苯肼对红细胞在体衰老过程中微观流变特性的影响

在Brunara等人用苯肼使动物造成急性溶血性贫血的方法基础上,建立一种由急性溶血性贫血后,而诱发家兔幼红细胞增多的非正常生理状态的红细胞在体衰老模型,继而研究新生红细胞从产生到死亡死亡过程,即衰老过程的流变学特性的变化规律。通过对新生红细胞的压积、变形、取向及与之相应的全血的粘度、血沉等指标的连续60多天的监测,发现红细胞在衰老过程中的微观流变学特性确实有明显改变。红细胞在体衰老过程中微观流变特性逐渐变差。     

A complement-resistant HeLa cell line (T638) is blocked at the step of C3 deposition

A complement-resistant line of HeLa cells (T638) was derived by serial passage of complement-susceptible HeLa cells in anti-beta 2-microglobulin (b2m) antiserum and complement. The T638 line maintained stable complement resistance when passed for an additional 1500 generations in the absence of antiserum and complement. T638 cells expressed equivalent levels of cell-associated b2m as did the parent HeLa cell line. Furthermore, T638 cells were resistant to killing by complement and anti-HeLa antiserum with specificity for molecules other than b2m. These results indicate that the resistance of T638 cells does not simply reflect loss of anti-b2m binding antigens. We next investigated the mechanism of resistance of T638 cells to complement-mediated killing. Antibody-sensitized HeLa and T638 cells both consumed CH50 activity completely from normal human serum; cytotoxicity was not mediated via the alternative complement pathway. HeLa and T638 cells caused equivalent utilization of C4 from normal human serum in the presence of antibody. Consumption of C2, greater with T638 than with HeLa cells during incubation in serum, was complete when cells bearing purified C1 and limited C4 were incubated with C2. T638 cells bound more 3H-C4 than HeLa cells during incubation in serum, but binding of 3H-C3 by T638 cells was fourfold to fivefold less than by HeLa cells. Finally, we investigated the rate of decay in the capacity of C142 on HeLa and T638 to cleave and deposit 3H-C3. The T1/2 for decay of C142-mediated binding of 3H-C3 on HeLa was 3.9 min, whereas minimal C3 deposition was detected on T638 cells at all time points. These results show that T638 cells evade complement-mediated lysis despite activating early components of the classical complement pathway. The mechanism of resistance is a failure to form an effective C3 convertase.     

Serum resistance of metacyclic stage Leishmania major promastigotes is due to release of C5b-9

The mechanism of serum resistance for infective promastigotes of Leishmania major was investigated. Prior results suggested that the mechanism of resistance was mediated at a step after C3 deposition. Equivalent amounts of C3b were deposited on serum-susceptible, noninfective promastigotes harvested from log stage cultures (LOG) and on C-resistant, infective, metacyclic promastigotes (MP) purified from stationary stage cultures. Whereas binding of C9 to LOG was stable during incubation in serum, C9 binding to MP was minimal and unstable, because molecules bound initially to MP were released with continued incubation. Failure to bind C9 was not a result of inability to activate C; the kinetics of C3, C6, and C9 consumption were similar for LOG and MP. Deposition of C5b-7 on MP was stable, indicating that the initial steps in terminal complex formation were intact. Instead, the majority of C5b-9 formed on MP was spontaneously released into the serum as SC5b-9. Residual C5b-9 on MP was released with 1 M NaCl. These data show that developmental modification of the promastigote membrane during transition from a noninfective to an infective stage blocks insertion of lytic C5b-9 into the promastigote membrane.     

Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex

A bovine liver protein which catalyzes the transfer of triglyceride between membranes has previously been isolated from the lumen of the microsomal fraction. When further purified about 100-fold, two polypeptides of molecular mass 58,000 and 88,000 were identified (Wetterau, J. R., and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38, 205-222). We demonstrate here that the two polypeptides (referred to as 58-kDa and 88-kDa, respectively) are associated in a protein-protein complex, and that the triglyceride transfer activity is associated with this complex. Antibodies specific for either polypeptide immunoprecipitated both the 58-kDa and 88-kDa polypeptides as well as the lipid transfer activity. The 58-kDa subunit of the microsomal transfer protein complex was identified as protein disulfide-isomerase (PDI) (EC 5.3.4.1) by 1) a comparison of the amino-terminal sequence of PDI and the 58-kDa subunit of the transfer protein, 2) a comparison of the reverse phase high performance liquid chromatography peptide maps of CNBr digests of PDI and the lipid transfer protein, 3) immunoprecipitation competition experiments in which PDI was found to compete with the lipid transfer protein for immunoprecipitation by the anti-58-kDa polyclonal antibodies, 4) immunological cross-reactivity of the microsomal triglyceride transfer protein complex with polyclonal antibodies raised against PDI, and 5) the appearance of protein disulfide isomerase activity following the dissociation of purified microsomal transfer protein complex with guanidine HCl. In conclusion, the microsomal triglyceride transfer protein has a multi-subunit structure which is unique compared to other intracellular lipid transfer proteins which have been described to be single polypeptides. The unexpected finding that PDI is a component of the microsomal triglyceride transfer protein complex suggests a new previously undescribed role for protein disulfide isomerase.     

Distribution of Elements in Rat Peripheral Axons and Nerve Cell Bodies Determined by X-Ray Microprobe Analysis

X-ray microprobe analysis was used to determine concentrations (millimoles of element per kilogram dry weight) of Na, P, Cl, K, and Ca in cellular compartments of frozen, unfixed sections of rat sciatic and tibial nerves and dorsal root ganglion (DRG). Five compartments were examined in peripheral nerve (axoplasm, mitochondria, myelin, extraaxonal space, and Schwann cell cytoplasm), and four were analyzed in DRG nerve cell bodies (cytoplasm, mitochondria, nucleus, and nucleolus). Each morphological compartment exhibited characteristic concentrations of elements. The extraaxonal space contained high concentrations of Na, Cl, and Ca, whereas intraaxonal compartments exhibited lower concentrations of these elements but relatively high K contents. Nerve axoplasm and axonal mitochondria had similar elemental profiles, and both compartments displayed proximodistal gradients of decreasing levels of K, Cl, and, to some extent, Na. Myelin had a selectively high P concentration with low levels of other elements. The elemental concentrations of Schwann cell cytoplasm and DRG were similar, but both were different from that of axoplasm, in that K and Cl were markedly lower whereas P was higher. DRG cell nuclei contained substantially higher K levels than cytoplasm. The subcellular distribution of elements was clearly shown by color-coded images generated by computer-directed digital x-ray imaging. The results of this study demonstrate characteristic elemental distributions for each anatomical compartment, which doubtless reflect nerve cell structure and function.     

Hypersensitivity to very-low single radiation doses: Its relationship to the adaptive response and induced radioresistance

There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents. Phenomenologically, there are two ways in which these induced mechanisms operate. First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose. This has been termed the adaptive response. Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered. This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo. There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the adaptive response in the two-dose case and that it can be triggered by high and low LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents although exact homology remains to be tested. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified.     

Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors

Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS), which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K) channels and downregulating nicotinic acetylcholine receptors (nAChRs) in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function.     

ULTRASTRUCTURAL ANALYSIS OF MITOTIC SPINDLE ELONGATION IN MAMMALIAN CELLS IN VITRO : Direct Microtubule Counts

The mitotic spindle of many mammalian cells undergoes an abrupt elongation at anaphase. In both cultured rat kangaroo (strain PtK₁) and Chinese hamster (strain Don-C) fibroblasts, the distance from pole to pole at metaphase doubles during anaphase and telophase. In order to determine the organization and distribution of spindle microtubules during the elongation process, cells were fixed and flat embedded in Epon 812. Selected cells were photographed with the phase-contrast microscope and then serially sectioned perpendicular to the major spindle axis. Microtubule profiles were counted in selected sections, and the number was plotted with respect to position along the spindle axis. Interpretation of the distribution profiles indicated that not all interpolar microtubules extended from pole to pole. It is estimated that 55–70% of the interpolar microtubules are overlapped at the cell equator while 30–45% extend across the equator into both half spindles. This arrangement appeared to persist from early anaphase (before elongation) until telophase after the elongation process. Although sliding or shearing of microtubules may occur in the spindle, such appears not to be the mechanism by which the spindle elongates in anaphase. Instead, our data support the hypothesis that spindle elongation occurs by growth of prepositioned microtubules which "push" the poles apart.