Nicotinic acetylcholine receptor from rat brain. Solubilization, partial purification, and characterization

Nicotinic acetylcholine receptor protein (nAChR) has been solubilized from rat cerebral cortices by extracting a crude membrane fraction with the nonionic detergent Triton X-100 (polyoxyethylene-p-t-octylphenol). The solubilized nAChR was partially purified by affinity chromatography (Naja naja siamensis alpha-toxin affinity arm, linked to Sepharose 4B) and characterized by binding of 125I-labeled alpha-bungarotoxin. The reaction of labeled toxin and nAChR appears to be second order with a rate constant (k1) equal to 0.38 X 10(5) M-1 S-1 at 20 degrees. The toxin-nAChR complex dissociates with a dissociation rate constant (k-1) of 1.23 X 10(-5) S-1 at 20 degrees (t 1/2 = 15.6 h). The kinetically determined dissociation constant (Kd) for the complex is 3.24 X 10(-10) M. A variety of cholinergic ligands were studied for their ability to inhibit binding of labeled toxin. The results indicate that the brain receptor is indeed nicotinic. The s20, w and v of the toxin-nAChR complex in 0.1% Triton were determined by velocity sedimentation in D2O and H2O sucrose gradients. The values are 12.9 S and 0.80 cm3 g-1. The Stokes radius of the complex determined by gel filtration equals 7.5 nm. The Mr of the complex calculated from the hydrodynamic parameters, and corrected for bound detergent, equals 357,000.     

Synthesis of the second component of complement by long-term primary cultures of human monocytes

A method has been developed for preparation of confluent monolayers of human monocytes from small volumes of blood and for maintenance of these pure monocyte cultures for up to 16 wk in vitro. These cells phagocytosed 5.7 mum diameter latex beads, rosetted with erythrocytes coated with IgG or with C3, killed Listeria monocytogenes, and synthesized both lysozyme and the second component of complement. Lysozyme was secreted at a rate of approximately 50,000 mol/min per cell for at least 12 wk in cultures. The maximal rate of C2 synthesis and secretion was considerably less; i.e., approximately 30 mol/min per cell between the 2nd and 12th wk in culture. Monocytes produced little C2 during the first 6 days in culture after which a marked increase in the rate of C2 production was noted. This increase was coincident with morphologic evidence of monocyte maturation.     

Gonadoblastoma: clinicopathologic correlation in six patients

Six patients with a total of nine gonadoblastomas are presented; three--and possibly a fourth--had dysgerminomatous overgrowth which was massive in two patients. Calcification detected by abdominal films was present in three sufficient for preoperative diagnosis. All patients were found to have a Y stem line on peripheral leukocyte chromosome cultures except one patient, who had a 46 XX/45 XO karyotype. She was found to have Y chromatin bodies in the germ cells of her tumor which was in a normal ovary found at exploration for an ectopic pregnancy. Three were found in virilized phenotypic females investigated for amenorrhea, and two for therapy of pelvic masses due to dysgerminomatous overgrowth. Y chromatin studies are reported on gonadal tissue.     

Selenium and vitamin E and incidence of retained placenta in parturient dairy cows. II. Prevention in commercial herds with prepartum treatment

In a series of field experiments in Ohio involving 193 parturient cows of the Holstein and Guernsey breeds, the prophylactic efficacy of selenium and vitamin E was tested under field conditions. Herds initially were chosen because of a chronic problem with retained placenta which could not be related to a known etiological factor. Each herd was divided into three groups. Group A received an injection of 50 mg of sodium selenite 40 days prepartum and 680 units of alpha tocopherol acetate followed by the same treatment 20 days prepartum. Group B received a single injection of 50 mg of sodium selenite 20 days prepartum, and 680 IU of vitamin E. Group C served as the control. Incidence of retained placenta was reduced from a mean of 51.2% in control cows to 8.8% for animals injected with selenium and vitamin E. No differences in efficacy were between Group A and B, and it appears that the single 20 day prepartum injection of 50 mg of sodium selenite and 680 IU of alpha tocopherol acetate is an effective prophylactic for prevention of retained placenta.     

Concentration of Liquid Egg White by Vacuum Evaporation and Reverse Osmosis

Egg-white was concentrated by vacuum evaporation (VE) and reverse osmosis (RO). The treatments consisted of a control, RO process, VE process using 50°C water-jacket temperature and 60-min processing time (5060VE), and a VE process using 60°C water-jacket temperature and 45-minute processing time (6045VE). Solids contents for the concentrates were 17.82% for the RO. 17.41% for the 6045VE. and 18.98% for 5060VE. The VE product had better foaming properties and comparable gel qualities to that of the RO product. Foam enhancing and stabilizing additives improved the quality of all concentrates. Electrophoretic studies indicated a significant decrease in globulins A1 and A2 in the VE treatments.     

Immunochemical properties of a 60 kDa cell surface-associated heat shock-protein (Hsp60) from Helicobacter pylori

Western blot analysis (immunoblotting) of cell surface-associated proteins from Helicobacter pylori confirmed our previous findings that binding of human IgG is a common property (among H. pylori strains). Purification of the IgG-binding proteins (IGBP) was achieved by two purification steps, affinity chromatography on IgG-Sepharose and nickel chelate affinity chromatography. SDS-PAGE and immunoblotting analysis revealed a 60 kDa protein with affinity for peroxidase labeled human IgG. Solid phase binding assays showed that IgG binds to an immobilized protein (IGBP). The 60 kDa IGBP binds human IgG1, IgG3 and IgM. Binding could be inhibited by the kappa chain of the human IgG, but not with its Fc fragment, nor with IgA or IgM. In addition, rabbit polyclonal antibodies raised against the 60 kDa IGBP blocked IgG binding. Monoclonal antibodies, specific to the Hsp60 heat shock protein of H. pylori recognized the 60 kDa IGBP as revealed by immunoblotting analysis, both in crude preparations and in the purified fractions.     

Utilizing uncertainty factors in minimal risk levels derivation

While both 31P and 113Cd are present at locations of interest in many different macromolecular systems, heteronuclear-detected relaxation measurements on these nuclei have been restrained by limitations in either resolution or signal-to-noise ratio. We have developed heteroTOCSY-based methods to overcome both of these problems. Two-dimensional versions of these experiments were utilized to measure 31P T1 and T2 values in DNA oligonucleotides; the additional resolution offered by a second dimension allowed determination of these values for most of the 31P resonances in a DNA dodecamer. The results from the experiments indicated that there was little significant variation in T1 values for the different phosphates in the DNA dodecamer; however, the T2 values showed a clear pattern, with lower values in the interior of the sequence than at the ends of the helix. Furthermore, a significant correlation between 31P chemical shifts and T2 values was observed. One-dimensional, frequency-selective versions of these experiments were also developed for use on systems containing a smaller number of heteronuclear spins. These methods were applied to investigate the heteronuclear relaxation properties of 113Cd in 113Cd2LAC9(61), a Cys6Zn2 DNA-binding domain. Data from the experiments confirm biochemical evidence that more significant differences occur in the metal-protein interactions between the two metal-binding sites than has been previously identified for proteins containing this motif.     

Intracellular location of inapparently infecting Chlamydia in synovial tissue from patients with Reiter's syndrome

Culture of Chlamydia trachomatis from synovial tissues/fluids from Reiter's syndrome (RS) patients frequently yields negative results. However, we have identified chlamydial RNA at that site in such patients, suggesting that viable organisms may be present. Here we define the cellular location of chlamydia within the synovium via in situ hybridization. Using a chlamydial ribosomal RNA-directed probe, we show that synovial tissue from culture-negative RS patients gives strong hybridization which is often localized to a subsynovial cell layer, rather than to the synovial lining; in some cases, hybridizing cells are dispersed through the synovium. All hybridization signal is located within host cells, indicating that infectious extracellular elementary bodies are rare or absent. These data confirm the extensive intracellular presence of inapparent chlamydia in the synovia of RS patients and provide some insight into the usual culture negativity of synovial tissues for the organism.     

跟踪式热平衡温度测量

介绍了跟踪式热平衡温度测量的原理：当检测头与被测体靠近时，通过对流换热形成梯度为零的温度场，因而二者温度相等。这种梯度为零的温度场是通过对检测头的一端制冷（或加热）跟踪被测体的温度形成的．最后给出了静态和动态实验的测试结果。