Multilineage hematopoietic growth factor interleukin 3 and direct activators of protein kinase C stimulate phosphorylation of common substrates

In order to investigate early signal transduction events in myeloid cells, the phosphosubstrates of an interleukin 3 (IL 3)-dependent cell line, FDC-P1, have been analyzed. Using synthetic diacylglycerol as a direct activator of the unique calcium-phospholipid-dependent phosphotransferase protein kinase C (PK-C) and genetically engineered homogeneous IL 3, we have demonstrated a common element to signal transduction events associated with these stimulants. One novel substrate, p68 (68,000 kd), was rapidly phosphorylated in either IL 3- or diacylglycerol-stimulated cells. The phosphorylation of p68 was dose- dependent, with both the physiological ligand and diacylglycerol inducing the same maximal level of phosphorylation. Phosphorylation of p68 occurred in a time-dependent manner analogous to previously described kinetics of PK-C subcellular redistribution in the FDC-P1 cell line. The p68 substrate was also phosphorylated in a cell-free system under conditions designed to activate PK-C. Phosphoamino acid analysis demonstrated that the p68 molecule phosphorylated in intact cells as well as in a calcium-phospho-lipid-dependent cell-free system was phosphorylated on threonine residues, not tyrosine. These data support the hypothesis that the activation of PK-C that occurs after IL 3-receptor interaction which leads to the rapid phosphorylation of cellular proteins is an important element of the signal transduction mechanism in FDC-P1 cells. We propose that phosphorylation of the p68 molecule is a physiochemical marker for the activation of PK-C in myeloid cells, in response to the growth-promoting physiological ligand.     

Force sensing by the vascular protein von Willebrand factor is tuned by a strong intermonomer interaction

The large plasma glycoprotein von Willebrand factor (VWF) senses hydrodynamic forces in the bloodstream and responds to elevated forces with abrupt elongation, thereby increasing its adhesiveness to platelets and collagen. Remarkably, forces on VWF are elevated at sites of vascular injury, where VWF’s hemostatic potential is important to mediate platelet aggregation and to recruit platelets to the subendothelial layer. Adversely, elevated forces in stenosed vessels lead to an increased risk of VWF-mediated thrombosis. To dissect the remarkable force-sensing ability of VWF, we have performed atomic force microscopy (AFM)-based single-molecule force measurements on dimers, the smallest repeating subunits of VWF multimers. We have identified a strong intermonomer interaction that involves the D4 domain and critically depends on the presence of divalent ions, consistent with results from small-angle X-ray scattering (SAXS). Dissociation of this strong interaction occurred at forces above  ～ 50 pN and provided  ～ 80 nm of additional length to the elongation of dimers. Corroborated by the static conformation of VWF, visualized by AFM imaging, we estimate that in VWF multimers approximately one-half of the constituent dimers are firmly closed via the strong intermonomer interaction. As firmly closed dimers markedly shorten VWF’s effective length contributing to force sensing, they can be expected to tune VWF’s sensitivity to hydrodynamic flow in the blood and to thereby significantly affect VWF’s function in hemostasis and thrombosis.Force-sensing molecules are critically involved in a variety of biological processes, such as regulation of muscle gene expression or assembly of the cytoskeleton (1–4). In the vasculature, activation of the plasma glycoprotein von Willebrand factor (VWF) for hemostasis crucially depends on its distinct ability to sense hydrodynamic forces (5–7). These forces result from the interplay between hydrodynamic flow and VWF’s extraordinary length (8–10), which can exceed 15 μm in the plasma (6). VWF’s length arises from its linear multimeric nature. Linear multimers (concatamers) are composed of a variable number of dimers, which are linked N-terminally via disulfide bonds. Dimers, the smallest repeating subunits of VWF with a molecular mass of  ～ 500 kDa, consist of two monomers that are linked via C-terminal disulfide bonds (11, 12).Under static conditions, VWF was reported to adopt a collapsed conformation (6). When subjected to sufficiently high forces, as for instance at sites of vascular injury, vasoconstriction, or stenosis, VWF undergoes an abrupt transition from the collapsed to a stretched conformation (Fig. 1A) (6). This transition was shown to correlate with an increased adhesiveness to collagen and platelets (6, 13), enabling stretched VWF to recruit platelets to an injured vessel wall and to promote the formation of a platelet plug. VWF’s physiological importance is underlined by mutations that can cause von Willebrand disease (14), the most common hereditary bleeding disorder.Open in a separate windowFig. 1.Single-molecule force measurements on VWF dimers. (A) Illustration of VWF’s ability to sense hydrodynamic forces in blood vessels. When subjected to sufficiently high forces, for instance at sites of vascular injury, vasoconstriction, or stenosis, VWF undergoes an abrupt transition from a collapsed to a stretched conformation and promotes hemostasis. (B) Schematic representation of pulling recombinant VWF dimers. A ybbR-tag at the N terminus of one of the monomers allowed for covalent anchoring, and a Twin-Strep-tag at the N terminus of the other monomer enabled specific pulling via a Strep-Tactin functionalized AFM cantilever. (C) Denoised force–extension traces of dimers showing A2 unfolding peaks (blue arrows) at low (type I traces) or at high extension values (type II traces). Type I traces repeatedly exhibited a peak (dimer opening, green arrow) at higher force. The final peak (brown arrow) corresponds to the unbinding of the Twin-Strep-tag from Strep-Tactin. (D) Bimodal distribution of the position of the first A2 unfolding event.Down-regulation of VWF’s hemostatic potential is achieved by the cleavage of long concatamers into shorter ones by the enzyme ADAMTS13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13) (15). Notably, the specific cleavage site is buried in the A2 domain and exposed by A2 unfolding (8, 16). The interplay of force-induced A2 unfolding and enzymatic cleavage has been investigated in detail at single-molecule level (8). In this context, unfolding of an isolated A2 domain was shown to occur at forces of ∼ 7–14 pN at loading rates ranging from 0.35 to 350 pN⋅s⁻¹. In the presence of neighboring A1 and A3 domains, A2 unfolding was observed at slightly higher forces of ∼ 20 pN and comparable loading rates (17).VWF’s activation for hemostasis correlates with its elongation above a critical force threshold (6, 7). However, the current understanding of the underlying molecular mechanisms is limited. Clearly, A2 unfolding is likely to contribute significantly to the elongation of VWF, as the induced length increment of roughly 45 nm (at 20 pN) is almost as large as the end-to-end length of a static monomer (8, 17–19). Stabilization of all other domains in VWF through disulfide bonds was predicted (20), but lacks experimental evidence.Besides domain unfolding, separation of potential intramonomer and intermonomer interactions may play a crucial role for VWF’s elongation. For example, interactions between monomers may tune VWF’s force-sensing ability by promoting compactness, as suggested by various computational studies (6, 21, 22). Experimentally, self-association of VWF molecules was reported, but has not been assigned to individual domains (23, 24).In this study, we report on force-induced conformational changes of VWF and present a strong intermonomer interaction that is expected to tune VWF’s force-sensing ability in the bloodstream. Evidence for this interaction comes from force–extension traces of dimers, which were probed in atomic force microscopy (AFM)-based single-molecule force measurements. Complementarily, we characterized the static conformation of VWF by AFM imaging and small-angle X-ray scattering (SAXS). From the combination of force and imaging data, we gain a quantitative understanding of the mechanisms underlying the force-sensing ability of VWF.     

High frequency of N-ras activation in acute myelogenous leukemia

Using the NIH/3T3 cell transfection assay, activated cellular oncogenes have been detected in around 10% to 20% of human tumors. From a series of DNA preparations from tissues infiltrated with acute myelogenous leukemia (AML), 50% (3/6) caused transformation of NIH/3T3 cells. Thus AML appears to be the human tumor with the highest frequency of oncogenes detected by DNA transfection. In each case the oncogene involved was N-ras, a member of the ras gene family. Biologic and clinical parameters of AML patients with and without N-ras oncogenes in their tumors are discussed.     

Expression of cell surface receptors and oxidative metabolism modulation in the clinical continuum of sepsis

Background  

Infection control depends on adequate microbe recognition and cell activation, yet inflammatory response may lead to organ dysfunction in sepsis. The aims of this study were to evaluate cell activation in the context of sepsis and its correlation with organ dysfunction.     

Orthotopic liver transplantation in a patient with carbamyl phosphate synthetase deficiency and cystic fibrosis

A 15-year-old female with carbamyl phosphate synthetase deficiency, cystic fibrosis, and cystic fibrosis-related diabetes underwent orthotopic cadaveric liver transplantation. Metabolic control was maintained during the procedure with nutritional support and the use of intravenous sodium phenylacetate and benzoate. Her postoperative course was complicated by seizures and a transient decline in her pulmonary function tests, which returned to preoperative levels within one year of the transplant. Now, four years post-transplant, her quality of life has dramatically improved. There are only four Canadian centres with paediatric liver transplantation programs. However, expert medical care for adults with inborn error of metabolism is even more limited, suggesting that access to adult medical care is one of the many factors to be considered when liver transplantation is contemplated for patients with metabolically unstable conditions.