Potential of nitroimidazoles as markers of hypoxia in heart.
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Animals↗
Biomedical subjects
B Patel
Publications and source records attributed to B Patel.
Retinal and preretinal localisation of epidermal growth factor, transforming growth factor alpha, and their receptor in proliferative diabetic retinopathy.
A number of growth factors have been implicated in the development and perpetuation of preretinal fibrovascular membranes in patients with proliferative diabetic retinopathy (PDR). The aim of this study was to determine the potential role of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and their receptor (EGF-R) in PDR development. Immunostaining for EGF, TGF-alpha, and EGF-R was compared between normal retina, PDR retina, and PDR preretinal membranes. Weak staining for EGF and EGF-R was observed throughout the neural retina from non-diabetic eyes while weak to moderate staining for TGF-alpha was observed in the ganglion cell layer and the inner and outer nuclear layers. In contrast, intense staining for EGF and TGF-alpha and moderate staining for EGF-R were observed throughout the PDR retina. Immunoreactivity for EGF, TGF-alpha, and EGF-R was seen in the majority of the 11 excised membranes studied and, though variable, was generally greater than that observed in normal retinas. These results suggest an autocrine/paracrine role for EGF, TGF-alpha, and EGF-R in PDR.
Diabetic Retinopathy↗
Oxygen regulation of TGF-beta 1 mRNA in human hepatoma (Hep G2) cells.
Solid tumours comprise populations of cells whose behaviour is thought to be influenced by growth factors and the local oxygen environment. We investigated the effect of oxygen on steady state levels of transforming growth factor-beta 1 mRNA in cultured human hepatoma cells. Northern blot analysis demonstrated that hypoxia mediated an early and sustained induction of TGF-beta 1 mRNA above that seen at higher oxygen tensions. The data suggests that modulation of TGF-beta 1 expression by the local oxygen environment may be important in tumour development and progression.
Blotting, Northern↗
Antibodies to Toxoplasma gondii in eluates from filter paper blood specimens.
Blood and serum samples were tested directly for antibodies to Toxoplasma gondii, or as eluates from filter paper using the dye test, IgM enzyme-linked immunosorbent assay (ELISA) and IgM immunosorbent agglutination assay (ISAGA). The specificity and reproducibility of all three assays based on filter paper eluate was satisfactory. The sensitivity of testing eluates was 10 IU by the dye test and 53 EIU by IgM ELISA. IgM ISAGA findings, using eluate or sera, were comparable. Seroprevalence studies of congenital toxoplasmosis can be based on IgM measurement by ISAGA using eluates of filter paper stored infants' blood. Dye test and IgM ISAGA testing of eluate can be used to reduce the frequency of venepuncture when investigating infants for suspected congenital toxoplasmosis.
Animals↗
Dynamics of prolactin in amniotic fluid and extraembryonic coelomic fluid in early human pregnancy.
Concentrations of prolactin were measured by an immunoradiometric assay in 26 matched samples of amniotic fluid, extraembryonic coelomic fluid and maternal serum from 9 to 12 weeks of pregnancy and in a further 131 amniotic fluid samples from 9 to 20 weeks. Low levels of prolactin (median 40 MU/l) were present in amniotic fluid from 9 to 12 weeks. Levels in the coelomic fluid were higher (median level 371 MU/l; P < 0.0001) than in amniotic fluid. From 13 weeks, there was a rapid rise in amniotic fluid prolactin to reach a peak at 19 weeks (median level 99,850 MU/l). The pattern of increase of prolactin in amniotic fluid is similar to, but occurs 2 weeks later than that for insulin-like growth factor-binding protein-1, another major decidual product.
Amniotic Fluid↗
Sequence of the porcine apoA-I gene.
The nucleotide sequence of the swine apoA-I gene (encoding apolipoprotein) has been determined. The structure of the gene is similar to that of the human gene, and the exons exhibit a high degree of homology with those of the human gene. The porcine apoA-I gene is located adjacent to the apoC-III gene, as in humans.
Amino Acid Sequence↗
Synthesis and structure-activity relationships of a series of penicillin-derived HIV proteinase inhibitors containing a stereochemically unique peptide isostere.
A series of HIV-1 proteinase inhibitors was synthesized based upon a single penicillin derived thiazolidine moiety. Reaction of the C-4 carboxyl group with (R)-phenylalaninol gave amide 10 which was a moderately potent inhibitor of HIV-1 proteinase (IC50 = 0.15 microM). Further modifications based on molecular modeling studies led to compound 48 which contained a stereochemically unique statine-based isostere. This was a potent competitive inhibitor (Ki = 0.25 nM) with antiviral activity against HIV-1 in vitro (5 microM). Neither modification to the benzyl group in an attempt to improve interaction with the S2' pocket, nor introduction of a hydrogen bond donating group to interact with residue Gly48' resulted in improved inhibitory or antiviral activity.
Amino Acid Sequence↗
Cytolysis of leukemic B-cells by T-cells activated via two bispecific antibodies.
Bispecific monoclonal antibodies can be used in the activation of effector cells to lyse autologous tumor cells. We analyzed the activation of human T-cells in vitro with bispecific monoclonal antibodies, which were generated by hybridoma-hybridoma fusion. Preactivated allogeneic and autologous T-cells could be triggered to lyse tumoral B-cells in the presence of CD3 x CD19 bispecific antibodies. In addition, the combined use of two CD3 x CD19 plus CD28 x CD22 bispecific antibodies induced optimal interleukin 2 secretion by Jurkat T-cell acute lymphocytic leukemia cells in the presence of target B-cells. The same antibody combination was able to generate cytolytic effector cells without prior activation, when resting T-cells were cocultured with freshly isolated autologous leukemic B-cells in the presence of the bispecific antibodies. The results suggest that signals required to activate cytolytic T-cell precursors can be provided by the two bispecific antibodies. Although activation of resting T-cells can be achieved by CD3 x CD19 bispecific antibodies in association with monospecific bivalent CD28 antibodies, the second bispecific antibody, CD28 x CD22, further increases the specificity of the target cell dependent activation of T-cells. When used for immunotherapy of B-cell malignancies, the CD3 x CD19 and CD28 x CD22 bispecific antibody combination may avoid the need for ex vivo activated effector cells, because the antibodies may induce T-cell activation directly at the tumor site.
Animals↗
Organization of the human gene encoding the cytoskeletal protein vinculin and the sequence of the vinculin promoter.
The human vinculin gene contains 22 exons ranging in size from 71 base pairs (bp) to 303 bp (average 155 bp) with the exception of exon 22 which contains 144 bp of coding sequence and 1848 bp of 3'-untranslated sequence including two polyadenylation signals. There is a limited correlation between exon boundaries and functional domains within the vinculin molecule. The talin-binding domain in vinculin spans residues 1-258, and the first 6 exons encode residues 1-261. Similarly, the predicted boundaries of the central repeat domain (residues 259-589) are close to the boundaries of exons 7 and 12. Analysis of vinculin mRNAs in human uterus showed that alternative splicing of the gene is limited to exon 19, which encodes the 68 amino acids included in the muscle-specific isoform called metavinculin. We have determined 1.1 kilobases of sequence 5' of the transcription start site. The vinculin promoter lacks a TATA box, but does contain six Sp1 sites, and a CArG box at position -262 which forms the core of the serum response element found in immediate-early response genes. Expression of a vinculin promoter/CAT construct is serum-inducible in NIH3T3 cells demonstrating that the promoter does contain a functional serum response element.
3T3 Cells↗
The control of cellular adenine nucleotides: the discovery and metabolism of an oligomeric derivative of ATP and phosphoglyceric acid in mitochondrial intermembrane space.
The observation of large systematic variations in the purine nucleotides in perfused rat hearts inexplicable by known metabolic transformations led us to propose the existence of some unknown major form of nucleotide. Radiolabelling studies located a rapidly metabolized TCA/methanol-insoluble species in heart, kidney and liver which selective digestion and chromatography allied to 31P-nmr and mass spectrometry suggested to be a tetraphosphate derivative of adenosine and phosphoglyceric acid of the form p3Gri1p[pp5'p3Gri1p]npp5'A, oligo-phosphoglyceroyl-ATP. Partial chemical synthesis has confirmed this basic structure. We have shown that substantial amounts of oPG-ATP are located in liver mitochondrial intermembrane space tightly bound to a specific 3' phosphodiesterase which releases the monomer p3Gri1ppp5'A. Here we show that oPG-ATP is remarkably stabilized by Mg2+ ions and describe the 300-fold purification of the stable substrate-free form of the 3' phosphodiesterase; it has an apparent M(r) = 165k with possibly up to 4 subunits of M(r) = 42.5k, 41.9k, 40.5k and 38.9k. The potential function of oPG-ATP is briefly discussed.
Adenosine Triphosphate↗
Inhaled nedocromil sodium in the management of asthma.
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Administration, Inhalation↗
Transforming growth factor-alpha (TGF-alpha) in a semisolid dosage form: preservative and vehicle selection.
The selection of an ideal semisolid vehicle for growth factors presents a challenge. Some antimicrobial agents are known to delay wound healing. The objective of this investigation was to identify appropriate preservatives and vehicles for TGF-alpha. Criteria for acceptance are noninterference with the mitogenic activity of TGF-alpha as well as adequate product preservation. Vehicles considered were o/w creams, ointments, and a gel. Combinations of six preservatives were tested. Selection was determined using both microbial preservative challenge and TGF-alpha mitogenic assay. In the former, 10 species of microorganisms were inoculated into formulation samples. At selected time intervals, it was determined whether colonies decreased, increased, or remained constant. In the mitogenic assay, samples of either preservatives or formulation prototypes were introduced to TGF-alpha-stimulated fibroblast cell cultures. Mitogenesis was determined by measuring 3H-dThd uptake into newly synthesized DNA. As preservatives, sorbic acid and quaternium-15 appear to satisfy both selection criteria. A thermosetting gel appears most promising as vehicle.
Cell Division↗
The cytoskeletal protein talin contains at least two distinct vinculin binding domains.
We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and 125I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of 125I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2-6 x 10(-7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin.
3T3 Cells↗
Immunoglobulin-A detection and the investigation of clinical toxoplasmosis.
Current serological methods for the investigation of Toxoplasma gondii infection are unreliable for the diagnosis of congenital disease, reactivated infection associated with the acquired immune deficiency syndrome (AIDS), or the determination of the date of onset of infection. An enzyme-linked immunosorbent assay (ELISA) and an immunosorbent agglutination assay (ISAGA) were developed for the detection of toxoplasma-specific immunoglobulin-A (IgA) and used to investigate patients in these three categories. The IgA ISAGA and IgA ELISA were found to be reproducible and specific tests. The IgA ISAGA demonstrated enhanced sensitivity. Measurement of IgA in patients with toxoplasma-associated lymphadenopathy of known duration and AIDS patients with toxoplasma infection was of limited value. Detection of specific IgA by ISAGA was more sensitive than conventional methods for the diagnosis of congenital toxoplasmosis. We recommend the investigation of infant sera with IgA ISAGA. IgA estimation is not indicated in other clinical situations.
Acquired Immunodeficiency Syndrome↗
Development of obsessive-compulsive symptoms during clozapine treatment.
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Adult↗
Food safety and inspection service update on food safety of animals derived from biotechnology experiments.
Recent progress in the field of biotechnology and the production of transgenic livestock has raised a question regarding the need for the regulation of these animals. There is also the need to regulate nontransgenic animals resulting from transgenic animal research. It is anticipated that several governmental agencies will be involved in regulatory issues pertaining to these animals. The United States Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) will ultimately be responsible for ensuring that transgenic animals intended for human consumption are wholesome, unadulterated, and properly labeled. The FSIS has implemented a program for the regulation of slaughtering nontransgenic animals resulting from transgenic animal experiments. However, the FSIS has not yet approved any transgenic livestock for slaughter. Scientists from the FSIS, in conjunction with other government agencies, are currently developing guidelines for the slaughter of transgenic animals.
Animals↗
A chick skeletal-muscle alpha-actinin gene gives rise to two alternatively spliced isoforms which differ in the EF-hand Ca(2+)-binding domain.
A chick non-muscle alpha-actinin cDNA probe encoding the EF-hand region of molecule was used to screen a lambda gt10 chick brain cDNA library from 14-day embryos. A partial 2.1-kb alpha-actinin cDNA was isolated (8W cDNA) which encoded a protein identical to chick skeletal-muscle alpha-actinin, except in the C-terminal part of the first EF hand. In the variant, the 22 residues found in the skeletal-muscle isoform were replaced by a stretch of 26 unique residues. Analysis of the structure of the skeletal-muscle alpha-actinin gene showed that the region of divergence was encoded by two exons which are alternatively spliced. Quantitative reverse transcriptase/polymerase chain reaction (RT/PCR) was used to investigate the levels of the alpha-actinin transcripts in various tissues. The skeletal-muscle alpha-actinin variant was expressed at low levels in brain, liver and spleen, but could not be detected in skeletal muscle. Surprisingly, skeletal-muscle alpha-actinin mRNA was also expressed in brain, liver and spleen. The RT/PCR products were authenticated by using diagnostic restriction enzyme sites and by sequencing. The splice variant derived from the skeletal-muscle alpha-actinin gene was also detected in a variety of cDNA libraries from both adult and embryonic tissues by PCR. Although a transcript encoding this alpha-actinin splice variant is expressed in non-muscle tissues, neither of the two EF-hands would be predicted to be functional, making it unlikely to be a typical non-muscle isoform which are calcium-sensitive with respect to binding actin. The two vertebrate non-muscle alpha-actinins sequenced to date also have a spacer of five amino acids between the two EF hands, whereas in the variant, the spacer is just four residues in length. Further analysis will be required before this alpha-actinin isoform, which we refer to as SKv, can be classified as muscle or non-muscle alpha-actinin. We propose a new nomenclature to describe the various alpha-actinin genes and their transcripts.
Actinin↗
Effect of direct control of electroosmosis on peptide and protein separations in capillary electrophoresis.
The separations of peptide and protein mixtures in capillary zone electrophoresis (CZE) at various solution conditions were studied with the direct control of electroosmosis. The zeta potential at the aqueous/capillary interface and the resulted electroosmosis in the presence of an electric field were directly controlled by using an additional electric field applied from outside of the capillary. The controlled electroosmotic flow affected the migration time and zone resolution of peptide and protein mixtures. The changes in the magnitude and polarity of the zeta potential caused the various degrees of peptide and protein adsorption onto the capillary through the electrostatic interactions. The separation efficiencies of peptide and protein mixtures were enhanced due to the reduction in peptide and protein adsorption at the capillary wall. The direct manipulations of the separation efficiency and resolution of peptide and protein mixtures in CZE were demonstrated by simply controlling the zeta potential and the electroosmotic flow with the application of an external electric field.
Adrenocorticotropic Hormone↗