Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the absence of a calcium signal or microtubule assembly

Mouse egg activation, which includes release from meiotic metaphase II arrest, results from fertilization-induced increase in intracellular calcium concentration ([Ca²⁺]_i). However, during egg activation caused by exposure to the protein synthesis inhibitor, cycloheximide, [Ca²⁺]_i did not change. Although eggs fertilized in the presence of microtubule inhibitors remain arrested at metaphase, eggs treated for 32 hr with cycloheximide and the microtubule inhibitor, colcemid, formed nuclei. In untreated eggs aged in culture for 24 hr, the microtubule spindles became deformed. These eggs formed nuclei after exposure to cycloheximide, but not the calcium ionophore A23187. Our results indicate that eggs in which protein synthesis is inhibited are released from metaphase without an increase in [Ca²⁺]_i, and despite disruption of the Spindle. © 1995 Wiley-Liss, Inc.     

Na_2SO_3和NaHCO_3对叶绿体CF_1－ATPase活力作用的机制

Ｎａ２ＳＯ３对热－ＤＴＴ活化的游离ＣＦ１及类囊体膜上ＣＦ１－ＡＴＰａｓｅ活力均有显著的促进作用,ＮａＨＣＯ３亦有明显的促进作用。Ｎａ２ＳＯ３和ＮａＨＣＯ３的促进作用与它们解除Ｍｇ２＋的抑制作用有关。从ＮａＨＣＯ３和Ｎａ２ＳＯ３及它们与Ｍｇ２＋之间的竞争性关系,表明三者是结合在酶的同一部位上。Ｎａ２ＳＯ３可明显降低热－ＤＴＴ活化的游禹ＣＦ１－ＡＴＰａｓｅ催化反应的活化能,这可能与促进产物ＡＤＰ的释放有关。     

A novel Cl− conductance in cultured chick cardiac myocytes: Role of intracellular Ca2+ and cAMP

Cl^– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na⁺and K⁺-free internal and external solutions, the basal whole-cell current was predominantly a Cl^– current. Cl^–-sensitive current (I _Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl^–] _o when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl^–] < 40 mm, and was not saturated with a physiological Cl^– gradient. The Cl^– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl^– current. The cAMP- or forskolin-activated and basal Cl^– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P _Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca²⁺] _i , the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl^– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca²⁺] _i .This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance.     

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Furhter validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.     

Some improvements in the quality of NAA procedures and the reliability of the results

After considering the need for quality control in NAA, the concept of quality in NAA procedures themselves is discussed, and some important factors identified. Two approaches to improve quality are then described in more detail. The first concerns the unique ability of NAA using different isotopic reactions and different modes (INAA/RNAA) to provide independent data sets in the same laboratory, thus allowing internal validation or crosschecking. The second discusses the need for chemical yield measurements in RNAA and the advantages of the radioisotopic tracer technique. Some recent advances and further possibilities for this use of tracers are listed.     

Developments in tomographic methods for biological trace element research

Neutron-induced γ-ray emission tomography for quantitative determination of the concentration and distribution of elements in a selected plane through a biological specimen is briefly explained and applied by way of illustration to the analysis of gallstones. A system capable of carrying out studies of the binding site of⁷⁵Se in different matrices using time differential perturbed angular correlation spectroscopy is also briefly described. Developments in the detector technology of positron emission tomography have allowed small-diameter imaging devices to be built for in vivo preclinical evaluation of new tracers in small animals and are discussed in the context of a proposed experiment combining the techniques mentioned above.     

Two conformational states of Candida rugosa lipase.

The structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 A resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain-the flap. Comparison of this structure with the previously determined "open" form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole do not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the "open" conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis-trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap.     

New technique in optical resolutions

The present paper illustrates the development of an advanced technique in optical resolution. Both of the amphetamine enantiomers can be obtained by a two-step distillation in nearly quantitative yield without any loss of the resolving agent. It is proved that the second-order interactions (H-bond) are sufficient for separation of enantiomers by distillation. © 1994 Wiley-Liss, Inc.