Transfer ofF′ fromEscherichia coli K 12 toEscherichia coli B and to strains ofParacolobacter andKlebsiella

The transfer of theF episome fromEscherichia coli K 12 toE. coli B,Paracolobacter andKlebsiella was studied. The frequency of transfer of the episomal markers toE. coli B was very low. The large majority ofE. coli B cells which had received the episomal markerslac ⁺ orgal ⁺ were F^–, which indicates that the episomal markers were stably integrated on the chromosome. Recombinants from K 12 F⁺ × B F^– crosses were mostly F^–. These results suggest that the multiplication of theF-factor ofE. coli K 12 is restricted inE. coli B. The transfer of theF-lac ⁺ Ad ⁺ episome fromE. coli K 12 toParacolobacter andKlebsiella strains was in most cases only possible when donor and acceptor strain were plated together on selective media. Stable incorporation of episomal markers was also found withParacolobacter coliforme. Paracolobacter aerogenoides andKlebsiella aerogenes strains could be infected withF-lac ⁺ Ad ⁺. The episomal markers were not incorporated and the episomes were easily lost, which indicates that these strains contained theF factor in the autonomous state.     

The separation of voltage-dependent photoemfs and conductances in Rudin-Mueller membranes containing magnesium porphyrins

It is shown that the photoemf in a pigmented membrane is specific to the magnesium-porphyrin conductance channel. A null current method was devised to measure directly the voltage dependence of the photoemf and the magnesium-porphyrin conductance. Their voltage dependence is in agreement with the hypothesis that the magnesium-porphyrin cation is the majority carrier     

High affinity targets of protein kinase inhibitors have similar residues at the positions energetically important for binding

Inhibition of protein kinase activity is a focus of intense drug discovery efforts in several therapeutic areas. Major challenges facing the field include understanding of the factors determining the selectivity of kinase inhibitors and the development of compounds with the desired selectivity profile. Here, we report the analysis of sequence variability among high and low affinity targets of eight different small molecule kinase inhibitors (BIRB796, Tarceva, NU6102, Gleevec, SB203580, balanol, H89, PP1). It is observed that all high affinity targets of each inhibitor are found among a relatively small number of kinases, which have similar residues at the specific positions important for binding. The findings are highly statistically significant, and allow one to exclude the majority of kinases in a genome from a list of likely targets for an inhibitor. The findings have implications for the design of novel inhibitors with a desired selectivity profile (e.g. targeted at multiple kinases), the discovery of new targets for kinase inhibitor drugs, comparative analysis of different in vivo models, and the design of "a-la-carte" chemical libraries tailored for individual kinases.     

The novel GABA adamantane derivative (AdGABA): design, synthesis, and activity relationship with gabapentin

A facile preparation of 2-aminomethyl-2-tricyclo[3.3.1.1(1,7)]decaneacetic acid hydrochloride 5 (AdGABA) is described. The synthesis of AdGABA involves the hydrogenation of 2-cyano-2-tricyclo[3.3.1.1(1,7)]decaneacetic acid 11, which was synthesized by two different synthetic routes. AdGABA was found to antagonize the pentylenetetrazole (PTZ) and semicarbazide (SCZ) induced tonic convulsions and exhibits analgesic activity in the hot plate test on mice. Although its mechanism of action is quite similar to that proposed previously for gabapentin (interaction with the alpha2delta subunit of the voltage gated Ca2+ channels), further studies were undertaken in order to clarify the precise mechanism of the anticonvulsant and analgesic effects of AdGABA on a molecular level.     

J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1

J-domain cochaperones confer functional specificity to their heat shock protein (HSP)70 partner by recruiting it to specific substrate proteins. To gain insight into the functions of plastidic HSP70s, we searched in Chlamydomonas databases for expressed sequence tags that potentially encode chloroplast-targeted J-domain cochaperones. Two such cDNAs were found: the encoded J-domain proteins were named chloroplast DnaJ homolog 1 and 2 (CDJ1 and CDJ2). CDJ2 was shown to interact with a approximately 28-kDa protein that by mass spectrometry was identified as the vesicle-inducing protein in plastids 1 (VIPP1). In fractionation experiments, CDJ2 was detected almost exclusively in the stroma, whereas VIPP1 was found in low-density membranes, thylakoids, and in the stroma. Coimmunoprecipitation and mass spectrometry analyses identified stromal HSP70B as the major protein interacting with soluble VIPP1, and, as confirmed by cross-linking data, as chaperone partner of CDJ2. In blue native-PAGE of soluble cell extracts, CDJ2 and VIPP1 comigrated in complexes of >669, approximately 150, and perhaps approximately 300 kDa. Our data suggest that CDJ2, presumably via coiled-coil interactions, binds to VIPP1 and presents it to HSP70B in the ATP state. Our findings and the previously reported requirement of VIPP1 for the biogenesis of thylakoid membranes point to a role for the HSP70B/CDJ2 chaperone pair in this process.     

Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis

Cilia, as motile and sensory organelles, have been implicated in normal development, as well as diseases including cystic kidney disease, hydrocephalus and situs inversus. In kidney epithelia, cilia are proposed to be non-motile sensory organelles, while in the mouse node, two cilia populations, motile and non-motile have been proposed to regulate situs. We show that cilia in the zebrafish larval kidney, the spinal cord and Kupffer's vesicle are motile, suggesting that fluid flow is a common feature of each of these organs. Disruption of cilia structure or motility resulted in pronephric cyst formation, hydrocephalus and left-right asymmetry defects. The data show that loss of fluid flow leads to fluid accumulation, which can account for organ distension pathologies in the kidney and brain. In Kupffer's vesicle, loss of flow is associated with loss of left-right patterning, indicating that the 'nodal flow' mechanism of generating situs is conserved in non-mammalian vertebrates.     

Control of dendritic diversity

The dendritic trees of different neuronal types display an astonishing diversity in structure and function. How this diversity is generated remains incompletely understood. However, recent studies have revealed some of the underlying mechanisms by which intrinsic programs of cell-type specification and extrinsic factors exert their effects on the dendritic cytoskeleton to regulate patterns of growth and branching.     

Fused pyrimidine based inhibitors of phosphodiesterase 7 (PDE7): synthesis and initial structure-activity relationships

A series of fused pyrimidine based inhibitors of PDE7 have been derived from an earlier screening lead 1. The synthesis, structure-activity relationships (SAR) and selectivity against several other PDE family members are described.