Adhesion of lymphocytes to bladder cancer cells: the role of the alpha(E)beta(7) integrin

The alpha(E)beta(7)integrin (defined by CD103) is expressed by most intra-epithelial lymphocytes (IEL) but by fewer than 2% peripheral blood lymphocytes (PBL). An important ligand for this molecule is the epithelial cell adhesion molecule E-cadherin. Loss of E-cadherin is associated with increased invasion and metastasis in bladder cancer. This study examines the role of the alpha(E)beta(7)-E-cadherin interaction in lymphocyte targeting of bladder cancer cells. Lymphocytes were activated in vitro by mixed lymphocyte reaction (MLR) and CD103 was upregulated by treatment with transforming growth factor beta (TGFbeta). The CD103(+) lymphocytes were used in a flow cytometric adhesion assay with bladder cancer cell lines, differing in expression of E-cadherin and intercellular adhesion molecule-1 (ICAM-1). Antibody blockade was used to confirm the relative importance of CD103 and ICAM-1 to intercellular adhesion. Lymphocytes with upregulated CD103 compared to control lymphocytes showed enhanced adhesion to an E-cadherin expressing bladder cancer cell line ( P=0.0003). This increased adhesion could be abrogated by anti-CD103 adhesion blockade. For ICAM-1 expressing bladder cells, adhesion of lymphocytes could be markedly reduced using anti-ICAM-1 blockade. In conclusion, the upregulation of CD103 by lymphocytes increases adhesion to E-cadherin expressing bladder cancer targets. Loss of E-cadherin in bladder cancer progression may provide a mechanism both for increased invasion and effective immune evasion.     

99mTc-Labeling of a hydrazinonicotinamide-conjugated LTB(4) receptor antagonist useful for imaging infection and inflammation

This report describes the (99m)Tc labeling of a hydrazinonicotinamide (HYNIC)-conjugated LTB(4) receptor antagonist (SG380). The ternary ligand technetium complex [(99m)Tc(SG38)(tricine)(TPPTS)] (RP517) was prepared using a non-SnCl(2)-containing formulation ((2001) J. Pharm. Sci. 90, 114-123). Unlike other HYNIC-conjugated small biomolecules, SG380 is lipophilic and has low solubility in the kit matrix. Using a combination of a solubilizing agent (Lysolecithin) and a cosolvent (ethanol), we have developed a new formulation for routine preparation of RP517. Using this formulation, RP517 can be prepared in high radiochemical purity (RCP > 90%) and remains stable in the kit matrix for at least 6 h. We also prepared the corresponding (99)Tc analogue, [(99)Tc]RP517. An HPLC concordance experiment using RP517 and [(99)Tc]RP517 showed that the same technetium complex was prepared at both the tracer and macroscopic levels. The LC-MS data are completely consistent with the 1:1:1:1 composition for Tc:SG380:tricine:TPPTS.     

Age-related changes in human bone proteoglycan structure. Impact of osteogenesis imperfecta

Proteoglycans (PGs) are a family of molecules that undergo extensive post-translational modifications that include addition of glycosaminoglycan (GAG) chains as well as N- and O-linked oligosaccharides to the protein core. PG composition and structure have been reported to alter with age. To test whether the post-translational modifications to PGs can serve as in vitro surrogate end point markers for chronological age, the extent of GAG modifications was determined for PGs derived from normal human bone cells of 14 donors (age range, fetal to 60 years). Isolated cells were steady state radiolabeled with (35)SO(4)(2-) and [(3)H]GlcN. For biglycan and decorin, iduronate content was linearly correlated with age (increased 1.5x between fetal and age 60 years). For the syndecan-like heparan sulfate PG, the N-sulfation of post-natal cells increased over 3.5-fold until reaching a plateau during the 4th decade of life. The amount of O-linked oligosaccharides was also found to decrease as a function of increasing normal donor age, whereas the specific activity of the metabolic precursor pool remained constant regardless of donor age. These age-related changes in post-translational modifications were then used to demonstrate that osteoblasts derived from patients with osteogenesis imperfecta did not exhibit facets of a pre-mature aging, but rather were arrested in a fetal-like phenotypic state. A growth matrix rich in thrombospondin altered PG metabolism in osteoblastic cells, resulting in the production and secretion of the fetal-like (rich in O-linked oligosaccharides) forms of decorin and biglycan. This effect was qualitatively different from the effect of transforming growth factor-beta, which predominantly altered GAGs rather than O-linked oligosaccharides. No other Arg-Gly-Asp protein (fibronectin, vitronectin, type I collagen, osteopontin, and bone sialoprotein) showed any detectable effect on PG metabolism in bone cells. These results indicate that a proper matrix stoichiometry is critical for metabolism of PGs.     

Kinetic effects of myosin regulatory light chain phosphorylation on skeletal muscle contraction

Kinetic analysis of contracting fast and slow rabbit muscle fibers in the presence of the tension inhibitor 2,3-butanedione monoxime suggests that regulatory light chain (RLC) phosphorylation up-regulates the flux of weakly attached cross-bridges entering the contractile cycle by increasing the actin-catalyzed release of phosphate from myosin. This step appears to be separate from earlier Ca(2+) regulated steps. Small step-stretches of single skinned fibers were used to study the effect of phosphorylation on fiber mechanics. Subdivision of the resultant tension transients into the Huxley-Simmons phases 1, 2(fast), 2(slow), 3, and 4 reveals that phosphorylation reduces the normalized amplitude of the delayed rise in tension (stretch activation response) by decreasing the amplitudes of phase 3 and, to a lesser extent, phase 2(slow). In slow fibers, the RLC P1 isoform phosphorylates at least 4-fold faster than the P2 isoform, complicating the role of RLC phosphorylation in heart and slow muscle. We discuss the functional relevance of the regulation of stretch activation by RLC phosphorylation for cardiac and other oscillating muscles and speculate how the interaction of the two heads of myosin could account for the inverse effect of Ca(2+) levels on isometric tension and rate of force redevelopment (k(TR)).     

DNA-binding interactions and conformational fluctuations of Tc3 transposase DNA binding domain examined with single molecule fluorescence spectroscopy

The fluorescent dye tetramethylrhodamine (TMR) was conjugated to a synthetic peptide containing the sequence-specific DNA binding domain of Tc3 transposase. Steady-state and single molecule fluorescence spectroscopy was used to investigate protein conformational fluctuations and the thermodynamics of binding interactions. Evidence is presented to show that the TMR-Tc3 conjugate exists in at least two conformational states. The most stable conformation is one in which the TMR fluorescence is quenched. Upon binding to DNA, the total fluorescence from TMR-Tc3 increases by three- to fourfold. Single molecule measurements of TMR-Tc3 bound to DNA shows that this complex also fluctuates between a fluorescent and quenched form. The fluorescent form of the conjugate is stabilized when bound to DNA, and this accounts for part of the increase in total fluorescence. In addition, the inherent photodynamics of the dye itself is also altered (e.g., fluorescent lifetime or triplet yield) in such a way that the total fluorescence from the conjugate bound to DNA is enhanced relative to the unbound form.     

Identification of bovine heart cytochrome c oxidase subunits modified by the lipid peroxidation product 4-hydroxy-2-nonenal

Bovine heart cytochrome c oxidase (CcO) was inactivated by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in a time- and concentration-dependent manner with pseudo-first-order kinetics. Cytochrome c oxidase electron transport activity decreased by as much as 50% when the enzyme was incubated for 2 h at room temperature with excess HNE (300-500 microM). HNE-modified CcO subunits were identified by two mass spectrometric methods: electrospray ionization mass spectrometry (ESI/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). All of the experimentally determined molecular masses were in excellent agreement with published sequence values with an accuracy of approximately 1 part per 10000 mass units for subunits smaller than 20 kDa and approximately 1 part per 1000 mass units for the three subunits larger than 20 kDa. Both MS methods detected six CcO subunits with an increased mass of 156 Da after reaction with HNE (subunits II, IV, Vb, VIIa, VIIc, and VIII); this result indicates a single Michael-type reaction site on either a lysine or histidine residue within each subunit. Reaction of HNE with either subunit VIIc or subunit VIII (modified approximately 30% and 50-75%, respectively) must be responsible for CcO inhibition. None of the other subunits were modified more than 5% and could not account for the observed loss of activity. Reaction of HNE with His-36 of subunit VIII is most consistent with the approximately 50% inhibition of CcO: (1) subunit VIII is modified more than any other subunit by HNE; (2) the time dependence of subunit VIII modification is consistent with the percent inhibition of CcO; (3) His-36 was identified as the HNE-modified amino acid residue within subunit VIII by tandem MS analysis.     

Roles of the complement system in human neurodegenerative disorders

Complement is an important component of the innate immune response with the capacity to recognize and clear infectious challenges that invade the CNS through a damaged blood brain barrier. For instance, the membrane attack complex is involved in cytotoxic and cytolytic activities while other smaller fragments lead to cell activation (chemotaxis) and phagocytosis of the intruders. It is noteworthy that there is a growing body of evidence that uncontrolled complement biosynthesis and activation in the CNS can contribute to exacerbate the neuronal loss in several neurodegenerative disorders. We provide here an insightful review of the double-edged sword activities of the local innate complement system in the CNS and discuss further the potential therapeutic avenues of delivering complement inhibitors to control brain inflammation.     

<Emphasis Type="Italic">Brassica</Emphasis> biotechnology: Progress in cellular and molecular biology

Summary Considerable progress has been accomplished in the cellular and molecular biology of Brassica species in the past few years. Plant regeneration has been increasingly optimized via organogenesis and somatic embryogenesis using various explants; with tissue culture improvements focusing on factors such as age of the explant, genotype, and media additives. The production of haploids and doubled haploids using microspores has accelerated the production of homozygous lines in the Brassica species. Somatic cell fusion has facilitated the development of interspecific and intergeneric hybrids in the sexually incompatible species of Brassica. Crop improvement using somaclonal variation has also been achieved. The use of molecular markers in marker-assisted selection and breeding, transformation technology for the introduction of desirable traits, and a comparative analysis of these as well as their future prospects are important parts of the current research that is reviewed.     

Antioxidant role of N-acetyl cysteine isomers following high dose irradiation

High dose, acute radiation exposure, as in radiation accidents, induces three clinical syndromes that reflect consequences of oxidative protein, lipid, and DNA damage to tissues such as intestine, lung, and liver. In the present study, we irradiated C57BL/6 mice with 18 Gy whole-body radiation (XRT) and evaluated N-acetyl cysteine (NAC) isomers LNAC and DNAC as potential radioprotectors under conditions that would model the gastrointestinal syndrome. We focused on tissues thought not immediately involved in the gastrointestinal syndrome. Both LNAC and DNAC protected the lung and red blood cells (RBC) from glutathione (GSH) depletion following radiation exposure. However, only LNAC also supplemented the spleen GSH levels following XRT. Protection from increased malondialdehyde (MDA) levels (lung) and increased 8-hydroxy-deoxyguanosine (8-oxo-dG) presence (liver) following XRT was observed with treatment by either isomer of NAC. These results imply that either NAC isomer can act as a radioprotectant against many aspects of oxidative damage; chirality is only important for certain aspects. This pattern would be consistent with direct action of NAC in many radioprotection and repair processes, with a delimited role for NAC in GSH synthesis in some aspects of the problem.