosystems 373A DNA Sequencer and the Dye Terminator sequencing methodology. We surveyed several common methods of DNA preparation including alkaline lysis, Triton-lysozyme lysis, CsCl density-gradient purification, and a commercial column matrix DNA purification kit manufactured by Qiagen. We found that a modified alkaline lysis preparation of P1 DNA was most successful for generating P1 DNA that could be sequenced directly. We also noted that the host bacterial strain from which the P1 DNA was purified dramatically affected the quality of sequencing templates. The bacterial strains NS3145 and NS3529, in which the Drosophila melanogaster and human P1 genomic libraries are harbored, routinely yielded poor-quality sequencing templates. However, the bacterial strain DH10B routinely yielded P1 DNA that was sequenced successfully. A bacterial mating scheme is presented that exploits gamma delta transposition events to allow the transfer of P1 clones from the library host strain to DH10B. Using either an SP6 or a T7 primer, an average of 350 base pairs of DNA sequence was obtained with an uncalled base frequency of approximately 2%. About 4% of P1 end sequences generated corresponded to unique Drosophila loci present in the Genbank database. These single-pass DNA sequences were used to design sequence-tagged site markers for physical mapping studies in both humans and Drosophila.

Animals↗
PubMed1993

A simple and efficient method for constructing high resolution physical maps.

This paper describes a simple and efficient walking method for constructing high resolution physical maps and discusses its applications to genome analysis. The method is an integration of three strategies: (1) use of a highly redundant library of 3Kb-long subclones; (2) construction of a multidimensional pool from the library; (3) direct application of a PCR (polymerase chain reaction)-based screening technique to the pooled library, with two PCR primers, one from the end of the subcloning vector and the other from the leading edge of the walk. This technique allows not only detection of each overlapping subclone but simultaneous determination of its orientation and the size of its overlap. The end of the subclone with the smallest overlap is sequenced and a primer is designed for the next step in the walk. Iteration of the screening procedure with minimum overlapping subclones results in completion of the high resolution map. Using this method, a 3Kb-resolution map was constructed from an 80Kb region of the bithorax complex of Drosophila melanogaster. The method is general enough to be applicable to DNA from other species, and simple enough to be automated.

Animals↗
PubMed1991

Optimized strategies for sequence-tagged-site selection in genome mapping.

The physical mapping of complex genomes is based on the construction of a genomic library and the determination of the overlaps between the inserts of the mapping clones in order to generate an ordered, cloned representation of nearly all the sequences present in the target genome. Evaluation of the relative efficiency of experimental procedures used to accomplish this goal must minimally include a comparison of the fraction of the genome covered by the ordered arrays (or "contigs"), the average size of the contigs, and the cost, in terms of time and resources, required to generate the map. Sequence-tagged-site (STS) content mapping is one strategy that has been proposed and is being utilized for this type of experiment. This paper describes three STS selection schemes and presents computer simulations of contig-building experiments based on these procedures. The results of these simulations suggest that a nonrandom STS strategy that uses paired probes requires one-third to one-fourth as many STS assays as are required in random and nonpaired approaches, and also results in a map that has both greater genome coverage and a larger average contig size. This strategy promises to reduce the time and cost required to build a high-quality physical map.

Base Sequence↗
PubMed1991

Large scale screen for transposon insertions into cloned genes.

We describe a method of screening for transposon insertions in or near Drosophila loci that correspond to cloned DNA sequences. We mobilize a modified P element transposon that carries a bacterial plasmid origin of replication and a drug-resistance marker. The genomic sequences flanking each transposon insertion site can then be rescued as a plasmid in Escherichia coli. Libraries of such plasmids, representing pools of transposon-mutagenized individuals, are used as hybridization probes against cloned sequences to determine whether a transposon has inserted next to a particular site in the genome. The number of loci that can be screened simultaneously by this procedure is quite large. We have screened an array of cDNA clones representing almost 700 distinct loci against libraries representing 760 mutagenized flies, and we obtained hybridization signals to 7 different cDNAs. Three of these events have been analyzed in detail and represent genuine insertions near genomic sequences that correspond to the cDNAs.

Animals↗
PubMed1991

Transposon-facilitated DNA sequencing.

We describe here a transposon-based DNA sequencing strategy that allows the introduction of sequencing priming sites throughout a target sequence by bacterial mating. A miniplasmid was designed to select against transposon insertions into the vector. Sites of transposon insertion are mapped by the polymerase chain reaction with bacterial overnight cultures providing the templates. A small set of plasmids with transposons spaced several hundred base pairs apart can then be sequenced. Sequencing primers corresponding to the transposon ends allow sequencing in both directions. Thus, the entire sequence of both strands can be easily determined.

Base Sequence↗
PubMed1990

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning.

We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.

Bacteriophage lambda↗
PubMed1989

Use of a new strategy to isolate and characterize 436 Drosophila cDNA clones corresponding to RNAs detected in adult heads but not in early embryos.

We describe a new strategy for producing tissue-specific cDNA libraries and subsequently identifying tissue-specific clones. This method was used to screen for cDNA clones corresponding to RNAs expressed in the Drosophila head that cannot be detected in the early embryo. RNA blots were used to assess the spatial and temporal patterns of expression of these RNAs. The ensemble of 436 head-not-embryo clones identified roughly 700 distinct RNAs that are differentially expressed in the Drosophila head. The RNA expression patterns can be classified into five major categories. it is argued that this ensemble of clones represents a large fraction of all genes differentially expressed in the adult head, but not detected in the early embryo. Many of these genes are likely to encode eye- and nervous system-specific products.

Aging↗
PubMed1987

A family of lambda phage cDNA cloning vectors, lambda SWAJ, allowing the amplification of RNA sequences.

This paper describes the construction and characterization of a family of lambda phage cDNA cloning vectors that allows high-efficiency directional cDNA cloning and selective amplification of either sense or antisense cRNA sequences. These vectors contain several unique restriction sites (EcoRI, XbaI, and SacI) positioned between two specific phage promoters, SP6 and T7. This system facilitates the in vitro preparation of single-stranded (ss) RNA molecules that should be useful in subtractive hybridization and in situ hybridization procedures. Using subtractive hybridization and this vector system, it should be possible to identify sequences present in one cDNA library and not another. In addition, it should be possible to construct subtracted cDNA libraries in these vectors and to generate high specific activity, ss, antisense cRNA probes directly from DNA prepared from the whole subtracted library or from individual clones.

Bacteriophage lambda↗
PubMed1983

The sensitization potential of D & C Yellow No. 11 in guinea pigs.

The certified drug and cosmetic dye substance known as D & C Yellow No. 11 [2-(2-quinolyl)-1,3 indandione] was tested for its dermal sensitization potential in guinea pigs. An induction regimen consisting of a single footpad injection of Complete Freund's Adjuvant diluted with antigen, and an intradermal injection of antigen, produced delayed-type hypersensitivity reactions. A high sensitization frequency was observed with 50 micrograms D & C Yellow No. 11 and the positive control 2,4-dinitrochlorobenzene (DNCB). The reaction severities of both DNCB and 50 micrograms D & C Yellow No. 11 were comparable. A dose-response profile was obtained with D & C Yellow No. 11 employing several concentrations (50, 250 and 500 micrograms/ml). Histologic examination of selected skin sites demonstrated a cellular inflammatory response which was consistent with delayed-type hypersensitivity.

Animals↗
PubMed1982

Characterization and localization of adenosine 3':5'-monophosphate-binding proteins in the nervous system of Aplysia.

Earlier work in Aplysia californica has indicated that sensitization of the gill reflex, a simple form of learning, is produced by cAMP-dependent protein phosphorylation which regulates the flux of ions in sensory neurons of the abdominal ganglion. These changes in ion flux result in the enhanced release of neurotransmitter from synapses of the sensory neurons which, in turn, mediate the behavior. Because it can be presumed that protein phosphorylation regulates the functioning of ion channel proteins, we have characterized cAMP-binding proteins photoaffinity labeled with 8-N3-cAMP and have found that, unlike other tissues, the nervous system contains a great variety of binding species. Also unlike other tissues, several of the binding proteins in neurons are associated with membrane, and these components are concentrated in fractions enriched in nerve endings. Selectivity of phosphorylation, not only between substrates in cytosol and membrane but also between different regions of the cell, is thus possible because of the variety of cAMP-binding proteins in neurons. We think that these membrane-associated binding proteins are the most likely candidates for the regulatory subunits of the cAMP-dependent kinases that control the functioning of ion channel proteins at the synapse.

Animals↗