Magnetic resonance study of exchangeable protons in human carbonic anhydrases.

A titratable exchangeable proton resonance assignable to a histidine imidazole ring N--H proton is observed approximately minus 15 ppm downfield from tetramethylsilane. The chemical shift of this resonance is affected by sulfonamide and anion inhibitors, and by removal of zinc or replacement of zinc by cobalt, indicating that the proton is located at or near the active site. The pH dependence of the chemical shift of this resonance, which is abolished by inhibitors, reflects the titration of a group with a pK-a of 7.3 in human carbonic anhydrase B and smaller than or equal to 7.1 in human carbonic anhydrase C. These pK-a values are interpreted to be due to the ionization of a neutral imidazole to form the imidazolate anion coordinated to zinc. A mechanism for enzymatic catalysis involving reversible deprotonation and coordination of a histidine to the metal is consistent with these studies.     

Nuclear magnetic resonance study of muscle water protons in muscular dystrophy of chickens

Using the pulsed nuclear magnetic resonance (NMR) spectroscopy, the spin-lattice (T1) and the spin-spin (T2) relaxations times of water protons from samples of pectoralis major muscles of normal (line 412) and homozygous dystrophic (line 413) chickens were measured. Both the T1 and T2 were significantly increased (P less than 0.05) in the dystrophic muscles. The mean values of the relaxation times are given +/- S.D. The T1 values were 654 +/- 22 msec in normal and 692 +/- 41 msec in dystrophic muscles. The T2 values for normal and dystrophic muscles were 39 +/- 4 msec and 52 +/- 7 msec, respectively. Although the water content of dystrophic muscles (78.9 +/- 0.6%) determined by gravimetric methods was significantly higher than normal muscles (74.9 +/- 1.1%), this difference in tissue hydration could not explain quantitatively the increase of T1 and T2 values in the dystrophic muscles. The results of the measurements of the relaxation times seem to suggest that there are changes in the composition and/or conformational state of the proteins.     

Magnetic resonance studies of intact human spermatozoa

A correlation has been established between the ability of sperms to reduce the nitroxide spin label--TEMPO and their metabolic activity. The rate of reduction of TEMPO is sensitive to the quantity and quality of sperms and therefore, this property can be developed into a quality rating method. Keeping this in mind, effects of various agents on the spin label reduction rates have been studied. The effect of cold shock on sperms has been found to be harmless and does not lead to loss in metabolic activity. The differential rates of spin label reduction after bubbling N2 and O2 gases through the samples indicate that the process of anaerobic glycolysis imparts higher electron donating capacity to spermatozoa. The method has been used to study influence of inhibitors of electron transport chain (ETC) such as rotenone, antimycin A, KCN and sodium azide which inhibit ETC at different levels.     

Study of anisotropy in nuclear magnetic resonance relaxation times of water protons in skeletal muscle.

The anisotropy of the spin-lattice relaxation time (T1) and the spin-spin relaxation times (T2) of water protons in skeletal muscle tissue have been studied by the spin-echo technique. Both T1 and T2 have been measured for the water protons of the tibialis anterior muscle of mature male rats for theta = 0, 55, and 90 degrees, where theta is the orientation of the muscle fiber with respect to the static field. The anisotropy in T1 and T2 has been measured at temperatures of 28, -5 and -10 degrees C. No significant anisotropy was observed in the T1 of the tissue water, while an average anisotropy of approximately 5% was observed in T2 at room temperature. The average anisotropy of T2 at -5 and -10 degrees C was found to be approximately 2 and 1.3%, respectively.     

Axial mechanical properties of fresh human cerebral blood vessels

Human cerebral blood vessels are frequently damaged in head impact, whether accidental or deliberate, resulting in intracranial bleeding. Additionally, the vasculature constitutes the support structure for the brain and, hence, plays a key role in the cranial load response. Quantification of its mechanical behavior, including limiting loads, is thus required for a proper understanding and modeling of traumatic brain injury--as well as providing substantial assistance in the development and application of preventive measures. It is believed that axial stretching is the dominant loading mode for the blood vessels, regardless of the nature of the insult. Eighteen arteries and fourteen veins were obtained from the cortical surface of the cerebral temporal lobe of patients undergoing surgery. These vessels were stretched to failure in the longitudinal direction, either quasi-statically or dynamically. The significance of specimen and experiment parameters was determined using multivariate analysis of variance (MANOVA) testing. Results demonstrate that the arteries were considerably stiffer than the veins, carrying approximately twice as much stress at failure but withstanding only half as much stretch. No significant rate dependence was measured over a strain rate range of more than four orders of magnitude (0.01 to 500 s -1).     

Magnetic cross-relaxation among protons in protein solutions.

The magnetic spin-lattice relaxation rates of solvent water nuclei are known to increase upon addition of diamagnetic solute protein. This enhancement of the relaxation rate is a function of magnetic field, and the orientational relaxation time of the protein molecules can be deduced from analysis of the field-dependent relaxation rates. Although the nature of the interactions that convey information about the dynamics of protein motion to the solvent molecules is not established, it is known that there is a contribution to the relaxation rates of solvent protons that plays no role in the relaxation of solvent deuterons and 17O nuclei. We show here that the additional interaction arises from a cross-relaxation process between solvent and solute protons. We introduce a heuristic three-parameter model in which protein protons and solvent protons are considered as two separate thermodynamic systems that interact across the protein-solvent interface. The three parameters are the intrinsic relaxation rates of each system and a cross-relaxation term. The sign of the latter term must always be positive, for all values of magnetic field, in order for magnetization energy to flow from the hotter to the cooler system. We find that the magnetic field-dependence of the cross-relaxation contribution is much like that of the remaining solvent proton relaxation, i.e., about the same as the deuteron relaxation field dependence. This finding is not compatible with the predictions of expressions for the cross-relaxation that have been used by other authors, but not applied to data over a wide range of magnetic field strength. The model predicts that the relaxation behavior of both the protein protons and the solvent protons is the sum of two exponentials, the relative contributions of which would vary with protein concentration and solvent isotopic composition in a fashion suggestive of the presence of two classes of protein protons, when there is in reality only one. This finding has immediate implications for the interpretation of published proton relaxation rates in complex systems such as tissues; these data should be reexamined with cross-relaxation taken into account.     

Surface plasmon resonance sensor for heparin measurements in blood plasma

Surface plasmon resonance (SPR) was used as an affinity biosensor to determine absolute heparin concentrations in human blood plasma samples. Protamine and polyethylene imine (PEI) were evaluated as heparin affinity surfaces. Heparin adsorption onto protamine in blood plasma was specific with a lowest detection limit of 0.2 U/ml and a linear window of 0.2–2 U/ml. Although heparin adsorption onto PEI in buffer solution had indicated superior sensitivity to that on protamine, in blood plasma it was not specific for heparin and adsorbed plasma species to a steady-state equilibrium. By reducing the incubation time and diluting the plasma samples with buffer to 50%, the non-specific adsorption of plasma could be controlled and a PEI pre-treated with blood plasma could be used successfully for heparin determination. Heparin adsorption in 50% plasma was linear between 0.05 and 1 U/ml so that heparin plasma levels of 0.1–2 U/ml could be determined within a relative error of 11% and an accuracy of 0.05 U/ml.     

NMR relaxation times of water protons in human colon cancer cell lines and clones

Established lines of human colon cancer cells from several sources (LS180, LS174T, HT29, SW480, SW1345) had water proton nuclear magnetic resonance (NMR) spin-lattice relaxation times (T1) of 460 +/- 45 msec to 982 +/- 9 msec and spin-spin relaxation times (T2) of 83 +/- 6 msec to 176 +/- 6 msec. Two clones derived from single cells of line LS174T were similar in T1 and T2 to the parent line. Differences among the cell lines were not totally a function of cellular hydration. Normal adult and fetal human primary colon cells were wetter and had higher T1 and T2 values than established cell lines. Relaxation times in this study substantiate variations seen for human colon tumors in earlier studies. Established cell lines maintained water relaxation times similar to tumor tissue values. Along with other morphological and biochemical criteria, the relaxation times suggest that these established human colon cancer cell lines may serve as a good experimental model for the study of human colon cancer.     

Induction of autoreactive cells by the preculture of human peripheral blood mononuclear cells with the autologous fresh plasma.

An AMLR in which precultured cells proliferated in response to fresh non-T cells is described. In our system, the responder is human peripheral blood mononuclear cells precultured in the autologous fresh plasma for up to 16 days, and the stimulator is fresh autologous non-T cells. Results suggested that there were two subpopulations of autoreactive cells obtained from the preculture; the high and low density small lymphocytes, both having ERF activity. The autoreactivity of low density cells was augmented when either macrophages or N-ERF-cells were depleted from PBM and thereafter precultures wre performed. A survey of the functional characteristics of the responding cells showed that the responding cells had NK activity against Molt-4 cells but had no significant ADCC activity against target CRBC. Mechanisms for the induction of autoreactive cells by the preculture in the presence of autologous fresh plasma are discussed.     

Nuclear magnetic resonance study of exchangeable protons in ferrocytochrome c

Five well-resolved exchangeable proton resonances have been observed in horse ferrocytochrome c in the low-field region between ?10.0 and ?12.0 ppm. A resonance at ?11.6 ppm is assigned to an amino proton of His 26 on the basis of its magnetic field position, pH dependence and its correlation with histidine at this position in eight species. A resonance at ?10.9 ppm observed in horse and donkey ferrocytochrome c is assigned to a hydrogen-bonded ?-amino proton of Lys 60. This resonance shifts upfield with increasing salt and decreasing pH, with the shifts increasing as one goes from chloride to bromide to iodide. With less assurance, a resonance at ?10.6 ppm is assigned to the indole amino of Trp 59 and a resonance at ?10.3 ppm to the amino proton of the His 18. These resonances have been used to study the binding of small anions to ferrocytochrome c, the results of which show that inorganic phosphate, ADP and ATP all bind in the immediate vicinity of His 26, and that the inorganic phosphate has the greatest effect upon the imidazole amino exchange time.     

Actinomycete virus in fresh water

An ecological study was made of the freshwater distribution of Actinomycete virus. One component of this, the virus of Actinoplanes, is highly specific and its pattern of occurrence mirrors that of its host. The occurrence of Streptomyces virus is more difficult to explain in ecological terms, but this is attempted. Micromonospora virus was not obtained and the significance of this is discussed. The Actinoplanes virus is characterized at the fine-structure level by a polyhedral-shaped head with a tail bearing a contractile sheath, the latter feature being previously undescribed for actinophage.     

Characterization of the human blood plasma proteome

We describe methods for broad characterization of the human plasma proteome. The combination of stepwise immunoglobulin G (IgG) and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of > 94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (< 30 pg/mL to approximately 30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods. More than 80% of the observed proteins demonstrate interactions with IgG and/or albumin, and the human plasma protein loss in the affinity chromatography/strong cation exchange/reversed-phase liquid chromatography-tandem mass spectrometry methodology was investigated in detail. The results of this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies of the identification of novel protein disease markers, as well as further studies of protein-protein interactions.