Effect of alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues.

α-Difluoromethylornithine (RMI 71.782), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (E.C. 4.1.1.17) $\text{in vitro}$, causes a rapid, long-lasting, dose-dependent decrease of ornithine decarboxylase activity in prostate and, to a lesser extent, in thymus and testis of rats when injected intraperitoneally. Repeated doses (100 mg/kg or 1 g/kg) of α-difluoromethylornithine given to rats for two weeks markedly decreased polyamine concentrations in several rat tissues and selectively slowed down growth of ventral prostate and of thymus.     

Swelling and contraction of potato mitochondria

Mitochondria isolated from potato tubers fail to undergo passive osmotic swelling when suspended in isotonic Na⁺ acetate or phosphate, in NaCl following addition of tripropyltin, or in Na⁺ nitrate following addition of an uncoupler. Swelling under each of these conditions in mitochondria from other sources has been attributed to the inward movement of Na⁺ on an endogenous Na⁺/H⁺ exchanger. Such a monovalent cation/H⁺ exchanger has also been implicated in respiration-dependent cation extrusion and contraction of swollen mitochondria. Potato mitochondria swollen in chloride and nitrate salts extrude ions and contract when respiration is initiated. The contraction reaction is slower and less efficient than that in beef heart mitochondria, but like the latter, is sensitive to uncouplers and stimulated by nigericin, butacaine, and Mg²⁺. These comparative studies suggest that a cation⁺/H⁺ exchanger is present in potato tuber mitochondria, but that it functions exclusively as a cation-extruding mechanism. They further suggest that cation⁺/H⁺ exchange activity is not identical in mitochondria from different sources and that these exchange components may have a directionality and regulatory features which differ with the metabolic needs of the source tissue.     

The interaction of methemoglobin and modified poly-(hydroxyethylmethacrylates) studied by spectroscopic methods

The interaction between methemoglobin (MetHb) and macroporous matrices on the basis of polymethacrylates was investigated by means of optical and e.p.r. spectroscopy. The spectroscopic data show that the adsorption of MetHb to imidazole-containing matrices occurs by complex formation between matrix-bound imidazole and the iron of the prosthetic group, with all 4 polypeptide chains of the MetHb molecule being included in the interaction. The adsorption to hydrophobic side chains containing matrices leads, via the protein-matrix interaction, to considerable disturbances of iron protoporphyrin IX in equilibrium or formed from protein-contacts, which are of general importance with respect to the functional variablity and control, respectively, of iron porphyrins in hemoproteins. In case of matrix containing n-hexyl groups deoxyHb is oxidized by O2 to MetHb, instead of being oxygenated to HbO2. Not all prosthetic groups are able to bind N-3. With the increase in hydrophobicity of the matrix a conformational change is enforced leading in the beta-chains to the direct interaction between iron and sulfur of cysteine (beta-cys 92), as it is proved in all cytochrome P-450 and other model compounds.     

Direct effects of ionizing radiation on integral membrane proteins. Noncovalent energy transfer requires specific interpeptide interactions

The 12 transmembrane alpha helices (TMHs) of human erythrocyte glucose transporter were individually cut by pepsin digestion as membrane-bound 2.5-3.5-kDa peptide fragments. Radiation-induced chemical degradation of these fragments showed an average target size of 34 kDa. This is 10-12 x larger than the average size of an individual TMH, demonstrating that a significant energy transfer occurs among these TMHs in the absence of covalent linkage. Heating this TMH preparation at 100 degrees C for 15 min reduced the target size to 5 kDa or less, suggesting that the noncovalent energy transfer requires specific helix-helix interactions. Purified phospholamban, a small (6-kDa) integral membrane protein containing a single TMH, formed a pentameric assembly in sodium dodecyl sulfate. The chemical degradation target size of this phospholamban pentamer was 5-6 kDa, illustrating that not all integral membrane protein assemblies permit intersubunit energy transfer. These findings together with other published observations suggest strongly that significant noncovalent energy transfer can occur within the tertiary and quaternary structure of membrane proteins and that as yet undefined proper molecular interactions are required for such covalent energy transfer. Our results with pepsin-digested glucose transporter also illustrate the importance of the interhelical interaction as a predominating force in maintaining the tertiary structure of a transmembrane protein.     

Movement correction of digital sequence angiographies of the retina]

Retinal hemodynamics can be quantified from videoangiographic image sequences by digital image processing. Intensity changes of dye dilution curves provide dynamics parameters of the local retinal blood flow. The measuring points of dye dilution curves have to be fixed on identical image contents in each image of a complete image sequence. To obtain measurements for every pixel on the retinal surface a motion-compensated image sequence is required. A new method adapted to the compensation of eye motion and movement artifacts in Scanning Laser Ophthalmoscopy in long image sequences (300-500 images) is presented in this paper. To inhibit error propagation of time sequential motion estimation, the eye movement is divided into two dynamic movements components. The method presented permits compensation for eye motion in retinal fluorescein angiographic sequences. Owing to the short calculation times, this algorithm can be used in clinical routine.     

Molecular dynamics study of a complex between the human histocompatibility antigen HLA-A2 and the IMP58-66 nonapeptide from influenza virus matrix protein.

The structure of the influenza-virus-matrix-protein (IMP) 58-66 nonapeptide, bound to the major-histocompatibility-complex-encoded human leukocyte antigen (HLA) A2 protein was studied by molecular dynamics simulation. Starting from the extra electron density map of peptides co-crystallized with HLA-A2, the nonapeptide IMP58-66 was docked residue by residue in the protein binding cleft. The complex was simulated for 100 ps in a shell of 1372 water molecules. The averaged simulated HLA-A2 conformation was found to be similar to the crystal structure (0.182 nm RMS deviation, for the backbone atoms of the alpha 1-alpha 2 domain). Nine out of the 14 hydrogen bonds observed in the antigen-binding site were reproduced in the simulation. The IMP58-66 peptide exhibits an extended conformation with kinks at positions 3 and 5. The side chains of residues 2, 3 and 9 develop van der Waals' interactions with hydrophobic pockets of HLA-A2, corresponding to polymorphic residues of the major-histocompatibility-complex-encoded proteins. Both the N-terminus and C-terminus of the nonapeptide were anchored in the antigen-binding groove by hydrogen bonds with conserved amino acids. The N-terminus was more flexible and contacts four HLA-A2 conserved tyrosines (Tyr7, Tyr59, Tyr159 and Tyr171) and Glu63 by direct or water-relayed hydrogen bonds. Water intercalation occurred only around the N-terminus of the peptide, the C-terminal carboxylate forming strong hydrogen bonds with polar residues (Tyr84 and Thr143) and a salt bridge with Lys146 all over the molecular dynamics simulation. This model is fully compatible with the recently published crystal structure of the HLA-B27 protein, complexed by a mixture of self nonapeptides.     

Prevention and therapy of experimental autoimmune neuritis by an antibody against T cell receptors-alpha/beta.

The mAb R73 directed to the TCR-alpha/beta of rat lymphocytes was tested for its therapeutic potential during the effector phase of experimental autoimmune neuritis (EAN) in Lewis rats. EAN can be actively induced by immunization with bovine peripheral nerve myelin, bovine P2 protein, or a peptide containing its neuritogenic epitope and serves as a model of the human Guilain-Barré syndrome. Adoptive transfer of activated P2-specific T lymphocytes also produces the monophasic disease (AT-EAN) characterized by inflammation and demyelination of peripheral nerves and highlights the central role of T lymphocytes in the pathogenesis of EAN. A single administration of the mAb R73 immediately after injection of activated P2-specific T line cells completely prevented the development of clinical and electrophysiologic signs of EAN in most animals and greatly alleviated the disease in the others. In further experiments mAb R73 was applied after the appearance of first clinical signs of EAN actively induced by immunization with a neuritogenic peptide or bovine peripheral nerve myelin. In both cases the anti-TCR-alpha/beta mAb reversed clinical signs of EAN and prevented the development of peripheral nerve dysfunction. In vivo and in vitro data suggest that impairment of Ag recognition and T cell function by occupancy of the TCR and R73-induced TCR-modulation rather than depletion of TCR-alpha/beta-bearing lymphocytes is the decisive mechanism underlying suppression of EAN that is apparent already within 48 h of the first R73 injection.     

Herpesvirus saimiri oncogene STP-C488 encodes a phosphoprotein.

The STP-C488 open reading frame of herpesvirus saimiri encodes an oncoprotein that has transforming and tumor-inducing activities independent of the rest of the herpesvirus genome. STP-C488 protein has an unusual, membrane-associated, fibrous structure and is located primarily in perinuclear compartments. We now report that STP-C488 is phosphorylated in vivo. The phosphorylated form, which accounted for about 15% of STP-C488 in transformed cells, migrated slightly more slowly through sodium dodecyl sulfate-polyacrylamide gels than unphosphorylated STP-C488. A serine residue near the amino terminus was shown to be the site of phosphorylation. However, phosphorylation was not required for transformation of Rat-1 cells by STP-C488.     

Role of cytoskeleton in cell shaping of developing mesophyll of wheat (Triticum aestivum L.).

The effects of oryzalin and cytochalasin B (CB) on microtubule and actin microfilament arrays and on cell shaping were investigated in developing wheat mesophyll. Excised immature leaf sections capable of differentiating were incubated with the drugs. The behavior of the cytoskeleton was monitored by fluorescence microscopy after labeling with fluorescent dyes. Brief incubation with oryzalin (40 min, 10 microM) caused disassembly of microtubules. Recovery of microtubule arrays was comparatively slow after removal of the drug. Cells failed to establish transverse cortical bands of microtubules and transverse hoops of wall reinforcement. They expanded isodiametrically rather than longitudinally without forming lobes typical of wheat mesophyll cells. Brief treatment with CB (60 min, 20 micrograms ml-1) appeared to disrupt the microfilament arrays. Filaments recovered rapidly after removal of CB, and cells were able to shape in an apparently normal fashion. Continuous incubation at comparatively low concentration of CB (4 micrograms ml-1) appeared to cause selective loss of the fine transverse cortical microfilament arrays. Cortical transverse microtubule arrays persisted, but failed to form distinct bands in the majority of the cells. Cells were able to elongate in an almost normal fashion, but no lobes were formed.     

Alpha-helical small molecular size analogues of neuropeptide Y: structure-activity relationships.

C-terminal analogues of neuropeptide Y (NPY) of small molecular size have been synthesized. The influence of chain length, single or multiple amino acid substitution, and segment substitutions on receptor binding, pre- and postsynaptic biological activity, and conformational properties have been investigated. Receptor binding and in vivo assays revealed biological activity for NPY Ac-25-36 that increased with increasing alpha-helicity. In attempts to stabilize the alpha-helical content, three independent types of modified NPY Ac-25-36 analogues were synthesized. Strong agonistic activities could be detected in a series of discontinuous analogues, which are constructs of N-terminal parts linked via different spacer molecules to C-terminal segments. One of the most active molecules was NPY 1-4-Aca-25-36 (Aca, epsilon-aminocaproic acid). For the first time conformational properties of a series of small NPY analogues have been investigated by CD, and correlated with biological activity and receptor binding. A C-terminal dodecapeptide segment of NPY with an amount of 50% substitution to the native C-terminal sequence of NPY was found to exhibit significant receptor binding.