Membrane structure and interactions with protein and DNA in bacteriophage PRD1

Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment. Biological membranes and their associated proteins present considerable difficulties for structural analysis. Although enveloped viruses have been imaged at about 9 A resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described. The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 A resolution, allows the first detailed analysis of a membrane-containing virus. The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper. Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA. The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets. The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell. In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit. In addition, the lipid headgroups show a surprising degree of order.     

The principle of temperature-dependent gating in cold- and heat-sensitive TRP channels

The mammalian sensory system is capable of discriminating thermal stimuli ranging from noxious cold to noxious heat. Principal temperature sensors belong to the TRP cation channel family, but the mechanisms underlying the marked temperature sensitivity of opening and closing ('gating') of these channels are unknown. Here we show that temperature sensing is tightly linked to voltage-dependent gating in the cold-sensitive channel TRPM8 and the heat-sensitive channel TRPV1. Both channels are activated upon depolarization, and changes in temperature result in graded shifts of their voltage-dependent activation curves. The chemical agonists menthol (TRPM8) and capsaicin (TRPV1) function as gating modifiers, shifting activation curves towards physiological membrane potentials. Kinetic analysis of gating at different temperatures indicates that temperature sensitivity in TRPM8 and TRPV1 arises from a tenfold difference in the activation energies associated with voltage-dependent opening and closing. Our results suggest a simple unifying principle that explains both cold and heat sensitivity in TRP channels.     

Expression of lysine-rich protein gene and analysis of lysine content in transgenic wheat

Expression vector pBPC102, which carries winged bean lysine-rich protein (wblrp) gene and dihydropicolinate synthase (DHDPS) gene, was transferred into hexaploid winter wheat cv. Jinghua No.l, Jing411, You899 and Yangnongl5 explants of immature inflorescence and immature embryos by particle bombardment. More than 100 transgenic plants were obtained under the selection of s-(2-aminoethyl)-L-cysteine (AEC). Confirmed transgenic plants of To and TI generation by PCR and PCR-Southern blotting analyses showed successful integration of wblrp gene into wheat genome. Analysis of transgenic plant lines of T2 by Northern dot-blotting showed good expression of wblrp gene in offspring seed. The content of free lysine in leaves, contents of bound lysine and total proteins in seeds of T2 transgenie wheat lines were determined and analyzed. Among 34 tested transgenic lines, levels of free lysine content in leaves of 9 transgenic lines are 2~3times higher than un-trans-formed wild-type cultivars. Among 17 analyzed transgenic lines, bound lysine content of 4 transgenic lines is more than 10% higher than that of wild-type cultivars. Our research suggests that introducing wblrp gene into wheat is an effective way to improve its nutrition quality.     

Differential distribution of TASK-1, TASK-2 and TASK-3 immunoreactivities in the rat and human cerebellum

In this work, the distributions of some acid-sensitive two-pore-domain K⁺ channels (TASK-1, TASK-2 and TASK-3) were investigated in the rat and human cerebellum. Astrocytes situated in rat cerebellar tissue sections were positive for TASK-2 channels. Purkinje cells were strongly stained and granule cells and astrocytes were moderately positive for TASK-3. Astrocytes isolated from the hippocampus, cerebellum and cochlear nucleus expressed TASK channels in a primary tissue culture. Our results suggest that TASK channel expression may be significant in the endoplasmic reticulum of the astrocytes. The human cerebellum showed weak TASK-2 immunolabelling. The pia mater, astrocytes, Purkinje and granule cells demonstrated strong TASK-1 and TASK-3 positivities. The TASK-3 labelling was stronger in general, but it was particularly intense in the Purkinje cells and pia mater.Received 25 February 2004; received after revision 19 April 2004; accepted 28 April 2004     

What’s new in the renin-angiotensin system?

Activation of the type 1 angiotensin II receptor (AT(1)R) is associated with the aetiology of left ventricular hypertrophy, although the exact intracellular signalling mechanism(s) remain unclear. Transactivation of the epidermal growth factor receptor (EGFR) has emerged as a central mechanism by which the G protein-coupled AT(1)R, which lacks intrinsic tyrosine kinase activity, can stimulate the mitogen-activated protein kinase signalling pathways thought to mediate cardiac hypertrophy. Current studies support a model whereby AT(1)R-dependent transactivation of EGFRs on cardiomyocytes involves stimulation of membrane-bound metalloproteases, which in turn cleave EGFR ligands such as heparin-binding EGF from a plasma membrane-associated precursor. Numerous aspects of the 'triple membrane-passing signalling' paradigm of AT(1)R-induced EGFR transactivation remain to be characterised, including the identity of the specific metalloproteases involved, the intracellular mechanism for their activation and the exact EGFR subtypes required. Here we examine how 'hijacking' of the EGFR might explain the ability of the AT(1)R to elicit the temporally and qualitatively diverse responses characteristic of the hypertrophic phenotype, and discuss the ramifications of delineating these pathways for the development of new therapeutic strategies to combat cardiac hypertrophy.     

Heterozygous missense mutations in BSCL2 are associated with distal hereditary motor neuropathy and Silver syndrome

Distal hereditary motor neuropathy (dHMN) or distal spinal muscular atrophy (OMIM #182960) is a heterogeneous group of disorders characterized by an almost exclusive degeneration of motor nerve fibers, predominantly in the distal part of the limbs. Silver syndrome (OMIM #270685) is a rare form of hereditary spastic paraparesis mapped to chromosome 11q12-q14 (SPG17) in which spasticity of the legs is accompanied by amyotrophy of the hands and occasionally also the lower limbs. Silver syndrome and most forms of dHMN are autosomal dominantly inherited with incomplete penetrance and a broad variability in clinical expression. A genome-wide scan in an Austrian family with dHMN-V (ref. 4) showed linkage to the locus SPG17, which was confirmed in 16 additional families with a phenotype characteristic of dHMN or Silver syndrome. After refining the critical region to 1 Mb, we sequenced the gene Berardinelli-Seip congenital lipodystrophy (BSCL2) and identified two heterozygous missense mutations resulting in the amino acid substitutions N88S and S90L. Null mutations in BSCL2, which encodes the protein seipin, were previously shown to be associated with autosomal recessive Berardinelli-Seip congenital lipodystrophy (OMIM #269700). We show that seipin is an integral membrane protein of the endoplasmic reticulum (ER). The amino acid substitutions N88S and S90L affect glycosylation of seipin and result in aggregate formation leading to neurodegeneration.     

A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling

PDGF-C is a member of the platelet-derived growth factor (PDGF) family, which signals through PDGF receptor (PDGFR) alphaalpha and alphabeta dimers. Here we show that Pdgfc(-/-) mice die in the perinatal period owing to feeding and respiratory difficulties associated with a complete cleft of the secondary palate. This phenotype was less severe than that of Pdgfra(-/-) embryos. Pdgfc(-/-) Pdgfa(-/-) embryos developed a cleft face, subepidermal blistering, deficiency of renal cortex mesenchyme, spina bifida and skeletal and vascular defects. Complete loss of function of both ligands, therefore, phenocopied the loss of PDGFR-alpha function, suggesting that both PDGF-A and PDGF-C signal through PDGFR-alpha to regulate the development of craniofacial structures, the neural tube and mesodermal organs. Our results also show that PDGF-C signaling is a new pathway in palatogenesis, different from, and independent of, those previously implicated.