Phosphorylation of membrane-bound guanylate cyclase of sea urchin spermatozoa

When Arbacia punctulata spermatozoa are incubated in seawater containing ammonium hydroxide (pH 8.8), the sperm plasma membrane-bound guanylate cyclase is dephosphorylated, its electrophoretic mobility increases (from an apparent molecular mass of 160 to 150 kD), and its enzymatic activity decreases 3.5-fold. Transfer of these cells into ammonium-free seawater (pH 7.4) results in the rephosphorylation of the cyclase, its reconversion to 160 kD, and recovery of the enzymatic activity lost upon dephosphorylation. This is the first direct demonstration that the activity of membrane-bound guanylate cyclase can be regulated by phosphorylation. A plasma membrane preparation is described that specifically supports the in vitro phosphorylation of the guanylate cyclase. This preparation will be useful in more detailed studies on the relationship between phosphorylation state and enzymatic activity of membrane-bound guanylate cyclase.     

Acquired drug resistance is accompanied by modification in the karyotype and nuclear matrix of a rat carcinoma cell line

A Walker 256 breast carcinoma cell line (WR) exhibiting a greater than 20-fold resistance to alkylating agents has been selected from a parent cell line (WS). Karyotypic heterogeneity was apparent, with a number of differences evident between WR and WS cells. The modal chromosome number for WS is 62; for WR, 54; double minutes were found only in WR, whereas spontaneous chromosomal aberrations were present in approx. 40% of the WS cells. No similar aberrations were observed in WR. Using SDS-gel electrophoresis and subsequent silver staining, differences in the profile of nuclear matrix proteins in WR and WS were observed. A diffuse band at approx. 70 kD in the WS was absent in WR cells. This protein was phosphorylated, together with a number of the other major matrix polypeptides. Levels of phosphorylated matrix proteins were approximately equivalent in both WR and WS cell lines, but matrix protein phosphorylation levels were approx. 2-fold higher than corresponding values for bulk nuclear proteins. Selective pressure of drug exposure has resulted in enhanced genetic stability in WR cells and observed karyotype differences are accompanied by modifications in the structural proteins of the nuclear matrix. Whether the observed differences are the cause or result of drug resistance remains to be established.     

Oxide-Based Solid-State Batteries: A Perspective on Composite Cathode Architecture

The garnet-type phase Li₇La₃Zr₂O₁₂ (LLZO) attracts significant attention as an oxide solid electrolyte to enable safe and robust solid-state batteries (SSBs) with potentially high energy density. However, while significant progress has been made in demonstrating compatibility with Li metal, integrating LLZO into composite cathodes remains a challenge. The current perspective focuses on the critical issues that need to be addressed to achieve the ultimate goal of an all-solid-state LLZO-based battery that delivers safety, durability, and pack-level performance characteristics that are unobtainable with state-of-the-art Li-ion batteries. This perspective complements existing reviews of solid/solid interfaces with more emphasis on understanding numerous homo- and heteroionic interfaces in a pure oxide-based SSB and the various phenomena that accompany the evolution of the chemical, electrochemical, structural, morphological, and mechanical properties of those interfaces during processing and operation. Finally, the insights gained from a comprehensive literature survey of LLZO–cathode interfaces are used to guide efforts for the development of LLZO-based SSBs.     

The cytolytic isolation of the cortex of the sea urchin egg

When the vitelline layer of sea urchin eggs (Lytechinus pictus) is disrupted by trypsin or dithiothreitol and the eggs are placed in an isosmotic medium devoid of Ca²⁺, cytolysis of the eggs occurs. During lysis the entire egg cortex peels off in one piece. Lysis is temperature and pH dependent and is inhibited by cytochalasin B. Cortices from unfertilized eggs contain seven major macromolecular components. A 42K-dalton component is believed to be actin, representing between 12 and 27% of the total protein. Cortices from fertilized eggs may contain between 50 and 65% actin. The actin appears to increase the strength of its attachment to the cortex after fertilization. This method of isolating the entire cortex may be useful for studying structural and enzymatic changes which may occur in the cortex during the cell cycle.     

The accessory function of murine intercellular adhesion molecule-1 in T lymphocyte activation. Contributions of adhesion and co-activation.

These studies demonstrate that the murine intercellular adhesion molecule-1 (ICAM-1) performs at least two roles in enhancing T cell activation. These two roles are evident in both of our experimental systems: with ICAM-1 expressed on the surface of transfected fibroblast cells, and with purified ICAM-1 immobilized on plastic. First, as has been documented by many investigators, ICAM-1 mediates adhesion between ICAM-1- and lymphocyte function-associated Ag-1 (LFA-1)-bearing cells. This adhesive interaction occurs even in the absence of T cell stimulation, although it is increased by addition of phorbol ester and calcium ionophore. Although ICAM-1 expression does markedly increase intercellular adhesion, the increase is significantly less than the improvement ICAM-1 expression makes in the Ag-presenting ability of MHC class II-transfected fibroblast cells. We have investigated whether this difference is due to LFA-1-mediated signaling, and we present data that demonstrates that although ICAM-1 does not deliver costimulatory signals required for T cell activation, the interaction of LFA-1 with ICAM-1 does synergize with TCR-transduced signals. This synergy is observed for ICAM-1 on live and on chemically fixed accessory cells, and for purified ICAM-1 molecules, but in all cases occurs only when the ICAM-1 and the TCR ligands are on the same surface. Finally, when the ICAM-1 is present on the surface of accessory cells, it enhances T cell activation by changing the Ag dose-dependence of the T cell, but when ICAM-1 and CD3 mAb are co-immobilized, ICAM-1 increases the peak response of the T cell without affecting the dose dependence of the response.     

In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.     

Resistance to receptor-mediated degradation of a murine epidermal growth factor analogue (EGF-Val-47) potentiates its mitogenic activity

In most cell types two classes of epidermal growth factor (EGF) receptors can be found: a major class that binds EGF with relatively low affinity and a minor class that binds with very high affinity. Structure-function studies have shown that mutations at amino acid 47 in the EGF molecule severely reduce its affinity for the EGF receptor but do not cause preferential binding to one or the other subclass of receptors. Using three EGF derivatives with a mutation at amino acid 47 (Ser-47, Leu-37-Tyr-47, and Val-47), we have investigated the relative contribution of the two receptor subclasses to the EGF-dependent mitogenic response. We show that mitogenicity correlates exclusively with occupancy of the high-affinity receptor and that full occupancy of this subclass is required for maximal stimulation. In addition we demonstrate that for the EGF-Val-47 analogue this requirement can be abrogated and half-maximal biological activity reached with a high-affinity receptor occupancy of only 8%. While the rate of internalization did not significantly differ between EGF-Val-47 and native mEGF, the analogue was much more resistant to degradation by cellular proteases and, after binding and receptor-mediated internalization, was released into the medium predominantly in an intact form. We propose that the increased mitogenicity of EGF-Val-47 is due to its prolonged half-life, resulting in continued occupancy of the high-affinity EGF receptor.     

KLOTHO allele status and the risk of early-onset occult coronary artery disease

We previously identified a functional variant of KLOTHO (termed "KL-VS"), which harbors two amino acid substitutions in complete linkage disequilibrium and is associated with reduced human longevity when in homozygosity. Klotho-deficient mice display extensive arteriosclerosis when fed a normal diet, suggesting a potent genetic predisposition. To determine whether klotho influences atherosclerotic risk in humans, we performed cross-sectional studies to assess the association between the KL-VS allele and occult coronary artery disease (CAD) in two independent samples of apparently healthy siblings of individuals with early-onset (age <60 years) CAD (SIBS-I [N=520] and SIBS-II [N=436]). Occult CAD was defined as the occurrence of a reversible perfusion defect during exercise thallium scintigraphy and/or as an abnormal result of an exercise electrocardiogram (SIBS-I, n=97; SIBS-II, n=56). In SIBS-I, the KL-VS allele conferred a relative odds of 1.90 (95% confidence interval 1.21-2.98) for occult CAD, after adjusting for familial intraclass correlations (P<.005). Logistic regression modeling, incorporating known CAD risk factors, demonstrated that the KL-VS allele is an independent risk factor (P<.019) and that the imposed risk of KL-VS allele status is influenced by modifiable risk factors. Hypertension (P<.022) and increasing high-density lipoprotein cholesterol (HDL-C) levels (P<.022) mask or reduce the risk conferred by the KL-VS allele, respectively, whereas current smoking (P<.004) increases the risk. Remarkably concordant effects of the KL-VS allele and modifying factors on the risk of occult CAD were seen in SIBS-II. These results demonstrate that the KL-VS allele is an independent risk factor for occult CAD in two independent high-risk samples. Modifiable risk factors, including hypertension, smoking status, and HDL-C level, appear to influence the risk imposed by this allele.     

Cooperative adhesion of ligand-receptor bonds

Cooperative (simultaneous) breakage of multiple adhesive bonds has been proposed as a mechanism for enhanced binding strength between adhesion molecules on apposing cell surfaces. In this report, we used the atomic force microscopy (AFM) to study how changes in binding affinity and separation rate of force-induced ligand-receptor dissociation affect binding cooperativity. The AFM force measurements were carried out using (strept)avidin-functionalized cantilever tips and biotinylated agarose beads under conditions where multiple (strept)avidin-biotin linkages were formed following surface contact. At slow surface separation of the AFM cantilever from the bead's surface, the (strept)avidin-biotin linkages appeared to rupture sequentially. Increasing the separation rate from 210 to 1950 nm/s led to a linear increase in the average rupture force. Moreover, force histograms revealed a quantized force distribution that shifted toward higher values with increasing separation rate. In measurements of streptavidin-iminobiotin adhesion, the force distribution also shifted toward higher values when the buffer was adjusted to a higher pH to raise the binding affinity. Together, these results demonstrate that the cooperativity of ligand-receptor bonds is significantly enhanced by increases in surface separation rate and/or binding affinity.     

芦芽山林线白杄生长季径向生长动态

高山林线作为树木分布的高度上限, 是全球范围最重要的植被过渡带之一, 其树木生长显著受到外界极端环境条件的影响。利用点状树木径向变化记录仪于2009年5-9月, 对山西省芦芽山林线组成树种白杄(Picea meyeri)生长季内树木径向生长进行了持续的动态监测。结果表明: 白杄茎干日变化主要受到树木蒸腾作用日变化的影响, 茎干呈现出白天脱水收缩与夜间吸水膨胀的循环变化; 生长季白杄径向生长可划分为3个不同的生长时段: 1)茎干水分恢复时段, 2)茎干快速生长时段, 3)茎干脱水收缩时段。在茎干水分恢复时段, 白杄茎干径向累积变化主要受到土壤含水量变化的影响。土壤温度是茎干快速生长时段影响茎干径向生长的主导环境因子, 同时它也影响着白杄茎干径向生长的开始。在茎干脱水收缩时段, 土壤温度、土壤含水量是影响茎干径向累积变化的主要环境因子。白杄径向生长最大速度出现在6月末, 其主要受到光周期(即白昼长短)影响, 是对林线处极端环境的一种适应。