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PubMed2006

Identification of enantioselective extractants for chiral separation of amines and aminoalcohols.

The major obstacle for the introduction of fractional reactive extraction as a chiral separation method in the chemical and pharmaceutical industries is the lack of versatile enantioselective extractants. Therefore, a rational approach is developed to transfer the extensive knowledge of chiral selectors reported in the literature on chiral recognition and other chiral separation techniques to extraction. Based on a similarity in separation mechanisms, it was expected that chiral selectors originating from a technique in which chiral recognition takes place in the liquid phase are most likely to function as enantioselective extractant. Using this approach, a selection of promising extractants was made from the literature and experimentally evaluated for the enantioseparation of aminoalcohols and amines. As a result, four enantioselective extractant systems, namely, dibutyl-L-tartrate with boric acid, N-(2-hydroxydodecyl)-L-hydroxyproline Cu(II) complex, N-dodecyl-L-hydroxyproline Cu(II) complex, and azophenolic crown ether, have been identified. The azophenolic crown ether system performed the best and demonstrated an enantioselectivity between 1.3-5.0 for five out of six test compounds. Identification of the enantioselective extractant systems was highly facilitated by the developed rational transfer approach that, although partially qualitative, appeared capable of reducing more than 50 encountered candidates to only three promising systems for further experimental evaluation. Therefore, it is expected that this approach can be successfully applied to identify enantioselective extractants for other classes of enantiomers as well.

Amines↗
PubMed1985

Development of T cell clones reactive to two defined restriction elements in conjunction with two defined epitopes of antigen.

A previously described pig insulin (PI)-specific T cell line of (B10 X B10.BR)F1 origin was assayed for its reactivity with species variants of insulin in the presence of antigen-presenting cells (APC) of various H-2 haplotypes. In addition to its reactivity with PI and bovine insulin (BI) in the context of syngeneic F1 (H-2b X k)-APC, a weak cross-reactivity was observed with parental B10 (H-2b)-APC and BI but not PI. The cross-reactive cells could be selected out by several restimulations with the combination of BI and B10-APC. From the resulting, strongly cross-reactive T cell line several interleukin 2-dependent sublines were developed which did not require antigen-specific restimulations for further propagation. All such sublines had retained the original cross-reactivity with BI and B10-APC but showed significant differences in their fine specificity patterns, which indicates that each subline represents a clonal population. One of the sublines was cloned by limiting dilution at one cell/culture with a cloning efficiency of 76%. Five of the clones that were tested for reactivity had the same cross-reactivity as the original subline and upon recloning at 0.1 cells/culture the pattern again remained unchanged. From an analysis of the two antigen combinations (PI/F1 and BI/B10) it can be concluded that single cells can react with different restriction elements in the context of distinct epitopes of insulin. The implications of this finding for the mechanism of T cell recognition are discussed and a model for the function of major histocompatibility complex molecules in T cell recognition is proposed.

Animals↗
PubMed2000

Molecular markers for the identification and global tracking of whitefly vector-Begomovirus complexes.

Recent unprecedented upsurges in populations of the whitefly Bemisia tabaci (Genn.) have drawn much attention to its worldwide importance as an insect pest and as the vector of emergent begomoviruses (Family: Geminiviridae; Genus: Begomovirus). Several begomoviruses that are considered 'new' and others previously regarded as minor pathogens have been linked to recent epidemics. Recent studies have revealed much variation in begomoviruses, despite the view that DNA-containing viruses do not rapidly accumulate mutations. Also, certain B. tabaci 'variants' are known that more effectively or selectively transmit certain begomoviruses and exhibit biotic differences that may influence their spread. Patterns of distribution and dissemination of begomoviruses transmitted by B. tabaci are poorly understood because standardized molecular-based tracking methods have not been available. Understanding virus/whitefly vector/host plant interrelationships in the context of emerging problems can be achieved only by linking predicted evolutionary histories with epidemiology using molecular phylogenetic approaches. Identification and validation of informative molecular sequences are essential initial steps in this process. Genus-wide degenerate polymerase chain reaction (PCR) primers have been developed to amplify and sequence the 'core' region of the coat protein open reading frame (ORF) (V1), permitting 'universal' detection and provisional virus identification by comparisons with described viral genotypes. In subsequent studies reported here, several potentially informative viral ORFs and a non-coding region are explored. Of particular use for expanding diversity studies are group- or virus-specific sequences that can be targeted by utilizing newly available core CP sequences, or additional conserved regions around which broad spectrum primers can be designed to target variable sequences in key ORFs or non-coding regions. Prospective markers under exploration were selected with a basis in the most highly conserved viral ORFs, CP (V1) and a portion of replication-associated protein (REP) (L1/C1), and a key non-coding sequence that contain sufficient variability and/or virus-specific sequences, and are consequently of potential epidemiological relevance. Because B. tabaci occurs as a cryptic species, or species complex, that exhibits biotic polymorphism, yet morphological invariance, traditional morphologically based identification is impossible. An overriding complication to establishing molecular markers for identifying whitefly vector variants is that whitefly sequences in general, have not been available. However, recent work has shown that a partial mitochondria cytochrome oxidase I (mt COI) sequence separates vector variants with a basis in geographical origin, suggesting it is useful for further exploring variability and the phylogenetic history of whiteflies on a large scale. Here, the utility of whitefly mt COI nucleotides (nt) sequences is illustrated for inferring relationships between B. tabaci collected from major world regions. Used collectively, these approaches permit investigations of the patterns of distribution and dissemination of begomovirus-whitefly vector complexes for the first time. Ultimately, more immediate recognition of exotic viruses and whitefly vectors and early detection of upsurges in vector populations and of emerging viruses will be possible.

Amino Acid Sequence↗
PubMed2003

The biological basis of a comprehensive grading system for the adverse effects of cancer treatment.

As described in the previous article in this issue by Trotti et al, there have been major changes in the philosophy and scope of the new National Cancer Institute comprehensive grading system for treatment-related toxicities, Common Terminology Criteria for Adverse Events version 3.0 (CTCAE v3.0). The most prominent changes are the merging of early and late effects criteria into a single uniform document and the development of criteria that cover all treatment modalities. In this article, we briefly outline the biological support for the new grading system in the context of our current knowledge base. The clinical consequences of radiotherapy in normal tissue have been classically grouped temporally, into early and late effects, using a somewhat arbitrary dividing line, 90 days after commencement of radiotherapy. This definition was developed in an era of standard fractionation used alone or in simple sequential programs involving other modalities. However, most patients are now managed with multiple highly integrated modalities, often augmenting tissue injury and limiting our ability to ascribe any given effect to a particular modality. The use of complex concurrent or hybrid (concurrent/sequential) schedules also undermines the usefulness of a simplistic temporally defined early-late construct. Moreover, there is growing recognition that chemotherapy and surgery produce inherent long-term biologic and clinical effects as well. Our basic understanding of the roles that surgery, chemotherapy, and radiation play in normal tissue response has expanded over the last decade because of vastly improved molecular techniques. The original biologic paradigm viewing acute and late tissue injury as a continuum of response and repair has been strengthened by these additional laboratory investigations. The expression of toxicity over time has been shown to be caused by a variety of cellular, tissue, environmental, and host factors. We continue to elucidate the roles of DNA damage, cytokines, chemokines, and associated inflammation, which lead in some cases to perpetuation of the wound-healing response, progressive tissue fibrosis, and vascular compromise. The continuum model of tissue injury supports the recent changes in the common toxicity grading system. It also provides insights into potential targets and strategies for modulating response, which may in turn lead to effective interventions for altering the therapeutic ratio.

Antineoplastic Agents↗
PubMed1992

TraK protein of conjugative plasmid RP4 forms a specialized nucleoprotein complex with the transfer origin.

Conjugative transfer of the self-transmissible IncP plasmid RP4 requires the product of the RP4 traK gene. By using the phage T7 expression system, the traK gene product was efficiently overproduced and purified to near homogeneity. traK encodes a basic protein (pI = 10.7) of 14.6 kDa that, as shown by DNA fragment retention assay, interacts exclusively with its cognate transfer origin. The apparent equilibrium constant K(app) for the complex of TraK and oriT-DNA was estimated to be 4 nM. Footprinting experiments using DNase I or hydroxyl radicals indicate that several TraK molecules interact specifically with an intrinsically bent region of oriT, covering a range of almost 200 base pairs. The TraK target sequence maps in the leading region adjacent to the relaxation nick site and recognition sequences involved in relaxosome formation but does not overlap them. Specific interactions between TraK and the DNA occur only on one side of the double helix. Electron microscopy of TraK-oriT complexes demonstrates that binding of TraK to its recognition region apparently shrinks the length of the target DNA, suggesting that the nucleic acid becomes wrapped around a core of TraK molecules. Formation of this structure could be favored by the presence of the sequence-directed bend in the TraK recognition region.

Amino Acid Sequence↗
PubMed2005

A modified K-means algorithm for circular invariant clustering.

Several important pattern recognition applications are based on feature vector extraction and vector clustering. Directional patterns are commonly represented by rotation-variant vectors Fd formed from features uniformly extracted in M directions. It is often desirable that pattern recognition algorithms are invariant under pattern rotation. This paper introduces a distance measure and a K-means-based algorithm, namely, Circular K-means (CK-means) to cluster vectors containing directional information, such as Fd, in a circular-shift invariant manner. A circular shift of Fd corresponds to pattern rotation, thus, the algorithm is rotation invariant. An efficient Fourier domain representation of the proposed measure is presented to reduce computational complexity. A split and merge approach (SMCK-means), suited to the proposed CK-means technique, is proposed to reduce the possibility of converging at local minima and to estimate the correct number of clusters. Experiments performed for textural images illustrate the superior performance of the proposed algorithm for clustering directional vectors Fd, compared to the alternative approach that uses the original K-means and rotation-invariant feature vectors transformed from Fd.

Algorithms↗
PubMed1989

Immunoregulation in clinical diseases: an overview.

The quality, magnitude, and persistence of each immune response results from the net balance of different influences that may amplify or decrease it. The complex interaction between such influences underlies the concept of immunoregulation. Comprehension of the complex mechanisms of immunoregulation has the potential to yield important insights into the genetic and the molecular basis of the physiology of immune responses and of the pathophysiology of a wide number of clinical diseases. The principal basic mechanisms of immunoregulation comprise the systems of recognition structures regulating antigen specificity of immune responses and the interconnecting systems involved in the overall regulation of immune responses. Such regulatory interconnecting systems include regulatory T cells, idiotypic network, isotopic network, and the intricate web of interactions between cytokines (cytokine network). In addition, there is now evidence that these highly complex and sophisticated regulatory systems are not only interrelated to each other, but are themselves regulated by signals originated outside the immune system (e.g., from the nervous or the endocrine apparatus). These alterations of immunoregulatory mechanisms are involved in the pathogensis of many groups of human diseases, such as immunodeficiencies, neoplastic and immunoproliferative disorders, many infectious diseases, atopic disease, and autoimmune disorders. A rational knowledge of immunoregulation will hopefully have great practical implications for future strategies of diagnosis and treatment of diseases in which deranged immune functions play a major role.

Autoimmune Diseases↗
PubMed1992

Thymidine kinase transcription is regulated at G1/S phase by a complex that contains retinoblastoma-like protein and a cdc2 kinase.

Transcription of the murine thymidine kinase gene, which is coregulated with the G1/S phase transition, is activated by changing the binding of protein complexes Yi1 and Yi2 to three upstream DNA motifs. Yi1 is replaced by Yi2 shortly before S phase. Yi1 contains a protein of 110 kDa that binds to the DNA motif sites and may be an underphosphorylated murine retinoblastoma protein, shown by its molecular mass, timing of its activity, and antibody recognition. An H1 kinase related to cdc2 cofractionates with both complexes. We propose that this kinase phosphorylates the murine retinoblastoma protein, releasing transcriptional inhibitions by Yi1 and permitting cell cycle progression. These results provide a cycle-related molecular target for such complexes. They are based on investigations of cycle control in uninfected cells. The Yi complexes are similar but not identical to complexes that include a cellular protein, E2F, that was originally found to bind to adenovirus DNA.

Animals↗
PubMed2000

MHC-linked olfactory receptor loci exhibit polymorphism and contribute to extended HLA/OR-haplotypes.

Clusters of olfactory receptor (OR) genes are found on most human chromosomes. They are one of the largest mammalian multigene families. Here, we report a systematic study of polymorphism of OR genes belonging to the largest fully sequenced OR cluster. The cluster contains 36 OR genes, of which two belong to the vomeronasal 1 (V1-OR) family. The cluster is divided into a major and a minor region at the telomeric end of the HLA complex on chromosome 6. These OR genes could be involved in MHC-related mate preferences. The polymorphism screen was carried out with 13 genes from the HLA-linked OR cluster and three genes from chromosomes 7, 17, and 19 as controls. Ten human cell lines, representing 18 different chromosome 6s, were analyzed. They were from various ethnic origins and exhibited different HLA haplotypes. All OR genes tested, including those not linked to the HLA complex, were polymorphic. These polymorphisms were dispersed along the coding region and resulted in up to seven alleles for a given OR gene. Three polymorphisms resulted either in stop codons (genes hs6M1-4P, hs6M1-17) or in a 16-bp deletion (gene hs6M1-19P), possibly leading to lack of ligand recognition by the respective receptors in the cell line donors. In total, 13 HLA-linked OR haplotypes could be defined. Therefore, allelic variation appears to be a general feature of human OR genes.

Alleles↗
PubMed1986

[Myocardial mechanical injury in acute ischemia: a pathophysiologic and histopathologic review].

The recognition of histopathologic substrates of myocardial contractile damage in human acute ischemia is still very poor, notwithstanding the impressive advances in the inherent clinical diagnostic technology and concepts. The first and foremost inotropic abnormality ensuing ischemia, easily taken for atonic in origin, actually consists of a pathologic contracture of the injured myocardium, depending upon abrupt fall of ATP, and defective extrusion calcium pump with persistence of actomyosin rigor-complexes. In sustained ischemia, further membrane damage exposes the myocell to massive calcium intrusion, with eventual precipitation of it and cell death (reperfusion stone-heart). In case of transient, "hit and run" ischemia, the "stunned" myocardium undergoes prolonged contractile abnormalities. In keeping with fundamentals in pathophysiology of contraction, ischemic myofibrils in human hyperacute infarct, showed spare I bands, accounting for contracture and followed by loss of the regular cross-striation register; then, groups of adjacent sarcomeres were seen to join into true "contraction" bands, with Z lines impinging upon A bands and obliterating the I bands. Coagulative denaturation of contractile proteins follows, presenting as irregular, amorphous degeneration stripes astride irreversibly damaged myocells. As such, these cells can be passively overstretched by the nearby functioning muscle. In turn, the fixed waviness of viable, acutely ischemic myocardium was thought to configure, histologically, the loss of ATP-dependent "plasticity" of myofilaments, in a state of contracture. The "relaxant effect" of inotropic-chronotropic-positive catecholamines, favoring diastole, has been also pointed out. The present microscopic findings are cogent to clinicopathologic problems of coronary ischemia-reperfusion, and sudden death from cardiogenic shock.

Adenosine Triphosphate↗
PubMed1977

Polynucleotide recognition by DNA alpha-polymerase.

In a survey of template-primer preference of a mouse myeloma DNA alpha-polymerase, the fastest rate of DNA synthesis was with poly(dT) as template and (rA)24 as primer. Such a preference for poly(dT).oligo(rA) was not observed with other DNA polymerases of mouse origin. DNA synthesis in this system resulted in formation of oligo(dA) chains, not template-length poly(dA); thus, the average enzyme molecule bound to a poly(dT).(rA)24 complex and initiated a new oligo(dA) chain many times during the incubation. Binding experiments revealed that the alpha-polymerase had high affinity for poly(dT). Although the alpha-polymerase did not bind to poly(dl) and failed to replicate it inreactions with a base pair complementary primer, poly(dl) was replicated after a (dT) block had been grafted to its 3'-end and the oligo(rA) primer had been added. In similar experiments, the (dT) block was found to be much more effective than other 3'-terminal blocks in promoting replication of denatured calf thymus DNA. The results indicate that specific base sequences may regulate initiation of DNA syntehsis by this alpha-polymerase.

Cell Line↗
PubMedDate not supplied

Protein tyrosine phosphatase SHP-1 specifically recognizes C-terminal residues of its substrates via helix alpha0.

The catalytic domain of protein tyrosine phosphatase SHP-1 possesses distinct substrate specificity. It recognizes the P-3 to P-5 residues of its substrates via the beta5-loop-beta6 region. To study the substrate specificity further, we determined the structure of the catalytic domain of SHP-1 (C455S) complexed with a less-favorable-substrate peptide originated from SIRPalpha. The complex has disordered N-terminal peptide structure and reduced interactions between the N-terminal peptide and the beta5-loop-beta6 region. This could be the basis for the lower affinity of peptide pY(427) for the catalytic domain of SHP-1. In addition, by comparing the SHP-1/less-favorable peptide complex structure with the SHP-1/substrate complex structures, we identified a novel substrate-recognition site in the catalytic domain of SHP-1. This site was formed by helix alpha0 and the alpha5-loop-alpha6 motif of SHP-1, and specifically bound residues at the P + 4 and further C-terminal positions of peptide substrates.

Binding Sites↗
PubMed1990

Neonatal hepatic hemangioendothelioma identified by real-time ultrasound: report of a case.

Sonographic findings emphasizing vascular imaging in the identification of a resectable huge hepatic hemangioendothelioma in a 1-day-old neonate are reported. The prominent sonographic features consisted of dilated hepatic veins, enlarged celiac trunk and hepatic artery, indicating arteriovenous shunting, in addition to the complex echo pattern of the mass itself. Moreover, sonographic visualization of marked caliber discrepancy between hepatic and splenic arteries at their origin from the celiac trunk readily provided another subjective evidence of hepatic artery dilatation, further reflecting the hypervascularity of this hepatic mass. Hence meticulous ultrasonic study of such hepatic masses with special attention to their enlarged feeding and draining vessels can provide a simple and noninvasive approach for early recognition of this vascular hepatic tumor in the immediate newborn period.

Hemangioendothelioma↗
PubMed1981

Vasculitis: an approach for physicians.

In the absence of a satisfactory classification, a sequential approach to the problems of vasculitis is recommended to the clinician. Clinical recognition will result from a presentation of: i. a well-defined syndrome; ii. a visually identifiable vasculitis (cutaneous or retina); iii--vasculitis-associated disease (connective tissue disease, infectious and neoplastic diseases); iv. multisystem syndrome. Aetiology will be sought from infectious, drug, autoimmune or tumour origin in the main. Helpful pathological features include vessel size, cellular infiltrate, granulomata, necrosis and immune deposits. Immunopathogenesis must clearly distinguish initiators (e.g. immune complexes) from amplifiers and regulators (e.g. fibrin, complement, protease inhibitors, phagocytes). Sites of pathology may be determined by such localising mechanisms as cryoglobulins, immune complex receptors and rheological factors. A rational approach to management dependent on awareness of immunopathology includes inflammatory mediator inhibitors, immunosuppression or plasmapheresis.

Antigen-Antibody Complex↗
PubMed2001

Development of stereoselective nonaqueous capillary electrophoresis system for the resolution of cationic and amphoteric analytes.

A stereoselective ion-pair nonaqueous capillary electrophoresis (NACE) method employing the partial filling technique with N-derivatized amino acids, e.g., (R)- and (S)-3,5-dinitrobenzoyl-leucine (DNB-Leu), as chiral selector for the separation of "pseudoenantiomeric" cinchona alkaloid derivatives and other structurally related basic compounds like the enantiomers of mefloquine is presented. Originating from NACE with cinchona alkaloid derivatives as chiral counterions, this method was developed by application of the reciprocity principle of chiral recognition, which was proven to be valid for stereoselective ion-pair capillary electrophoresis (CE). A variety of basic and amphoteric selectands (SAs) could be well resolved. Thereby, the separation was primarily based on stereoselective ion-pair formation of corresponding SA stereoisomers and mobility differences of free and complexed (ion-paired) SAs. Additionally, in the case of diastereomeric SAs, naturally existing mobility differences between the diastereomers played also a role, but was shown by control experiments with racemic DNB-Leu and without selector (SO) to be of minor contribution to overall separation selectivity. Due to its simplicity, speed, and good reproducibility, the established method can be utilized for fast screening of cationic as well as amphoteric chiral compounds, and therefore is a valuable tool in the development of new chiral selectors and chiral stationary phases. Small sample amounts of the SO (4-5 mg) and only analytical amounts of SAs are needed, and about 20-50 compounds per day can be tested.

Carbamates↗
PubMed1994

Nuclear magnetic resonance studies on complementary peptides.

From the original observation that the codons for the hydrophobic and hydrophilic amino acids on one strand of the DNA may be complemented by the codons for the hydrophilic and hydrophobic amino acids, respectively, on the complementary strand, arose the molecular recognition theory which forms the basis for much of the work involving complementary peptides. A number of examples have been documented where peptides with inverted hydropathic profiles have been shown to form complexes in high-affinity chromatography and solid matrix binding assays. Nevertheless, our current understanding of the molecular forces leading to the formation of these complexes is rather rudimentary, and it is highly desirable to have a detailed three-dimensional structure of a complex of interacting complementary peptides. In this article, we provide a brief review of the solution NMR work done by different laboratories in an attempt to study these interactions.

Amino Acid Sequence↗
PubMed2004

Diversity & overlap in the mechanisms of processing protein antigens for presentation to T cells.

The immune system needs to recognise target protein antigens from pathogens residing in both extracellular and intracellular locations. Intricate proteolytic processing events that follow antigen/ pathogen encounter provide the immune system with a complex display of a heterogeneous peptide mix, instrumental in the initiation of T cell immune responses, and allow the separation of extracellular and intracellular pathogen identification. However, recent evidence shows that this conventional dimorphism in the proteolytic processing of endogenous versus internalised antigen is less restrictive than originally recognized. The events that constitute the conventional major histocompatibility complex (MHC)-restricted processing pathways are accompanied by interesting deviations that provide novel adjuncts for the processing machinery to gain access to antigen in varied intracellular locations. This review discusses these aspects of classical and non-classical processing pathways for MHC-restricted protein presentation, which play significant roles in both optimising and diversifying the peptide repertoire available for immune recognition.

Antigen Presentation↗
PubMed1989

Most anti-human CD3 monoclonal antibodies are directed to the CD3 epsilon subunit.

The T cell receptor is a molecular complex compriSed of a clonally-restricted heterodimer (Ti) responsible for specific antigen recognition and a set of invariant CD3 peptides termed gamma, delta, epsilon, zeta and eta. The latter are believed to be involved in transmembrane signaling events given that monoclonal antibodies (mAb) directed to the native CD3 structure can trigger T cell activation. We show here that the vast majority of anti-human CD3 mAb are directed to an epitope(s) encoded in part or in total by the epsilon subunit since 15 of 18 independent mAb specifically react with a murine T cell line expressing the human CD3 epsilon chain at its cell surface. The WT31 mAb is also reactive with this cell line showing that its target epitope, originally assigned to the Ti structure, rather maps to the CD3 epsilon subunit. These findings suggest that the CD3 epsilon subunit is the most exposed of the native CD3 structures which are immunogenic and that cross-linking of the CD3 epsilon chain by mAb mediates the subsequent T cell activation via the T cell receptor complex.

Animals↗