Stoichiometric binding of diacylglycerol to the phorbol ester receptor

The major phorbol ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry.     

Cell surface changes in preimplantation mouse embryos during compaction investigated using FITC conjugated lectins after proteolytic enzyme treatment

Summary Fluorescein-conjugated lectins were used to examine the reappearance of glycoproteins on the surface of 8-cell mouse embryos after treatment with proteolytic enzymes. Embryos were decompacted in calcium free medium, treated with various proteases and the process of recompaction monitored. The most effective enzymes in delaying recompaction were subtilopeptidase A and proteinase K at 1 mg/ml; the initiation of recompaction was delayed by about 5 h and 90% recompaction by 14–18 h. Papain and -chymotrypsin were only effective in the absence of calcium. The reappearance of receptors for fluorescein-conjugated Con-A, MPA, RCA-I, FBP, BSL-II and DBA was examined photometrically at 0,8–10 and 17–18 h after proteinase K treatment. There was an increase in binding of MPA, RCA-I, FBP and BSL-II in control embryos during the period of the experiment, between approx. 61 and 80 h post coitum in which embryos passed from the 8-cell stage to the 16–32 cell stage. Con-A binding remained the same and that of DBA decreased. By the time that 50% of enzyme treated embryos had recompacted (8–10 h) binding of Con-A was similar to control embryos. Binding of FBP had almost reached control levels while that of BSL-II, DBA, RCA-I and MPA had reached 60–85% of control levels. When embryos were fully compact (17–18 h) Con-A, FBP and DBA were bound in equal or slightly greater amounts to enzyme treated as to control embryos, and receptors for BSL-II, MPA and RCA-I had recovered almost to control levels. The results clearly show that the recovery of glycoproteins on the surface of 8–16 cell embryos parallels recompaction, providing further evidence for the role of these molecules in compaction.     

Dose dependent responses of cattle to Theileria parva stabilate

Dolan T. T., Young A.S., Losos G.J., McMillan I., Minder Ch.E. and Soulsby K. 1984. Dose dependent responses of Theileria parva stabilate. International Journal for Parasitology14: 89–95. A tick derived stabilate of Theileria parva (Maguga) was titrated in a large group of Boran (Bos indicus) cattle of the same age, sex and origin. The infectivity data was analysed using the independent action model. The cattle were identified as heterogeneous in their response to infection with 75% showing one ID₅₀ (0.0014) and 25% showing another (0.01). The disease responses of the cattle given different dose levels were compared for a variety of parameters. The results obtained showed these parameters to be dose dependent including the time to onset of piroplasm parasitaemia. The stabilate is of large volume and can be used for controlled challenge in immunity studies and for comparison of susceptibility between cattle of different breeds and from different epidemiological backgrounds.     

A moving boundary model of acrosomal elongation

A sperm penetrates an egg by extending a long, actin-filled tube known as the acrosomal process. This simple example of biomotility is one of the most dramatic. In Thyone, a 90 m process can extend in less than 10 s. Experiments have shown that actin monomers stored in the base of the sperm are transported to the growing tip of the acrosomal process where they add to the ends of the existing filaments.The force that drives the elongation of the acrosomal process has not yet been identified although the most frequently discussed candidate is the actin polymerization reaction. Developing what we believe are realistic moving boundary models of diffusion limited actin fiber polymerization, we show that actin filament growth occurs too slowly to drive acrosomal elongation. We thus believe that other forces, such as osmotically driven water flow, must play an important role in causing the elongation. We conjecture that actin polymerization merely follows to give the appropriate shape to the growing structure and to stabilize the structure once water flow ceases.Work partially supported by the United States Department of Energy     

窦性节律下家兔心率与在位心室肌纤维有效不应期的关系

在自然呼吸和窦性节律下,用浮置式玻璃微电极引导在体单个左心室肌纤维动作电位,作为兴奋的指标,以其 0相触发产生期前的试验刺激,测定有效不应期(ERP)。38只家兔的测定结果表明,在R-R间期为205—330ms的范围内,随着心率加快(R-R间期缩短),ERP减小,而 ERP/RR 间期增大,说明 ERP 与心率直接有关。并且,在较快的心率时,ERP 相对延长。通过相关与回归分析,制出了能够删除心率影响的校正公式。静脉注射酒石酸锑钾(50mg/kg)发现,在窦性和起搏节律下,酒石酸锑钾均能轻度延长 ERP(P<0.001)。窦性节律下的校正后值与起搏节律的测定结果完全一致。证明校正后值能够用来比较处理前后不同心率条件下,各种药物、离子及其它因素对ERP 的影响。 本文的校正公式虽然只适用于同种动物和方法,但此校正公式的制作原理也可以广泛应用到其它多种动物。     

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and I h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED₅₀s of these antitumor effects afteri.v. administration of rHu-TNF were about 50 times as high as ED₅₀s afteri.t. administration. ED₅₀ ofi.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED₅₀ ofi.v. rHu-TNF for vascular effect was only 2–3 times as high as that for cytotoxicity. The whole body autoradiographies with [¹²⁵I] HSA giveni.v. to see the blood influx into tumor tissue and [¹⁴C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues.In conclusion, thein vivo antitumor effect of rHu-TNF giveni.t. ori.v., appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.     

Structural and developmental differences between three types of Na channels in dorsal root ganglion cells of newborn rats

Summary The changes in Na current during development were studied in the dorsal root ganglion (DRG) cells using the whole-cell patch-clamp technique. Cells obtained from rats 1–3 and 5–8 days after birth were cultured and their Na currents were compared. On top of the two types of Na currents reported in these cells (fast-FA current and slow-S current) a new fast current was found (FN). The main characteristics of the three currents are: (i) The voltages of activation are –37, –36, and –23 mV for the FN, FA and S currents, respectively. (ii) The activation and inactivation kinetics of FN and FA currents are about five times faster than those of the S current. (iii) The voltages at which inactivation reaches 50% are –139, –75 and –23 mV for the FN, FA and S currents, respectively.The kinetics and voltage-dependent parameters of the three currents and their density do not change during the first eight days after birth. However, their relative frequency in the cells changes. In the 1–3 day-old rats the precent of cells with S, FA, and mixed S+FN currents is 22, 18, and 60% of the cells, respectively. In the 5–8 day-old, the percent of cells with S, FA, and FN+S is 10, 66 and 22%. The relative increase in the frequency of cells with FA current during development can contribute to the ease of action potential generation compared with cells with FN currents, which are almost completely inactivated under physiological conditions. The predominance of FA cells also results in a significant decrease in the relative frequency of cells with the high-threshold, slow current.Antibodies directed against a part of the S₄ region of internal repeat I of the sodium channel (C ₁ ⁺ , amino acids 210–223, eel channel numbering) were found to shift the voltage dependence of FA current inactivation (but not of FN or S currents) to more negative potentials. The effect was found only when the antibodies were applied externally. The results suggest that FN, FA and S types of Na currents are generated by channels, which are different in the topography of the C ₁ ⁺ region in the membrane.     

Ca modulates outward current throughI K1 channels

Summary Inward-rectifier channels in cardiac cells (I _K1) stabilize the resting membrane potential near the K equilibrium potential. Here we investigate the role ofI _K1 in the regulation of action potentials and link this to the influx of Ca during beating. Inward Ca current alters the open-channel probability of outwardI _K1 current. Thus Ca ions depolarize cells not only by carrying an inward current but also by blocking an outward current.     

Emerging functional roles for the glycosyl-phosphatidylinositol membrane protein anchor

Conclusion Experimental evidence has accumulated over the past few years to suggest that the GPI protein anchor may play a broad role in the regulation of membrane protein function. The significant changes in the biophysical properties of proteins that are membrane-anchored through GPI in lieu of a hydrophobic transmembrane peptide indicates a variety phobic transmembrane peptide indicates a variety of potential new functions served by the anchor structure itself. Moreover, the number of structural variations within the family of GPI molecules indicates a further opportunity for subspecialization of such anchored proteins, especially regarding cellular localization, mobility, metabolism and susceptibility to enzymatically-induced release. It is likely that further exploration of the structure and function of the GPI anchor may reveal additional roles for this unusual mechanism of membrane-protein attachment.     

Reconstituted voltage-sensitive sodium channels from eel electroplax: Activation of permeability by quaternary lidocaine,N-bromoacetamide,and N-bromosuccinimide

Summary We have investigated the ion permeability properties of sodium channels purified from eel electroplax and reconstituted into liposomes. Under the influence of a depolarizing diffusion potential, these channels appear capable of occasional spontaneous openings. Fluxes which result from these openings are sodium selective and blocked (from opposite sides of the membrane) by tetrodotoxin (TTX) and moderate concentrations of the lidocaine analogue QX-314. Low concentrations of QX-314 paradoxically enhance this channel-mediated flux. N-bromoacetamide (NBA) and N-bromosuccinimide (NBS), reagents which remove inactivation gating in physiological preparations, transiently stimulate the sodium permeability of inside-out facing channels to high levels. The rise and subsequent fall of permeability appear to result from consecutive covalent modifications of the protein. Titration of the protein with the more reactive NBS can be used to produce stable, chronically active forms of the protein. Low concentrations of QX-314 produce a net facilitation of channel activation by NBA, while higher concentrations produce block of conductance. This suggests that rates of modifications by NBA which lead to the activation of permeability are influenced by conformational changes induced by QX-314 binding.