Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is a widespread receptor-coupled signalling system at the plasma membrane of most eukaryotic cells. The existence of an entirely separate nuclear phosphoinositide signalling system is suggested from evidence that purified nuclei synthesize PtdInsP2 and phosphatidylinositol 4-phosphate (PtdInsP) in vitro and that a transient decrease in the mass of these lipids occurs when Swiss 3T3 cells are cultured in the presence of insulin-like growth factor-1 (IGF-1). These IGF-1-dependent changes in inositol lipids coincide with an increase in nuclear diacyglycerol and precede translocation to the nucleus and activation of protein kinase C (refs 5, 6). Circumstantial evidence that links these changes with mitosis comes from the isolation of a 3T3 clone that expresses the type-1 IGF receptor and binds IGF-1 peptide but does not respond mitogenically or show transient mass changes in nuclear inositol lipids. A key question is how IGF-1 initiates the rapid breakdown of PtdInsP and PtdInsP2 in the nucleus. Here we present evidence that nuclei of 3T3 cells contain the beta-isozyme of phosphoinositidase C, whereas the gamma-isozyme is confined to the cytoplasm and that IGF-1 treatment stimulates exclusively the activity of nuclear phosphoinositidase C.     

Seasonality in human sleep.

The timing of sleep and sleep EEG parameters in 10 healthy male subjects were investigated in four seasons under controlled conditions. The phase of nocturnal sleep was delayed about one and a half hours in winter as compared to that in summer. The duration of stage 4 sleep decreased and REM sleep increased significantly in winter compared with summer. The seasonality in the timing of sleep can be explained by photoperiodic time cues, but the changes in sleep EEG parameters are difficult to explain in terms of photoperiod.     

国际发展趋势下实验力学的未来作用

考察了分析结构、结构构件以及技术系统中的位移、应变与应力的测量和实验方法在目前和未来的作用。现在,比以往任何时候都更有理由和有更广阔的领域需要用到实验力学方法;许多先进、现代化技术,新型测量设备,测量和赋值过程自动化系统可供使用;以先进的测量技术和计算机技术相结合而形成的各种杂交法已被提出来。利用这些方法,可以优化结构和产品,节约能源和原材料,提高产品质量,增强结构、技术系统的可靠、安全性,控制生产过程以及监督操作系统的安全性;利用这些方法,能够并且应当使技术系统可能发生的危险以及对环境的毁损降至最低程度。     

Prediction of the structure of a receptor-protein complex using a binary docking method.

To validate procedures of rational drug design, it is important to develop computational methods that predict binding sites between a protein and a ligand molecule. Many small molecules have been tested using such programs, but examination of protein-protein and peptide-protein interactions has been sparse. We were able to test such applications once the structures of both the maltose-binding protein (MBP) and the ligand-binding domain of the aspartate receptor, which binds MBP, became available. Here we predict the binding site of MBP to its receptor using a 'binary docking' technique in which two MBP octapeptide sequences containing mutations that eliminate maltose chemotaxis are independently docked to the receptor. The peptides in the docked solutions superimpose on their original positions in the structure of MBP and allow the formation of an MBP-receptor complex. The consistency of the computational and biological results supports this approach for predicting protein-protein and peptide-protein interactions.     

Clustering of voltage-dependent sodium channels on axons depends on Schwann cell contact.

In myelinated nerves, segregation of voltage-dependent sodium channels to nodes of Ranvier is crucial for saltatory conduction along axons. As sodium channels associate and colocalize with ankyrin at nodes of Ranvier, one possibility is that sodium channels are recruited and immobilized at axonal sites which are specified by the subaxolemmal cytoskeleton, independent of glial cell contact. Alternatively, segregation of channels at distinct sites along the axon may depend on glial cell contact. To resolve this question, we have examined the distribution of sodium channels, ankyrin and spectrin in myelination-competent cocultures of sensory neurons and Schwann cells by immunofluorescence, using sodium channel-, ankyrin- and spectrin-specific antibodies. In the absence of Schwann cells, sodium channels, ankyrin and spectrin are homogeneously distributed on sensory axons. When Schwann cells are introduced into these cultures, the distribution of sodium channels dramatically changes so that channel clusters on axons are abundant, but ankyrin and spectrin remain homogeneously distributed. Addition of latex beads or Schwann cell membranes does not induce channel clustering. Our results suggest that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that continuing axon-glial interactions are necessary to organize and maintain channel distribution during differentiation of myelinated axons.     

Short alanine-based peptides may form 3(10)-helices and not alpha-helices in aqueous solution.

Short alanine peptides, containing 16 or 17 residues, appear to form alpha-helices in aqueous solution. But the main spectroscopic analyses used on helical peptides (circular dichroism and nuclear magnetic resonance) cannot distinguish between an alpha-helix (in which the ith residue is hydrogen-bonded to residue i + 4; ref. 9) and the next most common peptide helix, the 3(10)-helix10 (i-->i + 3 hydrogen-bonding). To address this problem we have designed single and doubly spin-labelled analogues of alanine-based peptides in which the nitroxide spin label forms an unbranched side chain extending from the sulphur atom of a cysteine residue. Here we report the circular dichroism, Fourier-transform infrared and electron-spin resonance spectra of these peptides under helix-forming conditions. The infrared absorbance gives an amide I' band with a frequency that is substantially different from that observed for alpha-helices. The electron-spin resonance spectra of doubly labelled helices show that the ranking of distances between side chains, around a single turn (residues 4-8), is inconsistent with an alpha-helical structure. Our experiments suggest that the more likely peptide geometry is a 3(10)-helix.     

A calcium sensor in the sodium channel modulates cardiac excitability.

Sodium channels are principal molecular determinants responsible for myocardial conduction and maintenance of the cardiac rhythm. Calcium ions (Ca2+) have a fundamental role in the coupling of cardiac myocyte excitation and contraction, yet mechanisms whereby intracellular Ca2+ may directly modulate Na channel function have yet to be identified. Here we show that calmodulin (CaM), a ubiquitous Ca2+-sensing protein, binds to the carboxy-terminal 'IQ' domain of the human cardiac Na channel (hH1) in a Ca2+-dependent manner. This binding interaction significantly enhances slow inactivation-a channel-gating process linked to life-threatening idiopathic ventricular arrhythmias. Mutations targeted to the IQ domain disrupted CaM binding and eliminated Ca2+/CaM-dependent slow inactivation, whereas the gating effects of Ca2+/CaM were restored by intracellular application of a peptide modelled after the IQ domain. A naturally occurring mutation (A1924T) in the IQ domain altered hH1 function in a manner characteristic of the Brugada arrhythmia syndrome, but at the same time inhibited slow inactivation induced by Ca2+/CaM, yielding a clinically benign (arrhythmia free) phenotype.     

Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA

Heat-shock proteins are normal constituents of cells whose synthesis is increased on exposure to various forms of stress. They are interesting because of their ubiquity and high conservation during evolution. Two families of heat-shock proteins, hsp60s and hsp70s, have been implicated in accelerating protein folding and oligomerization and also in maintaining proteins in an unfolded state, thus facilitating membrane transport. The Escherichia coli hsp70 analogue, DnaK, and two other heat-shock proteins, DnaJ and GrpE, are required for cell viability at high temperatures and are involved in DNA replication of phage lambda and plasmids P1 and F. These three proteins are involved in replication in vitro of P1 DNA along with many host replication proteins and the P1 RepA initiator protein. RepA exists in a stable protein complex with DnaJ containing a dimer each of RepA and DnaJ. We report here that DnaK and DnaJ mediate an alteration in the P1 initiator protein, rendering it much more active for oriP1 DNA binding.     

Abnormal pattern detected in fragile-X patients by pulsed-field gel electrophoresis.

The fragile-X syndrome is the most frequent inherited form of mental retardation, with an incidence of 1 in 1,500 males. It is characterized by the presence of a fragile site at Xq27.3 induced in vitro by folate deprivation or by inhibitors of deoxynucleotide synthesis. Its mode of inheritance is unusual for an X-linked trait, with incomplete penetrance in both males and females. Some phenotypically normal males transmit the mutation to all their daughters who rarely express any symptoms, but penetrance is high in sons and daughters of these carrier women. Genetic and physical mapping of the Xq27-q28 region has confirmed that the disease locus is located at or very near the fragile site. Hypotheses proposed to account for the abnormalities in the inheritance of the disease include sequence rearrangements by meiotic recombination or a mutation that affects reactivation of an inactive X chromosome during differentiation of female germ cells. To detect such rearrangements, or methylation changes that may reflect a locally inactive X chromosome, we used pulsed-field gel analysis of DNA from fragile-X patients with probes close to the fragile-X locus. The probe Do33 (DXS465) detected abnormal patterns in fragile-X patients, but not in normal controls or in non-expressing male transmitters.