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Biomedical subjects

J Day

Publications and source records attributed to J Day.

At least 19 recordsLinked to original sources
PubMed2000

The refined structure of canavalin from jack bean in two crystal forms at 2.1 and 2.0 A resolution.

The structure of canavalin was refined to 2.1 and 2.0 A resolution in cubic and hexagonal crystals of space group P2(1)3 and P6(3), respectively. The threefold molecular symmetry is expressed in the symmetry of both crystals, where each identical subunit is an asymmetric unit. The canavalin subunit consists of two very similar domains, each comprised of a core subdomain having Swiss-roll topology with a loop subdomain that contains helices. The refined canavalin models resolved the discrepancy in amino-acid registers of the secondary-structural elements compared with phaseolin. The presence of strand Z in both domains of canavalin was confirmed and a new helix in the loop between strands A and B of each domain was observed. The models were analyzed in terms of the duplicated vicilin domains. Three strictly conserved residues, two glycines and a proline, were identified. The similarity between entire vicilin molecules is greater than that between separate domains of canavalin and phaseolin. Homology modeling of the sucrose-binding protein (SBP) from soybean showed a plausible trimeric assembly of subunits similar to that of vicilins.

Amino Acid Sequence↗
PubMed1999

Universal DNA microarray method for multiplex detection of low abundance point mutations.

Cancers arise from the accumulation of multiple mutations in genes regulating cellular growth and differentiation. Identification of such mutations in numerous genes represents a significant challenge in genetic analysis, particularly when the majority of DNA in a tumor sample is from wild-type stroma. To overcome these difficulties, we have developed a new type of DNA microchip that combines polymerase chain reaction/ligase detection reaction (PCR/LDR) with "zip-code" hybridization. Suitably designed allele-specific LDR primers become covalently ligated to adjacent fluorescently labeled primers if and only if a mutation is present. The allele-specific LDR primers contain on their 5'-ends "zip-code complements" that are used to direct LDR products to specific zip-code addresses attached covalently to a three-dimensional gel-matrix array. Since zip-codes have no homology to either the target sequence or to other sequences in the genome, false signals due to mismatch hybridizations are not detected. The zip-code sequences remain constant and their complements can be appended to any set of LDR primers, making our zip-code arrays universal. Using the K- ras gene as a model system, multiplex PCR/LDR followed by hybridization to prototype 3x3 zip-code arrays correctly identified all mutations in tumor and cell line DNA. Mutations present at less than one per cent of the wild-type DNA level could be distinguished. Universal arrays may be used to rapidly detect low abundance mutations in any gene of interest.

Alleles↗
PubMed1999

Multiplex PCR/LDR for detection of K-ras mutations in primary colon tumors.

Point mutations in codons 12, 13, and 61 of the K-ras gene occur early in the development of colorectal cancer and are preserved throughout the course of tumor progression. These mutations can serve as biomarkers for shed or circulating tumor cells and may be useful for diagnosis of early, curable tumors and for staging of advanced cancers. We have developed a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) method which identifies all 19 possible single-base mutations in K-ras codons 12, 13, and 61, with a sensitivity of 1 in 500 wild-type sequences. In a blinded study, 144 paraffin-embedded archival colon carcinomas were microdissected and K-ras mutations determined by both dideoxy-sequencing and multiplex PCR/LDR. Results were concordant for 134 samples. The ten discordant samples were re-evaluated using higher sensitivity uniplex PCR/LDR, and the original multiplex PCR/LDR result was confirmed in nine of these ten cases. Multiplex PCR/LDR was able to identify mutations in solid tumors or paraffin-embedded tissues containing a majority of wild-type stromal cells, with or without microdissection. The technique is well suited for large scale studies and for analysis of clinical samples containing a minority population of mutated cells.

Colorectal Neoplasms↗
PubMed1999

Conditions in rural Nepal for which depot-medroxyprogesterone acetate initiation is not recommended: implications for community-based service delivery.

The presence of medical conditions that might affect the use of depot-medroxyprogesterone acetate (DMPA) as a contraceptive method was assessed in a rural district in Nepal. A general health survey was conducted in nonpregnant and noncontracepting women aged 15-44 years to determine the presence of any health problems. The survey included a general assessment interview by nonphysicians, followed by formal medical histories and physical exams by female gynecologists. Possible pregnancy (nine cases) and abnormal uterine bleeding (one case) were the only conditions identified in which DMPA should not be used, based on the World Health Organization (WHO) Medical Eligibility Criteria for Contraceptive Use. Five additional cases of cardiovascular problems, in which DMPA initiation is not usually recommended, were also detected. Recently developed checklists based on the WHO criteria for DMPA use would have identified all of these health conditions. This checklist would allow the safe delivery of DMPA at the community health worker level, and increase the availability and accessibility of DMPA in rural Nepal.

Adolescent↗
PubMed1999

Pros and cons of the use of antihistamines in managing allergic rhinitis.

Histamine H1 -receptor antagonists are generally considered first-line therapy for the management of allergic rhinitis. Other than histamine antagonism, several of the second-generation antihistamines have also shown inhibitory effects on chemical mediators of inflammation such as leukotrienes, prostaglandins, and kinins. This article reviews the pharmacology, clinical use, and side effect profiles of the commonly used H1 -receptor antagonists.

Histamine H1 Antagonists↗
PubMed1999

Structure of orthorhombic crystals of beef liver catalase.

The growth mechanisms and physical properties of the orthorhombic crystal form of beef liver catalase were investigated using in situ atomic force microscopy (AFM). It was observed that the crystals grow in the <001> direction by an unusual progression of sequential two-dimensional nuclei of half unit-cell layers corresponding to the 'bottoms' and 'tops' of unit cells. These were easily discriminated by their alternating asymmetric shapes and their strong growth-rate anisotropy. This pattern has not previously been observed with other macromolecular crystals. Orthorhombic beef liver catalase crystals exhibit an extremely high defect density and incorporate great numbers of misoriented microcrystals, revealed intact by etching experiments, which may explain their marginal diffraction properties. To facilitate interpretation of AFM results in terms of intermolecular interactions, the structure of the orthorhombic crystals, having an entire tetramer of the enzyme as the asymmetric unit, was solved by molecular replacement using a model derived from a trigonal crystal form. It was subsequently refined by conventional techniques. Although the packing of molecules in the two unit cells was substantially different, with very few exceptions no significant differences in the molecular structures were observed. In addition, no statistically significant deviation from ideal 222 molecular symmetry appeared within the tetramer. The packing of molecules in the crystal revealed by X-ray analysis explained in a satisfying way the process of crystal growth revealed by AFM.

Animals↗
PubMed1998

Effects of nicotine on methadone self-administration in humans.

The effects of nicotine abstinence, ad libitum smoking, and 0, 2, and 4 mg nicotine gum on methadone self-administration were investigated. Five methadone-maintained patients with a history of smoking (18-30 cigarettes/day) were recruited as subjects. Upon arrival expired carbon monoxide levels were measured to confirm self-reported abstinence of 10-12 h. At 30 min prior to the methadone self-administration session, two response options were concurrently available. When a 64-button press requirement (FR64) was completed, 10 ml of 0.054 mg/ml methadone solution, or vehicle, was delivered. Immediately following, and 30, 60, 90, and 120 min after the self-administration session, expired carbon monoxide levels and typical symptoms of nicotine withdrawal were assessed. Relative to abstinence, subjects consumed more methadone following the 4-mg nicotine gum and ad libitum smoking conditions. Ratings of cigarette craving were significantly less following ad libitum smoking or administration of 4-mg nicotine gum, than following abstinence. Implications for understanding opioid and nicotine interactions are discussed.

Adult↗
PubMed1998

Refined structure of satellite tobacco mosaic virus at 1.8 A resolution.

The molecular structure of satellite tobacco mosaic virus (STMV) has been refined to 1.8 A resolution using X-ray diffraction data collected from crystals grown in microgravity. The final R value was 0.179 and Rfree was 0.184 for 219,086 independent reflections. The final model of the asymmetric unit contained amino acid residues 13 to 159 of a coat protein monomer, 21 nucleotides, a sulfate ion, and 168 water molecules. The nucleotides were visualized as 30 helical segments of nine base-pairs with an additional base stacked at each 3' end, plus a "free" nucleotide, not belonging to the helical segments, but firmly bound by the protein. Sulfate ions are located exactly on 5-fold axes and each is coordinated by ten asparagine side-chains. Of the 10,080 structural waters, 168 per asymmetric unit, about 20% serve to bridge the macromolecular components at protein-protein and protein-nucleic acid interfaces. Binding of RNA to the protein involves some salt linkages, particularly to the phosphate of the free nucleotide, but the major contribution is from an intricate network of hydrogen bonds. There are numerous water molecules in the RNA-protein interface, many serving as intermediate hydrogen bond bridges. The sugar-phosphate backbone contributes most of the donors and acceptors for the RNA. The helical RNA conformation is nearest that of A form DNA. The central region of a helical segment is most extensively involved in contacts with protein, and exhibits low thermal parameters which increase dramatically toward the ends. The visible RNA represents approximately 59% of the total nucleic acid in the virion and is derived from the single-stranded genome, which has folded upon itself to form helical segments. Linking of the helices and the free nucleotides in a contiguous and efficient manner severely restricts the disposition of the remaining, unseen nucleic acid. Using the remaining nucleotides it is possible to fold the RNA according to motifs that provide a periodic distribution of RNA structural elements compatible with the icosahedrally symmetrical arrangement seen in the crystallographic structure. The intimate relationship between protein and nucleic acid in STMV suggests an assembly pathway based on the cooperative and coordinated co-condensation of RNA with capsid protein dimers.

Amino Acid Sequence↗
PubMed1998

Comparison of the efficacy of budesonide and fluticasone propionate aqueous nasal spray for once daily treatment of perennial allergic rhinitis.

BACKGROUND: Intranasal corticosteroids, such as budesonide and fluticasone propionate, are widely prescribed in the treatment of perennial allergic rhinitis. Once daily budesonide dry powder and fluticasone propionate aqueous suspension have been found to provide similar efficacy in controlling symptoms of perennial allergic rhinitis. OBJECTIVE: The purpose of this study was to assess the efficacy and safety of treatment with once daily budesonide aqueous nasal spray. METHODS: This study involved a multicenter, blinded, randomized, parallel-group, placebo-controlled trial of adults with perrenial allergic rhinitis. Patients (n = 273) recorded daily nasal symptoms for 8 to 14 days (baseline) and 6 weeks (treatment). RESULTS: Budesonide decreased combined symptoms to a significantly greater extent than did fluticasone (P =.03); both treatments significantly decreased mean combined nasal symptoms scores compared with placebo. Of the 3 nasal symptoms assessed (ie, nasal blockage, runny nose, and sneezing), nasal blockage was significantly (P =. 009) more decreased with budesonide compared with fluticasone. Both treatments also significantly improved runny nose and sneezing compared with placebo. Improvement in combined nasal symptom scores of the budesonide-treated group reached statistical significance within 36 hours compared with placebo (P =.01); in those patients treated with fluticasone, significant improvement compared with placebo was first observed within 60 hours. Adverse events were mild and transient. CONCLUSIONS: Once daily budesonide aqueous nasal spray, 256 microgram, was significantly better in controlling the symptoms of perrenial allergic rhinitis than once daily fluticasone propionate, 200 microgram, especially nasal blockage. Both treatments were superior to placebo. Budesonide may have a faster onset of action than fluticasone.

Administration, Inhalation↗
PubMed1998

The neurosteroid pregnenolone sulfate infused into the nucleus basalis increases both acetylcholine release in the frontal cortex or amygdala and spatial memory.

The effects of an infusion (5 ng) of the neurosteroid pregnenolone sulfate into the nucleus basalis magnocellularis on acetylcholine release in the frontoparietal cortex and basolateral amygdala were evaluated during the 130 min post-injection in male Sprague-Dawley rats using in vivo microdialysis coupled "on line" with high performance liquid chromatography detection. One week later, the same animals were tested for spatial memory after another infusion of pregnenolone sulfate (5 ng) into the nucleus basalis. Results show that pregnenolone sulfate enhanced acetylcholine release by more than 50% of baseline concentrations in the two structures relative to a control injection. The duration of this effect was longer in cortex (130 min) than in amygdala (30 min). Furthermore, pregnenolone sulfate improved memory performance in a task based upon spatial recognition of a familiar environment. A significant positive correlation (r=0.49) was found between the recognition score in the spatial memory test and the levels of acetylcholine release in the frontoparietal cortex but not in the basolateral amygdala. Therefore, our results suggest that the nucleus basalis magnocellularis-cortical pathway could be in part responsible for the promnesic effect of pregnenolone sulfate. This neurosteroid acts as a negative modulator of the GABA(A) receptor complex and positively modulates the N-methyl-D-aspartate receptor, possibly resulting in a global stimulatory effect on central cholinergic neurotransmission.

Acetylcholine↗
PubMed1997

The crystallographic structure of the subtilisin protease from Penicillium cyclopium.

The major extracellular protease from the fungus Pencillium cyclopium was crystallized in the presence of p-phenylmethanesulfonyl fluoride (PMSF) and investigated by X-ray diffraction analysis. It was subsequently cloned and the amino acid sequence deduced from its cDNA. Although the sequence is only 49% identical to that of proteinase K of Tritirachium album, the three-dimensional structures of the two proteases are virtually identical. The model for P. cyclopium protease was refined by simulated annealing to an R of 18% at 1.7 A resolution. The greatest variation from the proteinase K polypeptide is in loop 114-134 and is due to the absence of a disulfide bridge in the P. cyclopium protease that is present in proteinase K. A difference was also observed in the orientation of the histidine in the catalytic triad, though this could be due to the presence of PMSF at the active site. The coordination geometry of the strongly bound calcium in the P. cyclopium protease is octahedral and uses some different protein ligands than does proteinase K. In the protease from P. cyclopium there is no cysteine near the active site, nor is there a second calcium binding site as is found in proteinase K, suggesting that neither is important to catalytic activity.

Amino Acid Sequence↗
PubMed1997

The open conformation of a Pseudomonas lipase.

BACKGROUND: . The interfacial activation of lipases results primarily from conformational changes in the enzymes which expose the active site and provide a hydrophobic surface for interaction with the lipid substrate. Comparison of the crystallization conditions used and the structures observed for a variety of lipases suggests that the enzyme conformation is dependent on solution conditions. Pseudomonas cepacia lipase (PCL) was crystallized in conditions from which the open, active conformation of the enzyme was expected. Its three-dimensional structure was determined independently in three different laboratories and was compared with the previously reported closed conformations of the closely related lipases from Pseudomonas glumae (PGL) and Chromobacterium viscosum (CVL). These structures provide new insights into the function of this commercially important family of lipases. RESULTS: . The three independent structures of PCL superimpose with only small differences in the mainchain conformations. As expected, the observed conformation reveals a catalytic site exposed to the solvent. Superposition of PCL with the PGL and CVL structures indicates that the rearrangement from the closed to the open conformation involves three loops. The largest movement involves a 40 residue stretch, within which a helical segment moves to afford access to the catalytic site. A hydrophobic cleft that is presumed to be the lipid binding site is formed around the active site. CONCLUSIONS: . The interfacial activation of Pseudomonas lipases involves conformational rearrangements of surface loops and appears to conform to models of activation deduced from the structures of fungal and mammalian lipases. Factors controlling the conformational rearrangement are not understood, but a comparison of crystallization conditions and observed conformation suggests that the conformation of the protein is determined by the solution conditions, perhaps by the dielectric constant.

Amino Acid Sequence↗
PubMed1997

Serum free prostate-specific antigen in the diagnosis of prostate cancer.

OBJECTIVE: To determine the value of the ratio of free prostate-specific antigen (fPSA) to total PSA (tPSA) in the diagnosis of benign prostatic hyperplasia (BPH) and prostate cancer in a cohort of patients undergoing prostatic transrectal ultrasonography (TRUS). PATIENTS AND METHODS: The study comprised 153 patients (99 with BPH and 54 with prostate cancer) undergoing diagnostic TRUS of the prostate. Patients with a tPSA of > 30 ng/mL were excluded from analysis. Free PSA was assayed using an immunoassay specific for unbound PSA (CanAg Diagnostics, Sweden). Total PSA was measured using the HybriTech Tandem-R PSA immunoradiometric assay in routine clinical use and this estimate was validated using the CanAg tPSA assay. RESULTS: The measurements of tPSA from both assay systems correlated closely. The f/tPSA ratios in patients with prostate cancer were significantly lower than in those with BPH (median values 0.152 and 0.2, respectively, P<0.01). In patients with prostate cancer, the median f/t PSA levels apparently declined with increasing tPSA levels but in those with BPH, the levels of tPSA were not significantly associated with the f/tPSA ratio; the ratios did not vary significantly with age in either group. A f/tPSA ratio at a threshold of 0.16 had positive and negative predictive values of 44% and 74%, respectively; the corresponding values for a tPSA of > 4 ng/mL were 30% and 52%. CONCLUSION: The f/tPSA ratio differs significantly between patients with BPH and cancer but because there is a considerable overlap of f/tPSA ratios between the groups, f/tPSA values alone were not sufficiently specific to be used as a single diagnostic test.

Biopsy↗
PubMed1997

Clinical, biochemical, and molecular investigations of a genetic isolate of growth hormone insensitivity (Laron's syndrome).

We have characterized the GH receptor mutation that is responsible for extreme short stature and GH insensitivity in a Bahamian genetic isolate. Heights of affected individuals ranged from -4.0 to -6.3 SD. Like others with Laron's syndrome, they had normal to high serum GH concentrations and low serum insulin-like growth factor I concentrations. Circulating levels of GH-binding protein activity were below limits of detection. Amplification of exons 2-7 and screening with single strand conformational polymorphism analysis located an abnormality in exon 7. Sequencing identified homozygosity for a C to T transition in the third position of codon 236. Reverse transcription and PCR amplification of complementary DNA from lymphocytes showed that this same sense mutation generated a new splice donor site 63 bp 5' to the normal exon 7 splice site. This novel site was used to the exclusion of the normal site in homozygotes. Both normal and variant messenger ribonucleic acid species were detected in heterozygotes. The predicted protein lacks 21 amino acids, including those defining the WS-like motif of the GH receptor extracellular domain. The high frequency of Laron's syndrome in this isolated island population probably reflects the introduction of the G236 splice mutation by a settler early in the 300-yr history of English settlement.

Adolescent↗
PubMed1997

Population genetics of the D4S139, D10S28, D17S74 and D17S79 VNTR loci among Asian, black, Caucasian, Hispanic and Native American populations from Seattle.

We studied four variable number of tandem repeat (VNTR) loci (D4S139, D10S28, D17S74, and D17S79) in five ethnic populations from the Seattle metropolitan area. DNA samples purified from randomly chosen individuals were digested with Hae III or Hinf I and probed with pH30, for D4S139; TBQ7 for D10S28; pCMM86 for D17S74 and pAC256 for D17S79. The allele frequencies, expected Hardy-Weinberg values, observed heterozygosities and genetic distances among the populations were obtained for all these loci. D4S139 restriction fragment lengths (RFLs) varied in size from 1.4 to 22 kilobase pairs (kbp). The observed heterozygosities (H) varied from 84% in Native American populations to 94% among Blacks. D10S28 RFLs varied in size from 650 base pairs (bp) to 10.1 kbp. H varied from 90% in Native Americans to 96% in Caucasians and Hispanics. D17S74 RFLs varied in size from 782 bp to 9.3 kbp. H varied from 87% in Asians to 92% among Blacks. D17S79 RFLs varied in size from 400 bp to 3 kbp. H varied from 87% in Hispanics to 95% in the Black population. The frequencies of genotypes of the loci conformed to Hardy-Weinberg equilibrium with the exception of the D17S79 in Hispanics and Native Americans. The genetic distances between the populations were also determined.

Alleles↗