Characterization of rat Leydig cell gonadotropin receptor structure by affinity cross-linking

The present study was intended to examine the structure of the rat Leydig cell gonadotropin receptor. Leydig cell suspensions were prepared by either collagenase digestion or mechanical disruption of the testes. The cells were incubated with 125I-human chorionic gonadotropin (hCG) following which the bound 125I-hCG was covalently cross-linked to the cell surface receptor using a cleavable (dithiobis(succinimidyl propionate] and a noncleavable (disuccinimidyl suberate) cross-linking reagent. The extracted cross-linked membrane proteins were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and subjected to autoradiographic analysis. Under nonreducing conditions, three radiolabeled bands, in addition to intact hCG and its alpha-subunit, were detected with apparent molecular weights of 184,000, 136,000, and 103,000. However, under reducing conditions, three radiolabeled bands migrated on the gel corresponding to molecular weights of 144,000, 106,000, and 75,000. The binding of 125I-hCG to the receptor was inhibited by hCG and luteinizing hormone, but not by a number of other peptides or proteins. The radiolabeled bands were not detectable in hCG down-regulated Leydig cells. Furthermore, a similar autoradiographic pattern of 125I-hCG-linked complexes was seen when the 125I-linked receptor complex was subjected to immunoprecipitation with anti-hCG antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, evidence was obtained indicating that these three labeled bands were derived from the same molecular species. The data suggests that the hCG receptor in Leydig cell is probably an oligomeric complex with a molecular weight of about 250,000, which is composed of three polypeptide chains of molecular weights 121,000, 83,000, and 52,000 held together through noncovalent forces. Additionally, collagenase treatment of Leydig cells does not appear to alter the autoradiographic pattern of the 125I-hCG-linked receptor.     

Testing the two-state model: anomalous effector binding to human hemoglobin

Three allosteric states are required to describe the relaxation of (carbon monoxy) hemoglobin following flash photolysis. Combined absorbance and fluorescence probes were used. The absorbance signals consist of a component corresponding to ligand recombination and a component for the R-T transition. The fluorescence of 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), an analogue of 2,3-diphosphoglycerate, shows rates and amplitudes correlated with the absorbance transients. Measurements were made at pII 6, 6.5, and 7.0 at CO partial pressures of 0.1 and 1 atm. The fractional photolysis was varied in each case to change the initial distribution of the R states. Data show an immediate absorbance change due to ligand dissociation, while the changes in the ligand isosbestic and the fluorescence signals occur with time constants of 80 microseconds (at pH 6.5). The signals then show a biphasic return to equilibrium, characteristic of the allosteric system. The measurements provide three independent probes of the kinetics of the substates of hemoglobin. Although the ligand binding data can be generally represented by a two-state model, the fluorescence data require T states with different affinities for HPT.     

Mechanism of action of Pseudomonas syringae phytotoxin, syringomycin. Interaction with the plasma membrane of wild-type and respiratory-deficient strains of Saccharomyces cerevisiae

The effects of the phytotoxin, syringomycin, produced by Pseudomonas syringae pv. syringae, were examined on cells of a wild-type and a respiratory-deficient (rho0) mutant of Saccharomyces cerevisiae. The growth of both strains in liquid culture was inhibited by 0.5 micrograms syringomycin per ml and higher. Uptake rates of tetraphenylphosphonium and dimethyloxazolidine ions in cell suspensions of both strains increased when 1.5 micrograms per ml syringomycin was added. These responses were kinetically and quantitatively similar in the two strains and indicated increases in electrical potential (cell interior negative) and pH differences (cell interior alkaline) across the plasma membrane. Glucose (0.1 M) enhanced the effect on the electrical potential, was required for the pH changes, and increased the cellular ATP levels. These results show that the effects of syringomycin are energy-dependent and are due to alterations of plasma membrane and not to mitochondrial function.     

Topographic modeling of free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer: bending of the 3'-terminal arm in tRNAfMet

Conformations of tRNAfMet, free and methionyl-tRNA synthetase bound forms, are analyzed by using singlet-singlet energy transfer as a spectroscopic ruler. tRNAfMet(8-13,3'-Flc), tRNAfMet(8-13,D-Etd), and tRNAfMet(3'-Flc,D-Etd) are prepared by sequential chemical modifications. The methionyl-tRNA synthetase binding affinity of these double-labeled tRNAfMets is similar to those of unmodified tRNAfMet. The fluorescence properties of the individual fluorophore in these tRNAs, including emission spectra, anisotropy, and quenching by methionyl-tRNA synthetase, are similar to those of single-labeled tRNAfMet. The transfer efficiencies of double-labeled tRNAfMets, as determined by both donor quenching and sensitized emission, showed efficient energy transfer in all cases. Random orientation being assumed, the apparent distances are 25 A between 8-13 and D20, 44 A between 8-13 and the 3'-terminus, and 49 A between the 3'-terminus and D20, respectively, in free tRNAfMet. Upon binding of methionyl-tRNA synthetase, the apparent distances are 25 A between 8-13 and D20, 45 A between 8-13 and the 3'-terminus, and 54 A between the 3'-terminus and D20, respectively. These results provide topographic models of these specific locations in free and methionyl-tRNA synthetase bound tRNAfMet and suggest that the immobilized 3'-terminal arm in the amino acid acceptor stem bends toward the inner loop of the L-shaped tRNA upon binding of methionyl-tRNA synthetase.     

Effects of 4-aminopyridine on the action potential and the after-hyperpolarization of cat spinal motoneurons

In cats under pentobarbital anaesthesia, intramotoneuronal administrations of 4-aminopyridine significantly prolong the falling phase of the antidromic action potential but have much less effect on the orthodromic action potential. 4-aminopyridine probably blocks the fast K channels involved in the repolarization of the membrane and indirectly activates ionic channels through enhancement of synaptic transmission, also suggested by the potentiation of excitatory postsynaptic potentials. In many cells, 4-aminopyridine depresses the amplitude and prolongs the time course of the after-hyperpolarization; therefore 4-aminopyridine may also partly block Ca2+-activated K+ channels.     

Studies on the phospholipid composition of pathogenic cell membranes of Mycoplasma hyopneumoniae

The membrane phospholipid and fatty acid compositions of Mycoplasma hyopneumoniae, a pathogen of porcine enzootic pneumoniae isolated in China, was studied by thin-layer chromatography and gas chromatography. The results showed that membrane phospholipids consisted predominantly of diphosphatidylglycerol. The percentage of C16 - C18 fatty acids comprised 79% of the total fatty acids, of which oleic acid as well as palmitic acid are the major fatty acids. Some differences were shown in fatty acid composition as compared with membranes of other species of Mycoplasma.     

Leptomeningeal carcinomatosis presenting as eosinophilic meningitis

The case of a 67-year-old man with underlying carcinomatous meningitis who presented with meningismus and cerebrospinal fluid (CSF) eosinophilia is reported. CSF eosinophilia can reflect a number of underlying conditions; however, carcinomatous meningitis is not generally considered. In this case, studies for bacterial, fungal and parasitic agents were negative. Cytologic examination of a lumbar puncture specimen revealed malignant epithelial cells in an inflammatory background. When unexplained eosinophilia is found in the CSF, a thorough search for coincident meningeal carcinomatosis should be undertaken.     

Improved method for production of antibodies against T-2 toxin and diacetoxyscirpenol in rabbits.

A new, improved approach for the production of antibodies against T-2 toxin and diacetoxyscirpenol (DAS) was developed. The method involves the use of immunogens which were prepared by conjugating O-carboxymethoxyl oxime (CMO) derivatives of both toxins to bovine serum albumin (BSA). Isomers a and b of CMO-T-2 toxin and isomer b of CMO-DAS were tested. Antibodies against both toxins were demonstrated as early as 4 weeks after immunization. a-CMO-T-2-BSA conjugate was a better immunogen than the b isomer, and the highest titers (6,000) were reached 14 weeks after immunization and one booster injection. Antibody titers for rabbits immunized with the b isomer of CMO-T-2 never reached more than 2,000. The specificity of antibodies obtained from rabbits after immunization with CMO-T-2-BSA was similar to that of hemisuccinate-T-2-BSA. Anti-b-T-2 antibodies had slightly higher cross-reactivity with H-T-2 toxin than did the antibody obtained from rabbits immunized with the conjugate of the a isomer. The relative cross-reactivities of anti-a-CMO-T-2 antibody with T-2, acetyl-T-2, H-T-2, T-2-triol, 3'-OH-T-2, and T-2 tetraol were 1, 4.5, 5.7, 250, 500, and 3,000, respectively. The relative cross-reactivities of anti-b-T-2 antibody with T-2, acetyl-T-2, H-T-2, and T-2 triol were 1, 2, 3, and 488, respectively. Antibodies against b-CMO-DAS showed a high degree of cross-reactivity with monoacetoxyscirpenols (MAS). The relative cross-reactivities of anti-B-DAS antibody with DAS, 4-MAS, 15-MAS, acetyl-deoxynivalenol, T-2-toxin, acetyl-T-2, and neosolaniol were 1, 4, 5, 76, 107, 147, and 266, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of cDNA clones for ligninase from Phanerochaete chrysosporium using synthetic oligonucleotide probes

Four cDNA clones for ligninase were isolated from the cDNA library (constructed into the PstI site of E. coli vector pUC9) representing 6 day-old lignin degrading culture of Phanerochaete chrysosporium by the use of three synthetic oligonucleotide probes corresponding to partial amino acid sequences of tryptic peptides of the ligninase. Each of the three probes, 14.1, 14.2 and 25, represents a mixture of 32 12- or 14-base long oligonucleotides. Three cDNA clones hybridized with probe 14.1 but not with probe 25 or 14.2, but one cDNA clone hybridized with all of the three probes. Differential hybridization studies showed that these clones are unique to 6-day poly(A) RNA, but not to 2-day poly(A) RNA.