High affinity uptake of L-glutamate and γ-aminobutyric acid inDrosophila melanogaster

Preparations having properties resembling those of synaptosomes have been isolated from whole fly homogenates ofDrosophila melanogaster using ficoll gradient floatation technique. These have been characterized by marker enzymes and electron microscopy and binding of muscarinic antagenist³H Quinuclidinyl benzilate. An uptake system for neurotransmitter, ã-Aminobutyric acid has been demonstrated in these preparations. A high affinity uptake system for L-glutamate has also been studied in these subcellular fractions. This uptake of glutamate is transport into an osmotically sensitive compartment and not due to binding of glutamate to membrane components. The transport of glutamate has an obligatory requirements for either sodium or potassium ions. Kinetic experiments show that two transport systems, withK _m values 0.33 X 10^-6M and 2.0 X 10^-6M, respectively, function in the accumulation of glutamate. ATP stimulates lower affinity transport of glutamate. Inhibition of glutamate uptake by L-aspartate but not by phenylalanine and tyrosine indicates that a common carrier mediates the transport of both glutamate and aspartate. β-N-oxalyl-L-β β-diamino propionic acid and kainic acid, both inhibitors of glutamate transport in mammalian brain preparations, strongly inhibited transport of glutamate inDrosophila preparations Comparison with uptake of ã-aminobutyric acid and glutamate in isolated larval brain is presented to show that the synaptosome-like preparations we have isolated are rich in central nervous system derived structures, and presynaptic endings from neuromuscular junctions.     

Purification and properties of the phosphorylated form of guanylate cyclase

Guanylate cyclase is dephosphorylated in response to the interaction of egg peptides with a spermatozoan surface receptor (Suzuki, N., Shimomura, H., Radany, E. W., Ramarao, C. S., Ward, G. E., Bentley, J. K., and Garbers, D. L. (1984) J. Biol. Chem. 259, 14874-14879). Here, the phosphorylated form of guanylate cyclase was purified to apparent homogeneity from detergent-solubilized spermatozoan membranes by the use of GTP-agarose, DEAE-Sephacel, and concanavalin A-Sepharose chromatography. To prevent dephosphorylation of the enzyme during purification, glycerol (35%) was required in all buffers. Following purification, a single protein-staining band of Mr 160,000 was obtained on sodium dodecyl sulfate-polyacrylamide gels. The final specific activity of the purified enzyme was 83 mumol of cyclic GMP formed/min/mg of protein at 30 degrees C, an activity 5-fold higher than that observed with the purified, dephosphorylated form of guanylate cyclase. A preparation containing protein phosphatase from spermatozoa, or highly purified alkaline phosphatase (from Escherichia coli), catalyzed the dephosphorylation of the enzyme; this resulted in a subsequent decrease in guanylate cyclase activity and a shift in the Mr from 160,000 to 150,000. The phosphate content of the high Mr form of the enzyme was 14.6 mol/mol protein whereas the phosphate content of the low Mr form was 1.6 mol/mol protein. All phosphate was localized on serine residues. The Mr 160,000 form of guanylate cyclase demonstrated positive cooperative kinetics with respect to MnGTP while the Mr 150,000 form displayed linear, Michaelis-Menten type kinetics. The phosphorylation state of the membrane form of guanylate cyclase, therefore, appears to dictate not only the absolute activity of the enzyme but also the degree of cooperative interaction between catalytic or GTP-binding sites.     

Receptor-mediated regulation of guanylate cyclase activity in spermatozoa

Two peptides, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), which activate sperm respiration and motility and elevate cyclic GMP concentrations in a species-specific manner, were tested for effects on guanylate cyclase activity. The guanylate cyclase of sea urchin spermatozoa is a glycoprotein and it is localized entirely on the plasma membrane. When intact sea urchin sperm cells were incubated with the appropriate peptide for time periods as short as 5 s and subsequently homogenized in detergent, guanylate cyclase activity was found to be as low as 10% of the activity of cells not treated with peptide. The peptides showed complete species specificity and analogues of one peptide (speract) caused decreases in enzyme activity coincident with their receptor binding properties. The peptides did not inhibit enzyme activity when added after detergent solubilization of the enzyme. When detergent-solubilized spermatozoa were incubated at 22 degrees C, guanylate cyclase activity declined in previously nontreated cells to the peptide-treated level. The rate of decline was dependent on temperature and protein concentration. When spermatozoa were first incubated with 32P, the decrease in guanylate cyclase activity was accompanied by a shift in the apparent molecular weight of a major plasma membrane protein (160,000-150,000) and a loss of 32P label from the 160,000 band. Other agents (Monensin A, NH4Cl) which were capable of stimulating sperm respiration and motility also caused decreases of guanylate cyclase activity when added to intact but not detergent-solubilized spermatozoa. The maximal decrease in guanylate cyclase activity occurred 5-10 min after addition of these agents. The enzyme response to Monensin A required extracellular Na+ suggestive that the ionophore caused the effect on guanylate cyclase activity by virtue of its ability to catalyze Na+/H+ exchange. These studies demonstrate that guanylate cyclase activity of sperm cells can be altered by the specific interaction of egg-associated peptides with their plasma membrane receptors.     

Receptor-operated calcium-channels in isolated rabbit jejunum.

Calcium channels were studied in isolated spontaneously rhythmic rabbit jejunum using the muscarinic agonist carbachol as stimulant. Carbachol failed to produce the characteristic phasic and tonic components of smooth muscle contractions. A variety of chemically distinct calcium antagonists, viz. bepridil, diltiazem, isradipine (PN 200-110), nifedipine, and verapamil, non-competitively inhibited the contractions. Diltiazem was most potent (-logIC50 = 8.30) and bepridil least potent (-logIC50 = 6.19) in inhibiting the contractions. The findings conclude with the presence of pharmacologically distinct receptor-operated calcium-channels, besides the potential-dependent calcium-channels, in the rabbit jejunum.     

Citrullus lanatus ‘sentinel’ (watermelon) extract reduces atherosclerosis in LDL receptor-deficient mice

Watermelon (Citrullus lanatus or C. lanatus) has many potentially bioactive compounds including citrulline, which may influence atherosclerosis. In this study, we determined the effects of C. lanatus, provided as an extract of the cultivar ‘sentinel,’ on hypercholesterolemia-induced atherosclerosis in mice. Male low-density lipoprotein receptor-deficient mice at 8 weeks old were given either C. lanatus ‘sentinel’ extract (2% vol/vol; n= 10) or a mixture of matching carbohydrates (2% vol/vol; n= 8) as the control in drinking water while being fed a saturated fat-enriched diet for 12 weeks ad libitum. Mice consuming C. lanatus ‘sentinel’ extract had significantly increased plasma citrulline concentrations. Systolic blood pressure was comparable between the two groups. Consumption of C. lanatus ‘sentinel’ extract led to lower body weight and fat mass without influencing lean mass. There were no differences in food and water intake and in urine output between the two groups. C. lanatus ‘sentinel’ extract administration decreased plasma cholesterol concentrations that were attributed to reductions of intermediate-/low-density lipoprotein cholesterol. Plasma concentrations of monocyte chemoattractant protein-1 and interferon-gamma were decreased and those of interleukin-10 were increased in mice consuming C. lanatus ‘sentinel’ extract. Intake of C. lanatus ‘sentinel’ extract resulted in reductions of atherosclerosis in both aortic arch and thoracic regions. In conclusion, consumption of C. lanatus ‘sentinel’ extract led to reduced body weight gain, decreased plasma cholesterol concentrations, improved homeostasis of pro- and anti-inflammatory cytokines, and attenuated development of atherosclerosis without affecting systolic blood pressure in hypercholesterolemic mice.     

Role of rapsyn tetratricopeptide repeat and coiled-coil domains in self-association and nicotinic acetylcholine receptor clustering

Rapsyn, a 43-kDa peripheral membrane protein of skeletal muscle, is essential for clustering nicotinic acetylcholine receptors (nAChR) in the postsynaptic membrane. Previous studies with rapsyn NH(2)-terminal fragments fused to green fluorescent protein, expressed in 293T cells along with nAChRs, establish the following: Rapsyn-(1-90), containing the myristoylated amino terminus and two tetratricopeptide repeats (TPRs), was sufficient for self-association at the plasma membrane; rapsyn-(1-287), containing seven TPRs, did not cluster nAChRs; whereas rapsyn-(1-360)(,) containing a coiled-coil domain (rapsyn-(298-331)), clustered nAChRs. To further analyze the role of rapsyn structural domains in self-association and nAChR clustering, we have characterized the clustering properties of additional rapsyn mutants containing deletions and substitutions within the TPR and coiled-coil domains. A mutant lacking the coiled-coil domain alone (rapsyn-(black triangle288-348)), failed to cluster nAChRs. Within the coiled-coil domain neutralization of the charged side chains was tolerated, while alanine substitutions of large hydrophobic residues resulted in the loss of nAChR clustering. Rapsyn self-association requires at least two TPRs, as a single TPR (TPR1 or TPR2 alone) was not sufficient. While TPRs 1 and 2 are sufficient for self-association, they are not necessary, as TPRs 3-7 also formed clusters similar to wild-type rapsyn. Fragments containing TPRs co-localized with full-length rapsyn, while the expressed coiled-coil or RING-H2 domain did not. These results are discussed in terms of a homology model of rapsyn, based on the three-dimensional structure of the TPR domain of protein phosphatase 5.     

Transdermal iontophoresis revisited

Iontophoresis evolved as a transdermal enhancement technique in the 20th century, primarily for the delivery of large and charged molecules. Significant achievements have been made in the understanding of underlying mechanisms of iontophoresis and these have contributed to the rational development of iontophoretic delivery systems. The major challenges in this area are the development of portable, cost effective devices and suitable semi-solid formulations that are compatible with the device and the skin. Some of the obstacles in transdermal iontophoresis can be overcome by combining iontophoresis with other physical and chemical enhancement techniques for the delivery of macromolecules. Iontophoresis also offers an avenue for extracting information from the body through the use of reverse iontophoresis, which has potential application in diagnosis and monitoring. The current research is focussed towards resolving the skin toxicity issues and other problems in order to make this technology a commercial reality.     

Mutations in QARS,Encoding Glutaminyl-tRNA Synthetase,Cause Progressive Microcephaly,Cerebral-Cerebellar Atrophy,and Intractable Seizures

Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.     

The InhA1 metalloprotease allows spores of the B. cereus group to escape macrophages

Bacteria of the Bacillus cereus group are resistant to the immune systems of various hosts and establish potent infections, implying that bacteria circumvent the bactericidal activity of host phagocytic cells. We investigated the fate of Bacillus spores after their internalization by macrophages. We found that these spores survive and escape from macrophages, and that the bacterial metalloprotease InhA1, the major component of the exosporium, is essential for efficient spore release from macrophages. InhA1 from Bacillus thuringiensis also enables Bacillus subtilis to escape from macrophages. Analysis of membrane permeability showed that the bacteria cause alterations in the macrophage membranes and that InhA1 is involved in these processes. Thus, InhA1 contributes to protect the bacteria against the host immune system. These findings provide further insight into the pathogenicity of B. cereus group members.