The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling

BACKGROUND: Human immunodeficiency virus (HIV) Nef protein accelerates virulent progression of acquired immunodeficiency syndrome (AIDS) by its interaction with specific cellular proteins involved in signal transduction and host cell activation. Nef has been shown to bind specifically to a subset of the Src family of kinases. The structures of free Nef and Nef bound to Src homology region 3 (SH3) domain are important for the elucidation of how the affinity and specificity for the Src kinase family SH3 domains are achieved, and also for the development of potential drugs and vaccines against AIDS. RESULTS: We have determined the crystal structures of the conserved core of HIV-1 Nef protein alone and in complex with the wild-type SH3 domain of the p59fyn protein tyrosine kinase (Fyn), at 3.0 A resolution. Comparison of the bound and unbound Nef structures revealed that a proline-rich motif (Pro-x-x-Pro), which is implicated in SH3 binding, is partially disordered in the absence of the binding partner; this motif only fully adopts a left-handed polyproline type II helix conformation upon complex formation with the Fyn SH3 domain. In addition, the structures show how an arginine residue (Arg77) of Nef interacts with Asp 100 of the so-called RT loop within the Fyn SH3 domain, and triggers a hydrogen-bond rearrangement which allows the loop to adapt to complement the Nef surface. The Arg96 residue of the Fyn SH3 domain is specifically accommodated in the same hydrophobic pocket of Nef as the isoleucine residue of a previously described Fyn SH3 (Arg96-->lle) mutant that binds to Nef with higher affinity than the wild type. CONCLUSIONS: The three-dimensional structures support evidence that the Nef-Fyn complex forms in vivo and may have a crucial role in the T cell perturbating action of Nef by altering T cell receptor signaling. The structures of bound and unbound Nef reveal that the multivalency of SH3 binding may be achieved by a ligand induced flexibility in the RT loop. The structures suggest possible targets for the design of inhibitors which specifically block Nef-SH3 interactions.     

Cyclin D1 overexpression in malignant lymphomas

Cyclin D1, the regulatory subunit of certain protein kinases thought to advance the G1 phase of the cell cycle, is now established as a proto-oncogene, with evidence indicating that its derangement may contribute to the uncontrolled cell growth characteristic of tumors. The chromosomal translocation t(11;14)(q13:q32), involving rearrangement of the BCL-1 locus, is closely associated with human lymphoid neoplasia affecting mantle cell lymphomas (MCL). Recently, the putative BCL-1 proto-oncogene turned out to be none other than the cyclin D1 gene. Although the observed break points in the BCL-1 locus are not tightly clustered, its rearrangement has been documented in 40-70% of cases of mantle cell lymphoma, whereas it only rarely occurs in other B cell lymphomas. Of note, all of the known break points leave the cyclin D1 coding region structurally intact and result in increased protein expression, implying that this may provide a highly sensitive and specific marker for MCL. Recent studies demonstrated that immunohistochemical detection in paraffin-embedded material, using a monoclonal antibody, is very useful for routine diagnosis. Current knowledge of cyclin D1 overexpression in malignant lymphomas, with emphasis on its clinicopathologic significance, is reviewed.     

Rapid and selective depletion of CD4+ T lymphocytes and preferential loss of memory cells on interaction of mononuclear cells with HIV-1 glycoprotein-expressing cells

Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanism by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G2/M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M. Here we describe the mechanism of the apigenin-induced G1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10-50 microM apigenin resulted in dose-dependent cell-cycle arrest at both the G0/G1 and G2/M phases as measured by flow cytometry. The G0/G1 arrest was more clearly defined by using HDF that were synchronized in G0 and then released from quiescence by replating at subconfluent densities in medium containing 10-70 microM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 microM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G1 phase rather than in a G0 quiescent state. The G1 arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex-kinase assays of apigenin-treated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kinase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 microM apigenin-treated cells. In conclusion, apigenin treatment produced a G1 cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein.     

Three-dimensional reconstruction of live embryos using roboticmacroscope images

To determine the three-dimensional (3-D) shape of a live embryo is a technically challenging task. The authors show that reconstructions of live embryos can be done by collecting images from different viewing angles using a robotic macroscope, establishing point correspondences between these views by block matching, and using a new 3-D reconstruction algorithm that accommodates camera positioning errors. The algorithm assumes that the images are orthographic projections of the object and that the camera scaling factors are known. Point positions and camera errors are found simultaneously. Reconstructions of test objects and embryos show that meaningful reconstructions are possible only when camera positioning and alignment errors are accommodated since these errors can be substantial. Reconstructions of early-stage axolotl embryos were made from sets of 33 images. In a typical reconstruction, 781 points, each visible in at least three different views, were used to form 1511 triangles to represent the embryo surface. The resulting reconstruction had a mean radius of error of 0.27 pixels (1.1 μm). Mathematical properties of the reconstruction algorithm are identified and discussed     

An experimental pilot study of tacalcitol activities during modulation of parakeratotic skin features

Establishing guidelines and experimental models preclinical and clinical evaluations of new agents for treatment, and/or prevention of human diseases has become a task of crucial importance. Psoriasis is such one disease holding great interest for dermatology owing to its high rate of incidence and complexity of treatment. However the absence of psoriatic lesions in animals and the inability to induce them, calls for experimental techniques both in vitro and in vivo. The purpose of this study was to evaluate experimentally the effects of tacalcitol on cell proliferation and differentiation process. Thereafter a human pilot study on psoriatic patients has been developed.     

Symbol rate and modulation level-controlled adaptivemodulation/TDMA/TDD system for high-bit-rate wireless data transmission

This paper proposes a time-division multiple-access/time-division duplex (TDMA/TDD)-based symbol rate and modulation level-controlled adaptive modulation system for high-bit-rate data transmission. The proposed system controls both the symbol rate and modulation level for the next transmission time slot according to the estimated carrier power to noise spectral density ratio (C/N₀) and delay spread for the time slot to achieve higher bit rate and higher transmission quality as well as higher delay-spread immunity. It is demonstrated by computer simulation and laboratory experiments that the proposed system can achieve a higher average bit rate with higher transmission quality in comparison with the fixed-rate quaternary phase-shift keying (QPSK) system and modulation level-controlled adaptive modulation system in both flat Rayleigh and frequency-selective fading environments. The simulated and experimental results also show that the proposed adaptive modulation techniques can be applied to 1-2-Mb/s indoor and outdoor microcellular systems with its delay spread of up to 250 ns and its terminal mobility of up to pedestrian speed without employing any special antifrequency-selective fading techniques, such as the adaptive equalizer and space diversity     

Motion Perception Using Analog VLSI

Motion perception is arguably a fundamental mechanism used by natural species to accomplish a number of tasks, such as navigating freely in an unknown environment. Traditional motion perception methods tend to be computationally intensive, requiring powerful computers and large memories. However, by copying biological mechanisms, such as elementary motion discrimination at the early stages of the visual processing paths, it should be possible to build small and efficient motion perception systems. This paper describes the manner in which a simple motion perception model based on the insect visual system has been implemented using mixed analog/digital VLSI. The device has been fabricated in a 2 micron double metal, double polysilicon process, and comprises 61 photo-detectors, and associated analog and digital circuitry. While not entirely successful in that component mismatches hamper the detection of dark-to-bright changes in contrast, the results clearly show the feasibility of using such a device in autonomous control systems.     

Clock stability characterization and measurement intelecommunications

Clock stability characterization and measurement for telecommunications pose peculiar issues and requirements. This paper aims to provide an overview on this subject. After briefly recalling the background work, the key features and issues of clock stability characterization and measurement in telecommunications are described. The timing signal reference model and the stability quantities adopted in the new international standards are introduced and the impact of the measurement configuration and of the time error sampling period on their behavior are elucidated. The measurement of clock stability in telecommunications is then addressed, and a standard practical measurement procedure is outlined. Several measurement results are provided to support the concepts expounded with experimental evidence. The results shown have been chosen among those obtained throughout the last three years by testing clerks of digital switching exchanges, clocks for synchronous digital hierarchy (SDH) equipment, and state-of-the-art stand-alone slave clocks for synchronization networks. They thus represent a survey of the actual performance of clocks currently deployed in telecommunications networks     

Phase I study of mitoxantrone plus etoposide with multidrug blockade by SDZ PSC-833 in relapsed or refractory acute myelogenous leukemia

PURPOSE: Expression of the multidrug resistance gene (MDR1) p170 protein is frequent in leukemic blasts from patients with relapsed acute myelogenous leukemia (AML). A phase I study using the nonimmunosuppressive MDR1 blocker SDZ PSC-833 (PSC) in combination with mitoxantrone (MITO) and etoposide (VP) was performed. PATIENTS AND METHODS: Starting doses (LVL0) of MITO (3.25 mg/m2/d on days 1 and 3 to 6) and VP (210 mg/m2/d on days 1 and 3 to 5) were 40% of the maximal-tolerated dose (MTD) from a prior study. A 1.5-mg/kg loading dose of PSC was followed by a 120-hour continuous infusion of 10 mg/kg/d on days 2 to 6. Blood samples for PSC, MITO, and VP pharmacokinetics (PK) were taken on days 1 and 3, and samples for MDR1 expression were taken on day 0. RESULTS: Severe mucositis developed in all patients at LVL0; therefore, MITO and VP doses were reduced to 2.5 and 170 mg/m2 (LVL-1) for the next seven patients, and this dose proved to be MTD. All LVL0 and three LVL-1 patients had transient elevations in the serum bilirubin level to > or = 4 mg/dL. Serum creatinine level increased to greater than 2 mg/dL in one case. There were no other grade 3 or 4 nonhematologic toxicities observed. The peripheral blood was cleared of leukemia in three LVL0 and four LVL-1 patients. The marrow was cleared of leukemic cells in one LVL0 and five LVL-1 patients, and a significant reduction in marrow leukemic infiltrate was observed in eight of 10. No patient achieved complete remission (CR), and all died of progressive disease (n = 8) or infection (n = 2). MDR1 expression was detected by fluorescent-activated cell sorter (FACS) analysis in five of seven cases. An elevated MDR1 mRNA level was detected by quantitative polymerase chain reaction (Q-PCR) in six of eight cases studied. Clearing of leukemia cells from the marrow occurred in four of six MDR1-positive and one of three MDR1-negative patients. Despite the fact that LVL0 doses had to be reduced due to toxicity, coadministration of PSC did not produce a consistent effect on MITO PK; however, it did repeatedly lead to increased levels of VP in the serum. CONCLUSION: We conclude that PSC-MITO-VP is a tolerable regimen with antileukemic activity. Addition of PSC necessitated a 66% reduction in MITO and VP doses from a prior study without PSC.     

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB), a neural member of the EGF family of differentiation factors, is implicated in neurite formation

Chicken acidic leucine-rich EGF-like domain containing brain protein (CALEB) was identified by combining binding assays with immunological screens in the chicken nervous system as a novel member of the EGF family of differentiation factors. cDNA cloning indicates that CALEB is a multidomain protein that consists of an NH2-terminal glycosylation region, a leucine-proline-rich segment, an acidic box, a single EGF-like domain, a transmembrane, and a short cytoplasmic stretch. In the developing nervous system, CALEB is associated with glial and neuronal surfaces. CALEB is composed of a 140/130-kD doublet, an 80-kD band, and a chondroitinsulfate-containing 200-kD component. The latter two components are expressed in the embryonic nervous system and are downregulated in the adult nervous system. CALEB binds to the extracellular matrix glycoproteins tenascin-C and -R. In vitro antibody perturbation experiments reveal a participation of CALEB in neurite formation in a permissive environment.