Regulation of Potassium-Dependent Kdp-ATPase Expression in the Nitrogen-Fixing Cyanobacterium Anabaena torulosa

The KdpB polypeptides in the cyanobacterium Anabaena torulosa were shown to be two membrane-bound proteins of about 78 kDa, expressed strictly under K(+) deficiency and repressed or degraded upon readdition of K(+). In both Anabaena and Escherichia coli strain MC4100, osmotic and ionic stresses caused no significant induction of steady-state KdpB levels during extreme potassium starvation.     

Comparative chlorine and temperature tolerance of the oyster crassostrea madrasensis: Implications for cooling system fouling

Crassostrea madrasensis is an important fouling oyster in tropical industrial cooling water systems. C. madrasensis individuals attach to surfaces by cementing one of their two valves to the substratum. Therefore, oyster fouling creates more problems than mussel fouling in the cooling conduits of power stations, because unlike the latter, the shell of the former remains attached to the substratum even after the death of the animal. However, there are no published reports on the tolerance of this species to chlorination and heat treatment. The mortality pattern and physiological behaviour (oxygen consumption and filtration rate) of three size groups (13 mm, 44 mm and 64 mm mean shell length) of C. madrasensis were studied at different residual chlorine concentrations (0.25, 0.5, 0.75, 1, 2, 3 to 5 mg l^{m 1}) and temperatures (30°C to 45°C). The effect of shell size (=age) on C. madrasensis mortality in the presence of chlorine and taking into account temperature was significant, with the largest size group oysters showing highest resistance. At 1 mg l^{m 1} residual chlorine, the 13 mm and 64 mm size group oysters took 504 h (21 d) and 744 h (31 d), respectively to reach 100% mortality. At 39°C, the 13 mm size group oysters took 218 min to reach 100% mortality, whereas the 64 mm size group oysters took 325 min. The oxygen consumption and filtration rate of C. madrasensis showed progressive reduction with increasing residual chlorine concentrations. However, the filtration rate and oxygen consumption responses of C. madrasensis were not significantly different between 30°C (control) and 37.5°C. There was a sharp decrease in the filtration rate and oxygen consumption at 38.5°C. A comparison of the present mortality data with previous reports on other bivalves suggests that the chlorine tolerance of C. madrasensis lies in between that of Perna viridis and Perna perna, while its temperature tolerance is significantly higher than that of the other two bivalve species. However, in power station heat exchangers, where simultaneous chlorine and thermal stresses are existent, C. madrasensis may have an edge over other common foulants, because of its high temperature tolerance.     

Hydrodynamic Determinants of Cell Necrosis and Molecular Delivery Produced by Pulsed Laser Microbeam Irradiation of Adherent Cells

Time-resolved imaging, fluorescence microscopy, and hydrodynamic modeling were used to examine cell lysis and molecular delivery produced by picosecond and nanosecond pulsed laser microbeam irradiation in adherent cell cultures. Pulsed laser microbeam radiation at λ = 532 nm was delivered to confluent monolayers of PtK₂ cells via a 40×, 0.8 NA microscope objective. Using laser microbeam pulse durations of 180–1100 ps and pulse energies of 0.5–10.5 μJ, we examined the resulting plasma formation and cavitation bubble dynamics that lead to laser-induced cell lysis, necrosis, and molecular delivery. The cavitation bubble dynamics are imaged at times of 0.5 ns to 50 μs after the pulsed laser microbeam irradiation, and fluorescence assays assess the resulting cell viability and molecular delivery of 3 kDa dextran molecules. Reductions in both the threshold laser microbeam pulse energy for plasma formation and the cavitation bubble energy are observed with decreasing pulse duration. These energy reductions provide for increased precision of laser-based cellular manipulation including cell lysis, cell necrosis, and molecular delivery. Hydrodynamic analysis reveals critical values for the shear-stress impulse generated by the cavitation bubble dynamics governs the location and spatial extent of cell necrosis and molecular delivery independent of pulse duration and pulse energy. Specifically, cellular exposure to a shear-stress impulse J?0.1 Pa s ensures cell lysis or necrosis, whereas exposures in the range of 0.035?J?0.1 Pa s preserve cell viability while also enabling molecular delivery of 3 kDa dextran. Exposure to shear-stress impulses of J?0.035 Pa s leaves the cells unaffected. Hydrodynamic analysis of these data, combined with data from studies of 6 ns microbeam irradiation, demonstrates the primacy of shear-stress impulse in determining cellular outcome resulting from pulsed laser microbeam irradiation spanning a nearly two-orders-of-magnitude range of pulse energy and pulse duration. These results provide a mechanistic foundation and design strategy applicable to a broad range of laser-based cellular manipulation procedures.     

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD^−/− mice. LCAD^−/− mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD^−/− mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD^−/− surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD^−/− lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.     

Design,synthesis and pharmacological evaluation of some novel indanone derivatives as acetylcholinesterase inhibitors for the management of cognitive dysfunction

The present study reports the effect of indanone derivatives on scopolamine induced deficit cholinergic neurotransmission serving as promising leads for the therapeutics of cognitive dysfunction. Eleven compounds 54–64 have been designed, synthesised and evaluated against behavioural alterations using step down passive avoidance protocol at a dose of 0.5?mg/kg with Donepezil (1) as the reference standard. All the synthesised compounds were evaluated for their in vitro acetylcholinesterase (AChE) inhibition at five different concentrations using mice brain homogenate as the source of the enzyme. Compounds 54, 56, 59 and 64 displayed appreciable activity with an IC₅₀ value of 14.06?µM, 12.30?µM, 14.06?µM and 12.01?µM, respectively towards acetylcholinesterase inhibition. The molecular docking study performed to predict the binding mode of the compounds suggested that these compounds could bind appreciably to the amino acids present at the active site of recombinant human acetylcholinesterase (rhAChE). The behavioural, biochemical and in silico pharmacokinetic studies were in concordance with each other.     

GKAP, a Novel Synaptic Protein That Interacts with the Guanylate Kinase-like Domain of the PSD-95/SAP90 Family of Channel Clustering Molecules

The molecular mechanisms underlying the organization of ion channels and signaling molecules at the synaptic junction are largely unknown. Recently, members of the PSD-95/SAP90 family of synaptic MAGUK (membrane-associated guanylate kinase) proteins have been shown to interact, via their NH₂-terminal PDZ domains, with certain ion channels (NMDA receptors and K⁺ channels), thereby promoting the clustering of these proteins. Although the function of the NH₂-terminal PDZ domains is relatively well characterized, the function of the Src homology 3 (SH3) domain and the guanylate kinase-like (GK) domain in the COOH-terminal half of PSD-95 has remained obscure. We now report the isolation of a novel synaptic protein, termed GKAP for guanylate kinase-associated protein, that binds directly to the GK domain of the four known members of the mammalian PSD-95 family. GKAP shows a unique domain structure and appears to be a major constituent of the postsynaptic density. GKAP colocalizes and coimmunoprecipitates with PSD-95 in vivo, and coclusters with PSD-95 and K⁺ channels/ NMDA receptors in heterologous cells. Given their apparent lack of guanylate kinase enzymatic activity, the fact that the GK domain can act as a site for protein– protein interaction has implications for the function of diverse GK-containing proteins (such as p55, ZO-1, and LIN-2/CASK).     

Identification of new HLA class I region genes by sample sequencing

 Although many human major histocompatibility genes have been identified, relatively few have been localized to the class I region. We searched for new class I region genes by sample sequencing, a process in which short stretches of random genomic sequence are generated from cosmids and then compared with sequences deposited in nucleotide databases. Four class I region cosmids were isolated for sample sequencing by screening a chromosome 6 specific cosmid library with probes derived from specific class I region genes or with overlapping class I region yeast artificial chromosomes. Cosmids were sonnicated to produce fragments of 0.5 – 1 kilobases, subcloned, and sequenced using an automated sequencer. Sequences were then compared with nucleotide sequences deposited in the GenBank databases using the BLASTN algorithm. A number of potential new class I region genes were identified, including a cDNA with similarity to the tre oncogene, the trans-activating factor SC1 (TCF19), and a member of the interferon inducible 1 – 8 gene family. These observations suggest that sample sequencing is an efficient method for identifying new class I region genes, which can be applied to other regions of the genome and to other species, and support previous observations that the class I region contains a variety of genes other than those encoding HLA antigens. Received: 10 December 1996 / Revised: 7 January 1997     

EhRho1, a RhoA-Like GTPase of Entamoeba histolytica, Is Modified by Clostridial Glucosylating Cytotoxins

Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi, which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[¹⁴C]glucose.     

Rod outer segment membrane guanylate cyclase type 1 (ROS-GC1) gene: Structure,organization and regulation by phorbol ester,a protein kinase C activator

At present there are two recognized members of the ROS-GC subfamily of membrane guanylate cyclases. They are ROS-GC1 and ROS-GC2. A distinctive feature of this family is that its members are not switched on by the extracellular peptide hormones; instead, they are modulated by intracellular Ca2+ signals, consistent to their linkage with phototransduction. An intriguing feature of ROS-GC1, which distinguishes it from ROS-GC2, is that it has two Ca2+ switches. One switch inhibits the enzyme at micromolar concentrations of Ca2+, as in phototransduction; the other, stimulates. The stimulatory switch, most likely, is linked to retinal synaptic activity. Thus, ROS-GC1 is linked to both phototransduction and the synaptic activity. The present study describes (1) the almost complete structural identity of 18.5 kb ROS-GC1 gene; (2) its structural organization: the gene is composed of 20 exons and 19 introns with classical GT/AG boundaries; (3) the activity of the ROS-GC1 promoter assayed through luciferase reporter in COS cells; and (4) induction of the gene by phorbol ester, a protein kinase C (PKC) activator. The co-presence of PKC and ROS-GC1 in photoreceptors suggests that regulation of the ROS-GC1 gene by PKC might be a physiologically relevant phenomenon.     

Structural and Functional Analyses Reveal Insights into the Molecular Properties of the Escherichia coli Z Ring Stabilizing Protein,ZapC

In Escherichia coli cell division is driven by the tubulin-like GTPase, FtsZ, which forms the cytokinetic Z-ring. The Z-ring serves as a dynamic platform for the assembly of the multiprotein divisome, which catalyzes membrane cleavage to create equal daughter cells. Several proteins effect FtsZ assembly, thereby providing spatiotemporal control over cell division. One important class of FtsZ interacting/regulatory proteins is the Z-ring-associated proteins, Zaps, which typically modulate Z-ring formation by increasing lateral interactions between FtsZ protofilaments. Strikingly, these Zap proteins show no discernable sequence similarity, suggesting that they likely harbor distinct structures and mechanisms. The 19.8-kDa ZapC in particular shows no homology to any known protein. To gain insight into ZapC function, we determined its structure to 2.15 Å and performed genetic and biochemical studies. ZapC is a monomer composed of two domains, an N-terminal α/β region and a C-terminal twisted β barrel-like domain. The structure contains two pockets, one on each domain. The N-domain pocket is lined with residues previously implicated to be important for ZapC function as an FtsZ bundler. The adjacent C-domain pocket contains a hydrophobic center surrounded by conserved basic residues. Mutagenesis analyses indicate that this pocket is critical for FtsZ binding. An extensive FtsZ binding surface is consistent with the fact that, unlike many FtsZ regulators, ZapC binds the large FtsZ globular core rather than C-terminal tail, and the presence of two adjacent pockets suggests possible mechanisms for ZapC-mediated FtsZ bundling.