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PubMed1990

Incidence of regional lymph node metastasis in operable osteogenic sarcoma.

Records of 182 consecutive patients with the diagnosis of operable osteogenic sarcoma, treated between 1954 through 1980 by the Breast, Bone and Mixed Tumors Department of the Instituto Nacional de Enfermedades Neoplasicas (Lima, Peru), were reviewed to study the incidence of regional lymph node metastases in this disease. All the patients included in this study had radical surgery, which means the complete resection of the bone where the tumor is located, including the proximal joint, which permitted excision of the regional lymph nodes. Nineteen patients (10.4%) had evidence of sarcoma metastatic to draining lymph nodes. A comprehensive analysis of the literature shows that the incidence of metastasis to the lymph nodes in this study, is higher than those cited in a review of the literature, probably due to the policy in the management of osteogenic sarcoma in our institution, during the period of study.

Adolescent↗
PubMed2001

High-throughput single molecule screening of DNA and proteins.

We report a novel imaging technology for real time comprehensive analysis of molecular alterations in cells and tissues appropriate for automation and adaptation to high-throughput applications. With these techniques it should eventually be possible to perform simultaneous analysis of the entire contents of individual biological cells with a sensitivity and selectivity sufficient to determine the presence or absence of a single copy of a targeted analyte (e.g., DNA region, RNA region, protein), and to do so at a relatively low cost. The technology is suitable for DNA and RNA through sizing or through fluorescent hybridization probes, and for proteins and small molecules through fluorescence immunoassays. This combination of the lowest possible detection limit and the broadest applicability to biomolecules represents the final frontier in bioanalysis. The general scheme is based on novel concepts for single molecule detection (SMD) and characterization recently demonstrated in our laboratory. Since minimal manipulation is involved, it should be possible to screen large numbers of cells in a short time to facilitate practical applications. This opens up the possibility of finding single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification.

Animals↗
PubMed1994

Assimilatory nitrate reductase: reduction and inhibition by NADH/NAD+ analogs.

Assimilatory nitrate reductase from Chlorella vulgaris catalyzes the rate-limiting step, the conversion of nitrate to nitrite, in nitrate assimilation. Initial rate studies of nitrate reductase activity, performed under optimum conditions of constant ionic strength (mu = 0.2) and pH (8.0) and using NADH as reductant, indicated the absence of substrate inhibition at NADH concentrations below 300 microM and NO3- concentrations less than 3 mM. Chlorella nitrate reductase exhibited a marked preference for NADH (Vmax = 9.2 mumol NADH/min/nmol heme and Km = 2.3 microM) as the physiological electron donor but could also utilize alpha-NADH (Vmax = 5.6 mumol NADH/min/nmol heme and Km = 131 microM) and NADPH (Vmax = 0.6 mumol NADPH/min/nmol heme and Km = 910 microM) though with significantly decreased efficiency. Examination of various NADH-analogs indicated that reduced nicotinamide hypoxanthine dinucleotide (NHDH) was used most efficiently (Vmax = 9.3 mumol NHDH/min/nmol heme and Km = 7.9 microM), while reduced nicotinamide mononucleotide (NMNH) was utilized least efficiently (Vmax = 0.07 mumol NMNH/min/nmol heme and Km = 676 microM). Overall, modifications to the nicotinamide moiety or the addition of a phosphate group were observed to result in the most significant decreases in Vmax, indicating poor reducing substrates. Product inhibition studies indicated both NAD+ (Ki = 2.2 mM) and NADP+ (Ki = 10.5 mM) to be competitive inhibitors of Chlorella NR. A variety of NAD+ analogs were also determined to act as competitive inhibitors with varying degrees of efficiency. 3-Pyridinealdehyde adenine dinucleotide was the most efficient inhibitor (Ki = 0.74 mM) while nicotinamide was the least efficient (Ki = 18.1 mM). Overall, changing substituents on the nicotinamide ring or its complete deletion produced the most effective inhibitors compared to NAD+. In contrast, changes in the adenine or ribose moieties produced less effective inhibitors when compared to NAD+. These results represent the most comprehensive analysis of the effect of modifications of the physiological reductant (NADH) and product (NAD+) on nitrate reductase activity.

Binding, Competitive↗
PubMed2000

Inactive systemic lupus erythematosus id associated with a normal stimulated Th(1)/Th(2) cytokine secretory pattern.

OBJECTIVES: To determine the Th(1)/Th(2)balance in systemic lupus erythematosus (SLE) patients with inactive disease. METHODS: A comprehensive analysis of peak secretion, overall cytokine production and secretory pattern of Th(1)and Th(2)cytokines from stimulated PBMC of 10 SLE patients with inactive disease and 10 age- and sex-matched controls. RESULTS: No significant differences were found in the peak and total secretion of all cytokines, as well as in the Th(1)and Th(2)secretory patterns and proliferative response between the two groups. CONCLUSION: Th(1)and Th(2) balance in inactive SLE is normal.

Adult↗
PubMed1998

erbB genes in the mouse uterus: cell-specific signaling by epidermal growth factor (EGF) family of growth factors during implantation.

We previously described spatiotemporal expression of various epidermal growth factor (EGF)-like ligands and receptor subtypes, ErbB1 and ErbB2, during the peri-implantation period. To better understand the roles of these ligands and their possible signaling schemes in implantation, it is important to define the status of all the ligands and receptor subtypes in the uterus/embryo. No information is available about uterine and embryonic status of ErbB3 or ErbB4 during implantation. We cloned mouse erbB3 and erbB4 cDNAs and examined their expression and bioactivity in the peri-implantation uterus (days 1-8). Two erbB3 (cytoplasmic and extracellular) and three erbB4 (two cytoplasmic and one extracellular) clones were generated. Both forms of the erbB3 clone showed similar transcript profiles, while different transcript profiles were obtained with erbB4 clones. The steady-state levels of erbB3 and erbB4 mRNAs in whole uterine poly(A)+ RNA samples showed little changes during the peri-implantation period, while their unique cell-specific accumulation was noted. erbB3 is predominantly expressed in the epithelial cells, although decidual and embryonic cells also accumulate this mRNA. In contrast, the erbB4 mRNA is primarily expressed in the submyometrial stroma and myometrial connective tissues during this period. Additionally, the extracellular form of the erbB4 clone detected signals in a subpopulation of stromal cells. Autophosphorylation and immunoprecipitation studies provided evidence that uterine ErbB3 and ErbB4 are biologically active. This study provides a comprehensive analysis of possible ligand-receptor signaling schemes for EGF-like ligands in implantation.

Animals↗
PubMed1996

Functional reconstitution of oxidase activity in X-linked chronic granulomatous disease by retrovirus-mediated gene transfer.

The feasibility of correction of the disease phenotype by gene gene transfer was investigated in cells of four patients with X-linked chronic granulomatous disease. These patients carry point mutations of the gp91-phox gene, encoding for the large subunit of the catalytic core of the phagocytic cell NADPH oxidase. A retroviral vector expressing the gp91-phox protein was constructed and used to transduce lymphoblastoid cell lines established from the patients. Several transduced lymphoblastoid cell clones were investigated for mRNA and protein expression, and for functional reconstitution of oxidase activity. Although extensive quantitative variability was detected among different clones, functional reconstitution of O2- production was obtained in most cases, with oxidase function within the same range as in B cell lines derived from normal individuals. The same vector was also used for transduction of hematopoietic precursors from bone marrow or peripheral blood either with or without enrichment for CD34+ cells. A comprehensive analysis was performed on differentiated myeloid colonies, to evaluate the efficiency of transduction, the levels of gp91-phox expression, and the extent of functional reconstitution of oxidase activity. A high efficiency of transduction was obtained in most experiments, with 60-100% of colonies containing proviral DNA. Among the transduced colonies, an extensive variability in the levels of expression of the transduced gene and of functional restoration of NADPH oxidase activity was observed. These results represent a step toward the development of a gene therapy protocol for these patients.

3T3 Cells↗
PubMed1993

Structure and organization of amplified DNA on double minutes containing the mdm2 oncogene.

We have been studying a transformed derivative of a mouse fibroblast line (3T3DM) that stably maintains double minute chromosomes (DMs). In this report we describe a comprehensive analysis of the structure of the DMs within this cell line, utilizing a combination of long-range mapping via pulsed-field gel electrophoresis, screening of DM-enriched genomic libraries, and DM sizing using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Our data indicate that the minute particles in these cells exist as a homogeneous population of circular molecules, roughly 4 Mb in size, upon which three genes are amplified. One of these is the mdm2 oncogene, which has also been found to be amplified in a number of human sarcomas. Further, we present evidence that these three genes are arranged as two identical inverted repeat units linked by spacer regions of heterogeneous size. This work has led to the first model for the structure of an entire double minute particle containing an amplified oncogene; this model provides clues to later events occurring in the gene amplification process in tumor cells.

3T3 Cells↗
PubMed1993

Comparison of a structural and a functional epitope.

A comprehensive analysis of the energetic importance of the 31 side-chains buried at the interface between human growth hormone (hGH) and the extracellular binding domain of its receptor (hGHbp) has been carried out to assess the roles of contact side-chains in modulating the affinity and kinetics of binding. Each side-chain in hGH was converted to alanine, and the kinetics and affinity were measured using a biosensor device. This detects binding of the mutated hormones to the immobilized hGHbp by changes induced in refractive index. The data generated on the biosensor match affinities obtained by radio-immune assay in solution. The study shows that only one-quarter of the side-chains buried at the interface can account for the majority of the binding energy. These residues cluster near the center of the structural epitope. The role of these side-chains is predominantly to slow dissociation because most of the effect of the alanine substitutions is to increase the off-rate, not to slow the on-rate. The hormone associates about 10,000 times slower than expected from random diffusion but 1000 times faster than may be expected if one imposes strict orientation restraints for a productive collision. Electrostatic interactions partly modulate association because mutations at Arg residues most affect association and together contribute a factor of about 20 to the on-rate. The data suggest that the hormone and receptor associate by diffusion and electrostatics to form an ensemble of weak collisional complexes. From these a bound complex is produced that is stabilized by only a small proportion of the contacts. We suggest that solvation energies and/or side-chains interactions within the free hormone or receptor may be so favorable that little energy is gained at most side-chains upon binding. The fact that the functional binding epitope is much smaller than the structural epitope suggests it may be possible to design smaller hormone mimics.

Amino Acid Sequence↗
PubMed1998

A role for CH...O interactions in protein-DNA recognition.

The concept of CH...O hydrogen bonds has recently gained much interest, with a number of reports indicating the significance of these non-classical hydrogen bonds in stabilizing nucleic acid and protein structures. Here, we analyze the CH...O interactions in the protein-DNA interface, based on 43 crystal structures of protein-DNA complexes. Surprisingly, we find that the number of close intermolecular CH...O contacts involving the thymine methyl group and position C5 of cytosine is comparable to the number of protein-DNA hydrogen bonds involving nitrogen and oxygen atoms as donors and acceptors. A comprehensive analysis of the geometries of these close contacts shows that they are similar to other CH...O interactions found in proteins and small molecules, as well as to classical NH...O hydrogen bonds. Thus, we suggest that C5 of cytosine and C5-Met of thymine form relatively weak CH...O hydrogen bonds with Asp, Asn, Glu, Gln, Ser, and Thr, contributing to the specificity of recognition. Including these interactions, in addition to the classical protein-DNA hydrogen bonds, enables the extraction of simple structural principles for amino acid-base recognition consistent with electrostatic considerations.

Base Composition↗
PubMed2000

Microsecond to minute dynamics revealed by EX1-type hydrogen exchange at nearly every backbone hydrogen bond in a native protein.

A previous comprehensive analysis of the pH dependence of native-state amide hydrogen (NH) exchange in turkey ovomucoid third domain (OMTKY3) yielded apparent opening and closing rate constants (k(op) and k(cl)) at 14 NH groups involved in global conformational changes. This analysis has been extended to 18 additional slowly exchanging NH groups. Quench-flow experiments were performed to monitor NH exchange in native OMTKY3 from neutral to very alkaline pH ( approximately 12) conditions. Above pH 10 the mechanism of exchange switched from one governed by a rapid equilibrium preceding the chemistry of exchange (i.e. EX2 exchange), to one where exchange was limited by the rate of opening (i.e. EX1 exchange). Kinetics of solvent exposure are now known for nearly all backbone NH groups in native OMTKY3, yielding rate constants that span five orders of magnitude, 0.004 to 200 s(-1).

Amides↗
PubMed2000

Prediction of transcription terminators in bacterial genomes.

This study describes an algorithm that finds rho-independent transcription terminators in bacterial genomes and evaluates the accuracy of its predictions. The algorithm identifies terminators by searching for a common mRNA motif: a hairpin structure followed by a short uracil-rich region. For each terminator, an energy-scoring function that reflects hairpin stability, and a tail-scoring function based on the number of U nucleotides and their proximity to the stem, are computed. A confidence value can be assigned to each terminator by analyzing candidate terminators found both within and between genes, and taking into account the energy and tail scores. The confidence is an empirical estimate of the probability that the sequence is a true terminator. The algorithm was used to conduct a comprehensive analysis of 12 bacterial genomes to identify likely candidates for rho-independent transcription terminators. Four of these genomes (Deinococcus radiodurans, Escherichia coli, Haemophilus influenzae and Vibrio cholerae) were found to have large numbers of rho-independent terminators. Among the other genomes, most appear to have no transcription terminators of this type, with the exception of Thermotoga maritima. A set of 131 experimentally determined E. coli terminators was used to evaluate the sensitivity of the method, which ranges from 89 % to 98 %, with corresponding false positive rates of 2 % and 18 %.

Algorithms↗
PubMed2000

Microbial genome analyses: comparative transport capabilities in eighteen prokaryotes.

Here, we present a comprehensive analysis of solute transport systems encoded within the completely sequenced genomes of 18 prokaryotic organisms. These organisms include four Gram-positive bacteria, seven Gram-negative bacteria, two spirochetes, one cyanobacterium and four archaea. Membrane proteins are analyzed in terms of putative membrane topology, and the recognized transport systems are classified into 76 families, including four families of channel proteins, four families of primary carriers, 54 families of secondary carriers, six families of group translocators, and eight unclassified families. These families are analyzed in terms of the paralogous and orthologous relationships of their protein members, the substrate specificities of their constituent transporters and their distributions in each of the 18 organisms studied. The families vary from large superfamilies with hundreds of represented members, to small families with only one or a few members. The mode of transport generally correlates with the primary mechanism of energy generation, and the numbers of secondary transporters relative to primary transporters are roughly proportional to the total numbers of primary H(+) and Na(+) pumps in the cell. The phosphotransferase system is less prevalent in the analyzed bacteria than previously thought (only six of 14 bacteria transport sugars via this system) and is completely lacking in archaea and eukaryotes. Escherichia coli is shown to be exceptionally broad in its transport capabilities and therefore, at a membrane transport level, does not appear representative of the bacteria thus far sequenced. Archaea and spirochetes exhibit fewer proteins with multiple transmembrane segments and fewer net transporters than most bacteria. These results provide insight into the relevance of transport to the overall physiology of prokaryotes.

Adenosine Triphosphatases↗
PubMed2001

Do water molecules mediate protein-DNA recognition?

A comprehensive analysis of interfacial water molecules in the structures of 109 unique protein-DNA complexes is presented together with a new view on their role in protein-DNA recognition. Location of interfacial water molecules as reported in the crystal structures and as emerging from a series of molecular dynamics studies on protein-DNA complexes with explicit solvent and counterions, was analyzed based on their acceptor, donor hydrogen bond relationships with the atoms and residues of the macromolecules, electrostatic field calculations and packing density considerations. Water molecules for the purpose of this study have been categorized into four classes: viz. (I) those that contact both the protein and the DNA simultaneously and thus mediate recognition directly; (II) those that contact either the protein or the DNA exclusively via hydrogen bonds solvating each solute separately; (III) those that contact the hydrophobic groups in either the protein or the DNA; and, lastly (IV) those that contact another water molecule. Of the 17,963 crystallographic water molecules under examination, about 6% belong to class I and 76% belong to class II. About three-fourths of class I and class II water molecules are exclusively associated with hydrogen bond acceptor atoms of both protein and DNA. Noting that DNA is polyanionic, it is significant that a majority of the crystallographically observed water molecules as well as those from molecular dynamics simulations should be involved in facilitating binding by screening unfavorable electrostatics. Less than 2% of the reported water molecules occur between hydrogen bond donor atoms of protein and acceptor atoms of DNA. These represent cases where protein atoms cannot reach out to DNA to make favorable hydrogen bond interactions due to packing/structural restrictions and interfacial water molecules provide an extension to side-chains to accomplish hydrogen bonding.

Base Sequence↗
PubMedDate not supplied

Sequence comparisons of intermediate filament chains: evidence of a unique functional/structural role for coiled-coil segment 1A and linker L1.

A comprehensive analysis of the sequences of all types of intermediate filament chains has been undertaken with a particular emphasis on those of segment 1A and linker L1. This has been done to assess whether structural characteristics can be recognized in the sequences that would be consistent with the role of each region in the recently proposed "swinging head" hypothesis. The analyses show that linker L1 is the most flexible rod domain region, that it is the most elongated structure (on a per residue basis), and that it is the most variable region as regards sequence and length. Segment 1A has one of the two most highly conserved regions of sequence in the rod domain (the other being at the end of segment 2B), with seven particular residues conserved across all chain types. It also contains one of the very few potential interchain ionic interactions that could be conserved across all chain types. However, the aggregation of chains in segment 1A is specified less precisely overall by interchain ionic interactions than are the other coiled-coil segments. The apolar residue contents in positions a and d of the heptad substructure are the highest of any coiled-coil segment in the intermediate filament family. Segment 1A also displays an amino acid composition atypical of not only coiled-coil segments 1B and 2B, but indeed of two-stranded coiled coils in general. Nonetheless, molecular modeling based on the crystal structure of the monomeric 1A fragment from human vimentin shows that coiled-coil formation is plausible. The most extensive regions of apolar/aromatic residues lie at the C-terminal end of segment 2B in the helix termination motif and in segment 1A in and close to the helix initiation motif. The predicted stability of the individual alpha-helices in segment 1A is greater than in those comprising segments 1B and 2B, though potential intrachain ionic interactions are either lacking or are minimal in number. Analysis of the 1A sequence and those regions immediately N- and C-terminal to it has shown that the capping residues are near optimal close to the previously predicted ends, thus adding to the likely stability of the alpha-helical structure. However, a second terminating sequence is predicted in 1A (about 10 residues back from the C-terminus). This allows the possibility of some unwinding of the alpha-helical structure of 1A immediately adjacent to linker L1 when the head domains no longer stabilize the coiled-coil structure. All of these data are consistent with the concept of a flexible hinge at L1 and with the ability of the two alpha-helical coiled-coil strands to separate under appropriate conditions and partly unwind at their C-terminal ends to allow the head domains a greater degree of mobility, thus facilitating function.

Amino Acid Motifs↗
PubMed2000

Purification and identification of novel Rab effectors using affinity chromatography.

Rab GTPases are central regulatory elements of the intracellular transport machinery of eukaryotic cells. To regulate vesicle docking and fusion as well as organelle dynamics Rab proteins interact with effector molecules in the GTP-bound active state. The identification of Rab effectors is, therefore, of primary importance for the mechanistic understanding of intracellular transport. Here we describe the experimental system we have developed to biochemically purify and identify effectors of the small GTPase Rab5. The method, which is based on an affinity chromatography procedure, results in the large-scale purification of Rab effectors in amounts sufficient for both their identification by microsequencing techniques and their functional characterization. In the case of Rab5, the procedure allows a comprehensive analysis of the downstream effectors and regulators of this GTPase. We expect this strategy to provide fundamental insights into the molecular mechanism of membrane transport but also to be applicable to several other GTPase-dependent biological functions.

Animals↗
PubMed1993

Class-specific immunoglobulins and antibodies to mycobacterial sonicates and autoantigens in leprosy patients and contacts.

A comprehensive analysis of the humoral immune response in leprosy patients and contacts was undertaken. Class-specific antibodies to four mycobacterial sonicates, three autoantigens and three haptens were estimated by ELISA. It was found that IgG levels varied more extensively than IgM or IgA and that total serum IgG was significantly higher in lepromatous bacterial index positive (LL+ve) and negative LL-ve) leprosy patients than in tuberculoid (TT/BT) patients and controls. The high levels of anti-mycobacterial antibodies found in untreated LL+ve patients were significantly reduced in LL-ve patients after effective chemotherapy. Considerable amount of anti-mycobacterial IgG was also detected in TT/BT patients. Each serum when assayed against sonicates of Mycobacterium leprae, Mycobacterium tuberculosis, ICRC bacilli and BCG gave a similar antibody profile suggesting that these antibodies were directed predominantly against cross-reactive antigens. Up to 75% of LL patients and 35% of TT/BT patients were found to be positive for antibodies to histone, collagen and fibronectin. However, antibodies to several haptens were not detected in any of the patients and controls studied. Taken together, these results suggested that the amount of IgG antibodies is directly correlated with the antigenic load in the system, and that there is no evidence for polyclonal activation. It may be speculated that the regulatory mechanism of antibody production is severely deranged in lepromatous leprosy patients.

Antibodies, Bacterial↗
PubMed2001

High-throughput proteomics: protein expression and purification in the postgenomic world.

Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic and comprehensive analysis of protein structure and function. Protein expression and purification are key processes in these studies, but have typically only been applied on a case-by-case basis to proteins of interest. Researchers are addressing the challenge of parallel expression and purification of large numbers of gene products through the principles of high-throughput screening technologies commonly used in pharmaceutical development. Some of the issues relevant to these approaches are discussed here.

Genome, Human↗
PubMed1996

RNA splice site utilization by simian immunodeficiency viruses derived from sooty mangabey monkeys.

Alternative splicing of the full-length, primary transcript into numerous subgenomic mRNAs is one way that lentiviral gene expression is regulated. Because the behaviors of different viral isolates might reflect in part differences in splicing, we Investigated the patterns of splice site utilization by simian immunodeficiency viruses (SIVs)-originally isolated from sooty mangabey monkeys (Cercocebus atys) We used reverse transcription-polymerase chain reaction (RT-PCR), molecular cloning, and DNA sequencing approaches to characterize SIVdeltaB670, a pathogenic and neurovirulent isolate, and SIVsmmH4, a related molecular clone. The majority of randomly selected SIVdeltaB670 and SIVsmmH4 partial cDNAs contained tat, rev, nef, and long terminal repeat (LTR) intron splice donor and acceptor sites positioned as expected based on the proviral sequence of SIVsmmH4. Nearly all (87%) of the partial cDNAs analyzed contained a spliced LTR intron. A greater number of partial cDNAs derived from SIVdeltaB670-infected cells contained putative alternatively spliced introns In comparison to SIVsmmH4, including two previously undocumented splice junctions involving the LTR intron splice donor. These data provide the first comprehensive analysis of splice site utilization by an isolate of SIV in comparison to a related molecular clone and the first characterization of SIVsmm splice site utilization.

Amino Acid Sequence↗