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PubMed1996

The human anion transport protein, band 3, contains a CD36-like binding domain for Plasmodium falciparum-infected erythrocytes.

Epitope mapping of a murine monoclonal antibody (mAb), 5H12, prepared against live Plasmodium falciparum-infected red blood cells indicated that the epitope consisted of amino acid residues 474-487 of the human anion transport protein, band 3. mAb 5H12 enhanced cytoadherence, but inhibited the CD36-like mediated rosetting. A synthetic peptide based on the sequence of the epitope (FSFCETNGLE) blocked both rosetting and cytoadherence, suggesting that this amino acid sequence may form the CD36-like receptor. The CD36-like region of band 3 was antigenically distinct from platelet or endothelial CD36.

Amino Acid Sequence↗

PubMed1991

Characterization of cDNA clones encoding rabbit and human serum paraoxonase: the mature protein retains its signal sequence.

Serum paraoxonase hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. High serum paraoxonase levels appear to protect against the neurotoxic effects of organophosphorus substrates of this enzyme [Costa et al. (1990) Toxicol. Appl. Pharmacol. 103, 66-76]. The amino acid sequence accounting for 42% of rabbit paraoxonase was determined by (1) gas-phase sequencing of the intact protein and (2) peptide fragments from lysine and arginine digests. From these data, two oligonucleotide probes were synthesized and used to screen a rabbit liver cDNA library. A clone was isolated and sequenced, and contained a 1294-bp insert encoding an open reading frame of 359 amino acids. Northern blot hybridization with RNA isolated from various rabbit tissues indicated that paraoxonase mRNA is synthesized predominately, if not exclusively, in the liver. Southern blot experiments suggested that rabbit paraoxonase is coded by a single gene and is not a family member of closely related genes. Human paraoxonase clones were isolated from a liver cDNA library by using the rabbit cDNA as a hybridization probe. Inserts from three of the longest clones were sequenced, and one full-length clone contained an open reading frame encoding 355 amino acids, four less than the rabbit paraoxonase protein. Each of the human clones appeared to be polyadenylated at a different site, consistent with the absence of the canonical polyadenylation signal sequence. Of potential significance with respect to the paraoxonase polymorphism, the derived amino acid sequence from one of the partial human cDNA clones differed at two positions from the full-length clone. Amino-terminal sequences derived from purified rabbit and human paraoxonase proteins suggested that the signal sequence is retained, with the exception of the initiator methionine residue [Furlong et al. (1991) Biochemistry (preceding paper in this issue)]. Characterization of the rabbit and human paraoxonase cDNA clones confirms that the signal sequences are not processed, except for the N-terminal methionine residue. The rabbit and human cDNA clones demonstrate striking nucleotide and deduced amino acid similarities (greater than 85%), suggesting an important metabolic role and constraints on the evolution of this protein.

Adult↗

PubMed2005

Definition of anti-tyrosinase MAb T311 linear determinant by proteome-based similarity analysis.

Using non-self discrimination as a driving force in generating peptide immunogenicity, we have developed a computer-assisted proteomic analysis in order to identify the protein antigenic regions that have evoked humoral response. The purpose of this study was to further validate the computational analysis for melanoma-associated antigens and, at the same time, to assess the efficacy of the methodology in defining antigenic regions of autoantigens associated to autoimmune diseases. To achieve this two-fold objective, we have examined the enzyme tyrosinase, a protein that represents an important autoantigen in patients with vitiligo or melanoma. Here, we report that the antigenic linear determinant of the monoclonal antibody (Mab) T311 raised against the melanoma/vitiligo tyrosinase autoantigen is located in the low similarity 15-mer amino acid sequence tyrosinase 233-247 IPYWDWRDAEKCDIC, within the fragment 237-247. These data confirm non-similarity to the host proteome as a factor that participates in shaping peptide immune reactivity and may be a first step towards designing tyrosinase antigenic peptides to be used for (i) direct neutralization of harmful melanocytes-attacking autoantibodies in vitiligo, or (ii) production of antibodies against tyrosinase-positive melanomas. Moreover tyrosinase peptide antigens might be used as key tools in studying the boundaries between self-tolerance and autoimmunity phenomena.

Amino Acid Sequence↗

PubMed2006

Saturating representation of loop conformational fragments in structure databanks.

BACKGROUND: Short fragments of proteins are fundamental starting points in various structure prediction applications, such as in fragment based loop modeling methods but also in various full structure build-up procedures. The applicability and performance of these approaches depend on the availability of short fragments in structure databanks. RESULTS: We studied the representation of protein loop fragments up to 14 residues in length. All possible query fragments found in sequence databases (Sequence Space) were clustered and cross referenced with available structural fragments in Protein Data Bank (Structure Space). We found that the expansion of PDB in the last few years resulted in a dense coverage of loop conformational fragments. For each loops of length 8 in the current Sequence Space there is at least one loop in Structure Space with 50% or higher sequence identity. By correlating sequence and structure clusters of loops we found that a 50% sequence identity generally guarantees structural similarity. These percentages of coverage at 50% sequence cutoff drop to 96, 94, 68, 53, 33 and 13% for loops of length 9, 10, 11, 12, 13, and 14, respectively. There is not a single loop in the current Sequence Space at any length up to 14 residues that is not matched with a conformational segment that shares at least 20% sequence identity. This minimum observed identity is 40% for loops of 12 residues or shorter and is as high as 50% for 10 residue or shorter loops. We also assessed the impact of rapidly growing sequence databanks on the estimated number of new loop conformations and found that while the number of sequentially unique sequence segments increased about six folds during the last five years there are almost no unique conformational segments among these up to 12 residues long fragments. CONCLUSION: The results suggest that fragment based prediction approaches are not limited any more by the completeness of fragments in databanks but rather by the effective scoring and search algorithms to locate them. The current favorable coverage and trends observed will be further accentuated with the progress of Protein Structure Initiative that targets new protein folds and ultimately aims at providing an exhaustive coverage of the structure space.

Amino Acid Sequence↗

PubMed2005

Impact of body mass index values on sperm quantity and quality.

Body mass index (BMI) has been demonstrated to affect female fertility; however, little information is available on the impact of BMI on male fertility or semen parameters. Therefore, the study objective was to determine the relationship between BMI and semen parameters, including sperm chromatin integrity. We analyzed data on semen samples from 520 men who were grouped based upon calculated BMI values (normal, 20-24 kg/m(2); overweight, 25-30 kg/m(2); obese, >30 kg/m(2)). The data collected included patient height and weight, semen volume, sperm concentration, percent sperm motility, percent sperm morphology (normal forms), and sperm chromatin integrity (DNA fragmentation index [DFI]). Data were analyzed by regression analysis and analysis of variance (ANOVA) with Tukey's test for multiple pairwise comparisons. The overall BMI mean (+/-SEM) was 27.5 (+/-0.49) kg/m(2). Linear regression revealed a significant (P < .05) and negative relationship between BMI and the total number of normal-motile sperm cells. ANOVA revealed a significant difference (P < .05) in the total number of normal-motile sperm cells among the different BMI groups. The number of normal-motile sperm cells per BMI group was as follows: normal, 18.6 x 10(6); overweight, 3.6 x 10(6); and obese, (0.7) x 10(6). All multiple pairwise comparisons were found to be significantly (P < .05) different. The overall DFI mean (+/-SEM) was 24.7 (+/-2.57). Linear regression revealed a significant (P < .05) and positive relation between BMI and DFI. Men presenting with a BMI greater than 25 kg/m(2) have fewer chromatin-intact normal-motile sperm cells per ejaculate. Therefore, to ensure maximum fertility potential, patients may be advised to reduce body weight.

Adult↗

PubMed1988

[Domain organization of alpha-actinin from the rabbit skeletal muscle].

Limited trypsinolysis of native alpha-actinin by trypsin was carried out. This procedure resulted in the formation of two large fragments with Mr of 30 and 70 kD which cover almost the whole subunit of alpha-actinin. Using the sedimentation equilibrium method, it was demonstrated that the bisubunit structure of alpha-actinin is provided for by C-terminal fragments of the subunits. Data from circular dichroism analysis suggest that the fragments formed are independent structural units, i.e., domains.

Actinin↗

PubMed1993

Automated fluorescence-based restriction fragment analysis.

We describe a procedure for the automated analysis of DNA restriction fragments in an agarose gel matrix. The procedure is based upon simultaneously digesting DNA with restriction enzymes and ligating a fluorochrome-labeled linker to the cut ends. The fluorochrome-labeled restriction fragments are loaded on a horizontal agarose gel scanning system (Applied Biosystems Model 362 GENE SCANNER Fluorescent Fragment Analyzer) for electrophoresis and data analysis. Mobility parameters for DNA fragment migration and detection sensitivity for the instrument are described. In addition, we demonstrate some unique applications, including electrophoresis and fragment size conversion of up to 72 cosmid restriction digests in 4 h, applicability of the system for subsequent DNA transfers and hybridizations, utility for establishing overlapping restriction maps of cosmids, and discrimination of restriction digest specific fragment ends following a double-digest protocol.

Base Sequence↗

PubMed1991

Metabolism during normal, fragmented, and recovery sleep.

Average metabolic data (O2 uptake and CO2 output) were obtained for each 3-min period during consecutive nights of normal, experimentally fragmented, and recovery sleep in a group of 12 normal young adult males. Naturally occurring arousals and awakenings resulted in a characteristic increase in metabolism on the baseline night. The placement of brief frequent experimental arousals on the following night resulted in significantly increased metabolism throughout the night and significantly decreased sleep restoration as measured by morning performance, mood, and alertness tests, even though total sleep time was minimally reduced. Metabolic variables were significantly decreased compared with baseline on the nondisturbed recovery night that followed the sleep fragmentation night. The data cannot be used to infer that increased metabolism during sleep causes nonrestorative sleep, but the direction and time course of metabolic change accompanying arousal are consistent with that hypothesis.

Adolescent↗

PubMed1987

Selective covalent labeling of cysteines in bovine serum albumin and in hepatoma tissue culture cell glucocorticoid receptors by dexamethasone 21-mesylate.

The specificity of protein labeling by an affinity label of glucocorticoid receptors, dexamethasone 21-mesylate (Dex-Mes), was investigated using bovine serum albumin (BSA) as a model. During the early stages of [3H]Dex-Mes labeling at pH 8.8, approximately 90% of the covalent bond formation occurred at the one non-oxidized cysteine (Cys-34) of BSA. The nonspecific labeling was equally distributed over the rest of the BSA molecule. [3H]Dex-Mes labeling of Cys-34 was totally, and specifically inhibited by nearly stoichiometric amounts of the thiol-specific reagent methyl methanethiolsulfonate (MMTS). Thus both Dex-Mes and MMTS appear to react very selectively with thiols under our conditions. In reactions with hepatoma tissue culture (HTC) cell glucocorticoid receptors, MMTS was equally efficient in preventing [3H]dexamethasone binding to receptors and [3H]Dex-Mes labeling of the 98-kDa receptor protein. These results indicate that Dex-Mes labeling of the glucocorticoid receptor involves covalent reaction with at least one cysteine in the steroid binding site of the receptor. Small (approximately 1600-dalton) fragments of the [3H]Dex-Mes-labeled 98-kDa receptor were generated by limit proteolysis with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease under denaturing conditions. Data from these fragments on 15% sodium dodecyl sulfate-polyacrylamide gels were consistent with all of the covalent [3H] Dex-Mes being located on one or a few cysteines in one approximately 15-residue stretch of the receptor. Further studies revealed no differences in the limit protease digestion patterns of activated and unactivated [3H]Dex-Mes-labeled receptors with trypsin, chymotrypsin, or V8 protease under denaturing conditions. These data suggest that activation does not cause any major covalent modifications of the amino acids immediately surrounding the affinity-labeled cysteine(s) of the steroid binding site.

Affinity Labels↗

PubMed1987

Expression of three genes for elongation factor 1 alpha during morphogenesis of Mucor racemosus.

Three genes, TEF-1, -2, and -3, encode elongation factor 1 alpha in Mucor racemosus. Neutral and alkaline S1 nuclease analyses revealed that the genetic organization is unique for each of the genes. The number and size of the intervening sequences vary in these closely related genes, which suggests that complex genetic rearrangements gave rise to the elongation factor 1 alpha gene family. Nucleotide sequence data from restriction fragments isolated from the 5' and 3' ends of TEF-2 and -3 confirmed the presence of a second intervening sequence in these genes. These data along with S1 nuclease mapping revealed a region at the 3' end of the three genes which was predicted to be transcribed but untranslated. Unique oligonucleotides containing 19 bases were synthesized to hybridize to this unique trailer region in the elongation factor 1 alpha transcripts. These oligonucleotides were used as probes in standard Northern analysis of RNA purified from M. racemosus cells of several morphological types. It was determined that all three genes were expressed in the cell morphological types studied. However, the accumulated level of transcript derived from each gene varied considerably, with TEF-1 mRNA present in approximately twofold greater quantity than the TEF-3 transcript and up to sixfold greater quantity than TEF-2. The level of TEF-1 and -2 mRNA varied little among the cell morphological types studied, whereas TEF-3 mRNA was present in twofold greater quantity in sporangiospores than in either germlings or yeast cells which had been induced to undergo morphogenesis to hyphae. These data suggest that there is differential expression of the genes encoding elongation factor 1 alpha in M. racemosus. At least one gene, TEF-3, shows a morphology-specific pattern of transcript accumulation.

DNA, Fungal↗

PubMed2002

Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting.

BACKGROUND: Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance), that calculates the relative abundance of genes in cDNA libraries. RESULTS: DADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of gene-specific restriction fingerprints in the resulting complex fragment mixtures. A specific cDNA cloning vector had to be constructed that governed missing or incomplete cDNA inserts which would generate misleading fingerprints in standard cloning vectors. Double stranded cDNA was synthesized using an anchored oligo dT primer, uni-directionally inserted into the DADA vector and cDNA libraries were constructed in E. coli. The cDNA fingerprints were generated in a PCR-free procedure that allows for parallel plasmid preparation, labeling, restriction digest and fragment separation of pools of 96 colonies each. This multiplexing significantly enhanced the throughput in comparison to sequence-based methods (e.g. EST approach). The data of the fragment mixtures were integrated into a relational database system and queried with fingerprints experimentally produced by analyzing single colonies. Due to limited predictability of the position of DNA fragments on the polyacrylamid gels of a given size, fingerprints derived solely from cDNA sequences were not accurate enough to be used for the analysis. We applied DADA to the analysis of gene expression profiles in a model for impaired wound healing (treatment of mice with dexamethasone). CONCLUSIONS: The method proved to be capable of identifying pharmacologically relevant target genes that had not been identified by other standard methods routinely used to find differentially expressed genes. Due to the above mentioned limited predictability of the fingerprints, the method was yet tested only with a limited number of experimentally determined fingerprints and was able to detect differences in gene expression of transcripts representing 0.05% of the total mRNA population (e.g. medium abundant gene transcripts).

Journal Article↗

PubMed1991

Biliary lithotripsy: correlation between gallbladder contractility before treatment and the success of treatment.

We perform biliary lithotripsy without adjuvant chemolitholytic agents, and therefore fragment clearance depends solely on the gallbladder's ability to evacuate its contents. We studied 205 patients to determine if gallbladder contractility before biliary lithotripsy is a predictor of treatment results and rate of fragment clearance. Percentage gallbladder contraction was calculated from the fractional difference in the sonographically measured gallbladder volumes before and after a fatty meal. Seventy-six patients (37%) were free of stones and fragments, and 129 patients (63%) had residual fragments at comparable follow-up intervals. Statistical analysis showed a significant difference in gallbladder contractility before biliary lithotripsy between the fragment-free group and the residual-fragment group (p = .008). Stone burden before treatment showed no significant difference between the groups (p = .074), but the number of stones was significantly less in the fragment-free group (p = .022). In the fragment-free group, a poor correlation (r = .047) was found between the percentage gallbladder contraction and the rate of fragment clearance. These data indicate that contractility of the gallbladder before treatment correlates with overall success of biliary lithotripsy but is not a predictor of the speed of fragment clearance.

Adolescent↗

PubMed2006

Supporting continuity of information in the patient transfer process should there be a minimum data set across care settings?

Existing clinical information systems do not facilitate the easy transfer of relevant clinical information; hence there are significant disparities in both the type and timeliness of information that accompanies a patient transfer between care settings. Patient data is commonly fragmented between various health care settings and providers, further contributing to a reduction in the quality of information that is shared. Delineating a transfer minimum data set would provide the basis for communicating consistent information between care settings.

Continuity of Patient Care↗

PubMed1989

Fragment A of diphtheria toxin causes pH-dependent lesions in model membranes.

Fragment A of diphtheria toxin has been shown to insert into lipid bilayers at low pH (Montecucco, C., Schiavo, G., and Tomasi, M. (1985) Biochem. J. 231, 123-128; Zhao, J.-M., and London, E. (1988) J. Biol. Chem. 263, 15369-15377). In this report, evidence is provided which demonstrates that fragment A, like diphtheria toxin, can also cause the release of a fluorescent dye (calcein) from vesicles under acidic conditions and that this release parallels fragment A insertion into the membrane. Although the permeability changes are not as large as those obtained with whole toxin (Jiang, G.-S., Solow, R., and Hu, V. W. (1989) J. Biol. Chem. 264, 13424-13429), molecular sieving experiments indicate that the lesion induced by fragment A increases in size with decreasing pH and reaches an upper limit of 30 A at pH 4.0. In addition to size differences, the lesion induced by fragment A releases calcein in a graded manner, whereas diphtheria toxin causes an all-or-none release. One possible interpretation of this result is that the fragment A lesion is transient in comparison to that induced by whole toxin. Although the molecular bases for the observed differences are not understood, these data suggest that fragment A interaction with the lipid bilayer may play a significant role in mediating its own translocation across membranes and that fragment B may aid this process by initiating, enlarging, and stabilizing the lesion formed.

Calcium↗

PubMed1984

Glucose-induced changes in somatostatin-14 and somatostatin-28 released from rat hypothalamic fragments 'in vitro'.

Previous data suggest that somatostatin is present and released from the hypothalamus in several molecular forms, basally and after K+ or electrical stimulation. In order to evaluate the proportions of somatostatin-14 (S14) and somatostatin-28 (S28) released during a stimulus which may be more closely related to the control of growth hormone secretion 'in vivo', we studied the molecular forms of somatostatin released from hypothalamic fragments ' in vitro', during incubations with different glucose concentrations (1.35 and 22mM), which we have previously shown to be inversely related to somatostatin release. Sephadex G-50 chromatography demonstrated that both forms are released in the same proportions (S14: 70%; S28: 30%) during incubation with different glucose concentrations; there is a parallel increase in both forms when low glucose is used. Although on a molar basis less S28 is released than S14, the higher potency, longer duration of action and higher affinity for pituitary receptors of S28 suggests that it may be of major physiological importance.

Animals↗

PubMed2005

Bioconjugates for tunable peptide fragmentation: free radical initiated peptide sequencing (FRIPS).

The free radical initiator Vazo 68 is coupled to a peptide and electrosprayed into an ion trap mass spectrometer. On collisional activation, the Vazo 68-peptide conjugate generates a free radical, which can be collisionally activated to cleave the peptide backbone. Mostly z-type fragments are formed, as in CAD of other radical peptides and ECD fragmentation. We present data for the Angiotensin II-Vazo 68 conjugate and discuss possible sites of H atom abstraction from the peptide. This experimental methodology for generating peptide fragments is a useful step toward the development of a completely gas-phase approach to protein sequencing.

Angiotensin II↗

PubMed2003

Scientific basis for the development of biokinetic models for radionuclide-contaminated wounds.

Radionuclide-contaminated wounds are of radiological concern because the wound provides a portal of entry of the radionuclide to the systemic circulation, and can also be a tissue at risk if sufficient dose is deposited at the wound site. Accordingly, a scientific committee established jointly by the US National Council on Radiation Protection and the International Commission on Radiological Protection has been developing an approach to describing the biokinetics of radionuclides deposited in wounds and calculating dose to the wound site. This paper focuses on the analyses, performed principally using experimental animal data, that have led to the development of a biokinetic model for deposited soluble radionuclides as well as more insoluble forms, such as colloids, particles and fragments. The available data for injected soluble materials have provided a basis for categorising 48 different elements (from Be to Cm and representing all of the chemical groups, except halogens and noble gases) into four distinct retention groups. In general, the data are adequate for developing a mechanistically based biokinetic model, whose application is exemplified for soluble radionuclides.

Animals↗

PubMed1997

Thiol-disulfide isomerization in thrombospondin: effects of conformation and protein disulfide isomerase.

Thiol-disulfide isomerization in thrombospondin may affect the function of this adhesive protein. Two assays were developed to analyze the determinants of thiol-disulfide exchange and to correlate this exchange with thrombospondin conformation. (1) A competitive immunoassay for the EDTA-conformation of thrombospondin was developed with monoclonal antibody D4.6. (2) The free thiol(s) in thrombospondin was labeled with [3H]N-ethylmaleimide (NEM) under various conditions (the presence or absence of calcium, temperature, and pH), and thrombin digests of the labeled protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Consistent with previous reports, thrombin digest fragments of 150, 120, 20, and 14 kD were observed, each with radioactivity under some condition, plus a 25-kD peptide that was not labeled. Sequence data for these fragments and comparisons of SDS-PAGE analyses under reducing and nonreducing conditions indicated that Cys974 was the free thiol. The appearance of thiol label in the 120-kD fragment was previously shown to be a consequence of thiol-disulfide exchange (J Biol Chem 265:17859,1990) and label was recovered in this peptide only under conditions (absence of calcium, 37 degrees C and pH 8.4) that led to the appearance of the EDTA-conformation of thrombospondin. Additional evidence for the correlation of EDTA-conformation and thiol-disulfide exchange was the enhanced conversion of thrombospondin to its EDTA-conformation in the presence of protein disulfide isomerase and the inability of thrombospondin pretreated with NEM to attain the EDTA-conformation. Flow cytometry with antibody D4.6 revealed platelet-associated thrombospondin in the EDTA-conformation in the presence of calcium, suggesting that the EDTA-conformation is a physiological conformation that does not necessarily require EDTA.

Antibodies, Monoclonal↗