Residues 88-109 of factor IXa are important for assembly of the factor X activating complex.

Activated platelets and phospholipid vesicles promote assembly of the intrinsic factor X (FX) activating complex by presenting high-affinity binding sites for blood coagulation FIXa, FVIIIa, and FX. Previous reports suggest that the second epidermal growth factor (EGF)-like domain of FIXa mediates assembly of the FX activating complex (Ahmad, S. S., Rawala, R., Cheung, W. F., Stafford, D. W., and Walsh, P. N. (1995) Biochem. J. 310, 427-431; Wong, M. Y., Gurr, J. A., and Walsh, P. N. (1999) Biochemistry 38, 8948-8960). To identify important residues, we prepared several chimeric FIXa proteins using homologous sequences from FVII: FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112), FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-127), and point mutants (FIXaR94D and FIXa(loop1)G94R). In the presence and absence of FVIIIa, a 2- to 10-fold reduced V(max) of FX activation (nm FXa min(-1)) was observed for FIXa(FVIIEGF2), FIXa(loop1), FIXa(loop2), and FIXa(loop1)G94R, whereas FIXa(loop3) and FIXaR94D were normal. For all of the FIXa proteins, K(m)((app)) values were normal as were EC(50) values for interactions with FVIIIa. However, K(d)((app)) (in nm) for the FX activating complex assembled on phospholipid vesicles was increased for FIXa(FVIIEGF2) (43.3 +/- 2.70), FIXa(loop1)(10.9 +/- 2.8), FIXa(loop2) (70.5 +/- 1.60), and FIXa(loop1)G94R (17.1 +/- 2.90) relative to FIXa(N) (3.9 +/- 0.11), FIXa(WT) (4.6 +/- 0.17), FIXa(loop3) (4.5 +/- 0.20), and FIXaR94D (2.2 +/- 0.09) suggesting that reduced V(max) is a result of impaired complex assembly. These data indicate that residues 88-109 (but not Arg(94)) are important for normal assembly of the FX activating complex on phospholipid vesicles.     

Aromatic residue position on the nonpolar face of class a amphipathic helical peptides determines biological activity

The apolipoprotein A-I mimetic peptide 4F (Ac-DWFKAFYDKVAEKFKEAF-NH(2)), with four Phe residues on the nonpolar face of the amphipathic alpha-helix, is strongly anti-inflammatory, whereas two 3F analogs (3F(3) and 3F(14)) are not. To understand how changes in helix nonpolar face structure affect function, two additional 3F analogs, Ac-DKLKAFYDKVFEWAKEAF-NH(2) (3F-1) and Ac-DKWKAVYDKFAEAFKEFL-NH(2) (3F-2), were designed using the same amino acid composition as 3F(3) and 3F(14). The aromatic residues in 3F-1 and 3F-2 are near the polar-nonpolar interface and at the center of the nonpolar face of the helix, respectively. Like 4F, but in contrast to 3F(3) and 3F(14), these peptides effectively inhibited lytic peptide-induced hemolysis, oxidized phospholipid-induced monocyte chemotaxis, and scavenged lipid hydroperoxides from low density lipoprotein. High pressure liquid chromatography retention times and monolayer exclusion pressures indicated that there is no direct correlation of peptide function with lipid affinity. Fluorescence studies suggested that, although the peptides bind phospholipids similarly, the Trp residue in 4F, 3F-1, and 3F-2 is less motionally restricted than in 3F(3) and 3F(14). Based on these results and molecular modeling studies, we propose that the arrangement of aromatic residues in class A amphipathic helical molecules regulates entry of reactive oxygen species into peptide-phospholipid complexes, thereby reducing the extent of monocyte chemotaxis, an important step in atherosclerosis.     

Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of alpha 1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells

Continuous exposure of DDT1 MF-2 smooth muscle cells to 10-100 microM norepinephrine results in a dramatic attenuation of the ability of norepinephrine to stimulate inositol phospholipid hydrolysis via alpha 1-adrenergic receptors (alpha 1-AR). In addition to the functional desensitization, norepinephrine exposure also reduces the number of accessible cell surface alpha 1-AR as assayed by [3H]prazosin binding at 4 degrees C. Desensitization of the cells with norepinephrine results in an increase in the phosphorylation of the Mr 80,000 alpha 1-AR ligand binding peptide (2.4 +/- 0.2 mol of 32P per mol of alpha 1-AR; n = 5) when compared to control cells (1.1 +/- 0.1 mol of 32P per mol of alpha 1-AR; n = 5). The time courses of these three processes are all comparable being half-maximal within 1-2 min. These norepinephrine-promoted effects can be prevented by the alpha 1-AR receptor antagonist phentolamine indicating that they are mediated via the alpha 1-AR. Treatment of cells with the vasoactive peptide bradykinin (10 microM) induces desensitization of alpha 1-AR function similar to that induced by tumor-promoting phorbol ester treatment (Leeb-Lundberg, L. M. F., Cotecchia, S., Lomasney, J. W., DeBernardis, J. F., Lefkowitz, R. J., and Caron, M. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5651-5655). Both treatments also result in phosphorylation of the alpha 1-AR, with stoichiometries of 1.7 +/- 0.1 (bradykinin; n = 5) and 3.6 +/- 0.1 (PMA; n = 5) mol of 32P/mol of alpha 1-AR. However, neither phorbol esters nor bradykinin reduce the number of accessible cell surface alpha 1-AR. Similar phosphopeptide maps are obtained from tryptic phosphopeptides generated from phosphorylated alpha 1-AR derived from cells treated with norepinephrine, phorbol 12-myristate 13-acetate, and bradykinin. Phosphoamino acid analysis reveals that the various agents induce phosphorylation on both serine and threonine residues. Thus, phosphorylation of receptors linked to the inositol phospholipid/Ca2+ signaling pathway may represent an important mechanism of regulation of receptor responsiveness.     

Early events in the transport of proteins into mitochondria. Import competition by a mitochondrial presequence.

Studies with a synthetic presequence peptide, F1 beta 1-20, corresponding to the NH2-terminal 20 amino acids of the F1-ATPase beta-subunit precursor (pF1 beta) show that although this peptide binds avidly to phospholipid bi-layers it does not efficiently compete for import of full-length precursor into mitochondria, Ki approximately 100 microM (Hoyt, D.W., Cyr, D.M., Gierasch, L.M., and Douglas, M.G. (1991) J. Biol. Chem. 266, 21693-21699). Herein we report that longer F1 beta presequence peptides F1 beta 1-32 + 2, F1 beta 1-32SQ + 2, and F1 beta 21-51 + 3 compete for mitochondrial import at 1000-, 250-, and 25-fold lower concentrations, respectively, than F1 beta 1-20. A longer peptide, F1 beta 1-51 + 3, was no more effective as an import competitor than F1 beta 1-32 + 2. Both minimal length and amphiphilic character appear required in order for F1 beta peptides to block mitochondrial import. Import competition by longer F1 beta peptides seems to occur at a step common to all precursors since they blocked import of precursors to F1-ATPase alpha- and beta-subunits and the ADP/ATP carrier protein. Dissipation of membrane potential (delta psi) across the inner mitochondrial membrane is observed in the presence of F1 beta-peptides, but this mechanism alone does not account for the observed import inhibition. F1 beta 1-32 + 2 and 21-51 + 3 block import of pF1 beta 100% at peptide concentrations which dissipate delta psi less than 25%. In contrast, experiments with valinomycin demonstrate that when mitochondrial delta psi is reduced 25% import of pF1 beta is inhibited only 25%. Therefore, at least 75% of maximal import inhibition observed in the presence of F1 beta 1-32 + 2 and F1 beta 21-51 + 3 does not result from dissipation of delta psi. Import inhibition by F1 beta-peptides is reversible and can be overcome by increasing the amount of full-length precursor in import reactions. F1 beta presequence peptides and full-length precursor are therefore likely to compete for a common import step. Presequence dependent binding of pF1 beta to trypsin-sensitive elements on the outer mitochondrial membrane is insensitive to inhibitory concentrations of F1 beta presequence peptide. We conclude that import inhibition by F1 beta presequence peptides is competitive and occurs at a site beyond initial interaction of precursor proteins with mitochondria.     

Calcium/calmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains

Regulatory domains of the multifunctional Ca2+/calmodulin-dependent protein kinase II were investigated utilizing synthetic peptides. These peptides were derived from the sequence between positions 281 and 319 as translated from the cDNA sequence of the rat brain 50-kDa subunit (Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A. F., Hanson, P., Schulman, H., and Rosenfeld, M. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5962-5966), which contain the putative calmodulin-binding region as well as potential autophosphorylation sites. Peptide 290 to 309 was found to be a potent calmodulin antagonist with an IC50 of 52 nM for inhibition of Ca2+/calmodulin-dependent protein kinase II. Neither truncation from the amino terminus (peptide 296-309) nor extension in the carboxyl-terminal direction (peptide 294-319) markedly affected calmodulin binding, whereas shortening the peptide from the carboxyl terminus (peptide 290-302) or from both ends (peptide 295-304) resulted in the elimination of this activity. Peptide 281-290 did not bind calmodulin, but was a good substrate for the enzyme, being phosphorylated at Thr-286. Several of the peptides inhibited the kinase in a partially competitive, substrate-directed manner, but were not themselves phosphorylated. These studies identify domains within Ca2+/calmodulin-dependent protein kinase II which may be involved in 1) inhibition of the kinase in the absence of calmodulin, 2) binding of calmodulin, and 3) the resulting activation. Additionally, it is suggested that phosphorylation of residues flanking these domains may be responsible for the known regulatory effects of autophosphorylation on the properties of the kinase.     

Parastrombidinopsis shimi n. gen., n. sp. (Ciliophora: Choreotrichia) from the coastal waters of Korea: morphology and small subunit ribosomal DNA sequence

The planktonic ciliate Parastrombidinopsis shimi n. gen., n. sp. is described from both living cells and quantitative protargol-stained (QPS) preparations and the sequence of the small subunit rDNA (SSU rDNA) is reported. This species is almost oval when the cells are alive; when stained, it is cylindrical for the upper two-fifths, half-bowl shaped for the middle two-fifths, and narrow rodshaped for the lower one-fifth. The ranges (and mean +/- standard deviation, n = 20) of cell length, cell width, and oral diameter of living cells were 112-221 microm (168 +/- 39), 88-176 microm (121 +/- 30), and 53-110 microm (80 +/- 14), respectively, while those of the QPS-stained specimens (n = 54) were 88-225 microm (162 +/- 29), 55-163 microm (102 +/- 19), and 53-98 microm (69 +/- 9), respectively. Thirty-six to 48 external oral polykinetids had cilia 25-40 microm long. However, unlike Strombidinopsis species sensu stricto, P. shimi has an external oral polykinetid zone that is an open circle. This species has two shorter polykinetids associated with the end of the oral polykinetid zone, deep in the oral cavity. Like Strombidinopsis species in the subclass Choreotrichia, 36-50 somatic kineties were equally spaced around the cell body and extended from the oral to the posterior regions with 68-105 dikinetids per kinety. Both kinetosomes of each kinetid bore cilia 3-10 microm long. Parastrombidinopsis shimi had 2 (1-4) ovoid macronuclei of 20-82 x 15-32 microm. When properly aligned, the sequence of the SSU rDNA of P. shimi (GenBank Accession No. AJ786648) was approximately 5% different from that of Strobilidium caudatum and 6% different from that of two Strombidinopsis species. Based both on morphology and gene sequence divergence, we establish this is as a new species in a new genus belonging to the family Strombidinopsidae.     

A protein G fragment from the salmonid viral hemorrhagic septicemia rhabdovirus induces cell-to-cell fusion and membrane phosphatidylserine translocation at low pH

The fusion-related properties of segments p9, p3, p4, and p9 + p2 surrounding the p2 phospholipid-binding domain of the protein G (pG) of the salmonid rhabdovirus of viral hemorrhagic septicemia (VHS) (Nu?ez, E., Fernandez, A. M., Estepa, A., Gonzalez-Ros, J. M., Gavilanes, F., and Coll, J. M. (1998) Virology 243, 322-330; Estepa, A., and Coll, J. M. (1996) Virology 216, 60-70), have been studied at neutral and fusion (low) pH values by using its derived peptides. Cell-to-cell fusion, translocation of phosphatidylserine, and inhibition of fusion of pG-transfected cells defined the p9 + p2 (fragment 11, sequence 56-110) as a fragment with higher specific activity for anionic phospholipid aggregation than the previously reported p2. While fragment 11, p2, and p3 showed interactions with anionic phospholipids, p9 and p4 showed no interactions with any phospholipids. When added to a cell monolayer model at low pH, fragment 11 induced pH-dependent cell-to-cell fusion and translocated phosphatidylserine from the inner to the outer leaflet of the membrane. At low pH and in the presence of anionic phospholipids, fragment 11 showed more than 80% beta-sheet conformation (IR and CD spectroscopies). Finally, anti-fragment 11 antibodies inhibited low pH-dependent pG-transfected cell-to-cell fusion. All of the data support the conclusion that fragment 11 is a primary determinant of some of the viral cell fusion events in VHSV.     

Hydroxyurea transport across the blood-brain and blood-cerebrospinal fluid barriers of the guinea-pig

Hydroxyurea is used in the treatment of HIV infection in combination with nucleoside analogues, 2'3'-didehydro-3'deoxythymidine (D4T), 2'3'-dideoxyinosine or abacavir. It is distributed into human CSF and is transported from the CSF to sub-ependymal brain sites, but its movement into the brain directly from the blood has not been studied. This study addressed this by a brain perfusion technique in anaesthetized guinea-pigs. The carotid arteries were perfused with an artificial plasma containing [14C]hydroxyurea (1.6 microm) and a vascular marker, [3H]mannitol (4.6 nm). Brain uptake of [14C]hydroxyurea (8.0 +/- 0.9%) was greater than [3H]mannitol (2.4 +/- 0.2%; 20-min perfusion, n = 8). CSF uptake of [14C]hydroxyurea (5.6 +/- 1.5%) was also greater than [3H]mannitol (0.9 +/- 0.3%; n = 4). Brain uptake of [14C]hydroxyurea was increased by 200 microm hydroxyurea, 90 microm D4T, 350 microm probenecid, 25 microm digoxin, but not by 120 microm hydroxyurea, 16.5-50 microm D4T, 90 microm 2'3'-dideoxyinosine or 90 microm abacavir. [14C]Hydroxyurea distribution to the CSF, choroid plexus and pituitary gland remained unaffected by all these drugs. The metabolic half-life of hydroxyurea was > 15 h in brain and plasma. Results indicate that intact hydroxyurea can cross the brain barriers, but is removed from the brain by probenecid- and digoxin-sensitive transport mechanisms at the blood-brain barrier, which are also affected by D4T. These sensitivities implicate an organic anion transporter (probably organic anion transporting polypeptide 2) and possibly p-glycoprotein in the brain distribution of hydroxyurea and D4T.     

Changes in somatostatin-like immunoreactivity in lungs from perinatal guinea pigs and the effects of somatostatin-14 on lung liquid production.

Somatostatin-like immunoreactivity was measured by radioimmunoassay with a monoclonal antibody in lungs from perinatal guinea pigs (62 +/- 2 days of gestation). Fetuses delivered by Caesarean section and dissected before breathing showed 4748 +/- 758 pg/lung (n = 25). Fetuses allowed to breathe (neonates) showed marked increases in activity: 7629 +/- 1355 pg/lung (n = 12) after breathing 30 seconds, and 10729 +/- 1064 pg/lung (n = 6) after breathing 3 minutes (2.3-fold increase, P < 0.005). Values then declined (5203 +/- 1050 pg/lung (n = 9) at 30 minutes; 1458 +/- 105 pg/lung (n = 4) at 60 minutes). Changes were similar in pg/g wet tissue. HPLC characterized the immunoreactive peptides as somatostatin-14 (SS-14) and somatostatin-28 (SS-28) in both fetuses and neonates (n = 11). SS-28 made up only 13.7 +/- 1.7% of the activity; this percentage did not change with breathing. The effects of synthetic SS-14 on lung liquid production were investigated in in vitro lungs from 42 fetal guinea pigs. All 21 preparations immersed in 10(-5)-10(-7) M SS-14 during the middle hour of 3 h incubations reduced production, often approaching zero after treatment (rates, ml/kg body weight per h, succeeding hours: 10(-5) M (n = 9), 3.09 +/- 0.68, 0.93 +/- 0.39, -0.05 +/- 0.60 (fall significant during and after treatment, P < 0.025-0.005); 10(-6) M (n = 6), 3.06 +/- 0.68, 1.29 +/- 0.58, 0.36 +/- 0.38 (P < 0.05-0.005); 10(-7) M (n = 6), 1.96 +/- 0.66, 1.11 +/- 0.34, 0.64 +/- 0.28 (P < 0.05-0.025).(ABSTRACT TRUNCATED AT 250 WORDS)     

Longitudinal oxygen gradients in cerebra micro vessels in acute anaemia in rats

Using a fine-tip oxygen microelectrodes the longitudinal gradients of oxygen tension (pO2) have been studied in small arterioles (with lumen diameter in control of 5 +/- 20 microm) and in capillaries of the rat brain cortex during stepwise decrease of the blood haemoglobin concentration [Hb] from control [Hb]--14.4 +/- 0.3 g/dl to 10.1 +/- 0.2 g/dl (step 1), 7.0 +/- 0.2 g/dl (step 2) and 3.7 +/- 0.2 g/dl (step 3). All data are presented as "mean +/- standard error". Oxygen tension was measured in arteriolar segments in two locations distanced deltaL = 265 +/- 34 microm, n = 30. Mean diameter of studied arterioles was 10.7 +/- 0.5 microm, n = 71. Length of studied capillary segments was about deltaL = 201 +/- 45 Mm, n = 18. The measured longitudinal pO2 gradient (deltapO2/deltaL) in arterioles amounted 0.03 +/- 0.01 mmHg/microm, n = 15 in control; 0.06 +/- 0.01 mmHg/microm, n = 16 (step 1); 0.07 +/- +/- 0.01 mmHg/microm, n = 14 (step 2); 0.1 +/- 0.01 mmHg/microm, n = 30 (step 3). In the capillaries, the deltapO2/deltaL amounted to: 0.07 +/- 0.01 mmHg/microm, n = 17 (control); 0.09 +/- 0.02 mmHg/microm, n = 16 (step 1); 0.08 +/- 0.01 mmHg/microm, n = 15 (step 2); 0.1 +/- 0.02 mmHg/microm, n = 18 (step 3). An over threefold decrease in the system blood oxygen capacity did not result in significant changes (p > 0.05) of the deltapO2/deltaL in capillaries that might result in relatively homogeneous oxygen flux from blood to tissue in acute anaemia. The longitudinal gradients of blood O2 saturation (deltaSO2/deltaL) in studied arterioles and capillaries were obtained using oxygen dissociation curve (ODC) of haemoglobin in the system blood. The gradients deltaSO2/deltaL in capillaries was shown to be threefold higher than the corresponding gradients in arterioles. The data show that anatomic capillaries are the main source of oxygen to brain tissue as in control and in hypoxic conditions. Sufficient oxygen delivery to brain tissue in acute anaemia is maintained by compensatory mechanisms of cardiovascular and respiratory systems. The data presented are the first measurements of the longitudinal pO, gradients in capillaries and minute cortical arterioles at acute anaemia.     

Albumin binding as a general strategy for improving the pharmacokinetics of proteins

Plasma protein binding can be an effective means of improving the pharmacokinetic properties of otherwise short lived molecules. Using peptide phage display, we identified a series of peptides having the core sequence DICLPRWGCLW that specifically bind serum albumin from multiple species with high affinity. These peptides bind to albumin with 1:1 stoichiometry at a site distinct from known small molecule binding sites. Using surface plasmon resonance, the dissociation equilibrium constant of peptide SA21 (Ac-RLIEDICLPRWGCLWEDD-NH(2)) was determined to be 266 +/- 8, 320 +/- 22, and 467 +/- 47 nm for rat, rabbit, and human albumin, respectively. SA21 has an unusually long half-life of 2.3 h when injected by intravenous bolus into rabbits. A related sequence, fused to the anti-tissue factor Fab of D3H44 (Presta, L., Sims, P., Meng, Y. G., Moran, P., Bullens, S., Bunting, S., Schoenfeld, J., Lowe, D., Lai, J., Rancatore, P., Iverson, M., Lim, A., Chisholm, V., Kelley, R. F., Riederer, M., and Kirchhofer, D. (2001) Thromb. Haemost. 85, 379-389), enabled the Fab to bind albumin with similar affinity to that of SA21 while retaining the ability of the Fab to bind tissue factor. This interaction with albumin resulted in reduced in vivo clearance of 25- and 58-fold in mice and rabbits, respectively, when compared with the wild-type D3H44 Fab. The half-life was extended 37-fold to 32.4 h in rabbits and 26-fold to 10.4 h in mice, achieving 25-43% of the albumin half-life in these animals. These half-lives exceed those of a Fab'(2) and are comparable with those seen for polyethylene glycol-conjugated Fab molecules, immunoadhesins, and albumin fusions, suggesting a novel and generic method for improving the pharmacokinetic properties of rapidly cleared proteins.     

Metabolic response to [13C]glucose and [13C]fructose ingestion during exercise

Seven healthy male volunteers exercised on a cycle ergometer at 50 +/- 5% VO2max for 180 min, on three occasions during which they ingested either water only (W), [13C]glucose (G), or [13C]fructose (F) (140 +/- 12 g, diluted at 7% in water, and evenly distributed over the exercise period). Blood glucose concentration (in mM) significantly decreased during exercise with W (5.1 +/- 0.4 to 4.2 +/- 0.1) but remained stable with G (5.0 +/- 0.4 to 5.3 +/- 0.6) or F ingestion (5.4 +/- 0.5 to 5.1 +/- 0.4). Decreases in plasma insulin concentration (microU/ml) were greater (P less than 0.05) with W (11 +/- 3 to 3 +/- 1) and F (12 +/- 4 to 5 +/- 1) than with G ingestion (11 +/- 2 to 9 +/- 5), and fat utilization was greater with F (103 +/- 11 g) than with G ingestion (82 +/- 9 g) and lower than with W ingestion (132 +/- 14 g). However F was less readily available for combustion than G; over the 3-h period 75% (106 +/- 11 g) of ingested G was oxidized, compared with 56% (79 +/- 8 g) of ingested fructose. As a consequence, carbohydrate store utilizations were similar in the two conditions (G, 174 +/- 20 g; F, 173 +/- 17 g; vs. W, 193 +/- 22 g). These observations suggest that, during prolonged moderate exercise, F ingestion maintains blood glucose as well as G ingestion, and increases fat utilization when compared to G ingestion. However, due to a slower rate of utilization of F, carbohydrate store sparing is similar with G and F ingestions.     

Effects of protein I of Neisseria gonorrhoeae on neutrophil activation: generation of diacylglycerol from phosphatidylcholine via a specific phospholipase C is associated with exocytosis

Upon engagement of chemoattractant receptors, neutrophils generate inositol trisphosphate and diacylglycerol (DG) by means of a phosphatidylinositol-specific phospholipase C (PI-PLC) which is regulated by a GTP-binding protein(s). We have previously reported (Reibman, J., H. M. Korchak, L. B. Vosshall, K. A. Haines, A. M. Rich, and G. Weissmann. 1988. J. Biol. Chem. 263:6322-6328) a biphasic rise in DG after exposure of neutrophils to the chemoattractant FMLP: a rapid (less than or equal to 15 s) phase ("triggering") and a slow (greater than or equal to 30 s) phase ("activation"). These derive from distinct intracellular lipid pools. To study the source of rapid and slow DG, we have used a unique probe, protein I, a porin that is the major outer membrane protein of Neisseria gonorrhoeae. Treatment of neutrophils with protein I inhibits exocytosis and homotypic cell adhesion provoked by FMLP without inhibiting assembly of the NADPH oxidase responsible for O2-. generation. DG turnover in PMN labeled with [3H]arachidonate and [14C]glycerol was profoundly altered by protein I. Whereas the rapid peak of DG was only modestly diminished (FMLP vs. FMLP plus protein I = DG labeled with [3H]arachidonic acid (3H-a.a.-DG): 142 +/- 14% SEM vs. 125 +/- 22%; DG labeled with the glycerol backbone with [14C]glycerol (D-14C-G): 125 +/- 10% SEM vs. 107 +/- 8.5% SEM), the slow rise in both 3H-a.a.-DG and D-14C-G was essentially abolished. Moreover, treatment of neutrophils with 4-4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which, like protein I, inhibits exocytosis without affecting O2-. generation also inhibited slow DG. However, protein phosphorylation and dephosphorylation (47phox, 66phox) were unaffected in the absence of slow DG. To determine the source of the slow DG, we have analyzed radiolabeled phospholipid (PL) turnover after FMLP +/- protein I (P.I.). Treatment of PMN with FMLP (0.1 microM) resulted in breakdown of phosphatidylcholine (PC), beginning at 30 s, and reaching a nadir at 60 s (3H-PC = 59 +/- 10.2% SEM of resting, 14C-PC = 57 +/- 6.4%). Protein I (0.25 microM) significantly inhibited PC turnover after FMLP ([3H]PC = 95 +/- 5.6% and [14C]PC = 86 +/- 8.4% of resting at 60 s), but failed to alter the metabolism of 3H- or 14C-phosphatidylinositol after FMLP (91 +/- 19.6 and 88 +/- 16.5% vs. 92 +/- 9.2 and 91 +/- 16% at 60 s).(ABSTRACT TRUNCATED AT 400 WORDS)     

Species of coccidia (Apicomplexa: Eimeriidae) in shrews from Alaska, U.S.A., and northeastern Siberia, Russia, with description of two new species

Fecal samples (n = 636) from 10 species of shrews collected in Alaska (n = 540) and northeastern Siberia (n = 96) were examined for the presence of coccidia (Apicomplexa: Eimeriidae). Five distinct oocyst morphotypes were observed. Three types were consistent with oocysts of previously recognized coccidia species from other shrew hosts. These were Eimeria inyoni, E. vagrantis, and Isospora brevicauda, originally described from the inyo shrew (Sorex tenellus), dusky shrew (S. monticolus), and northern short-tailed shrew (Blarina brevicauda), respectively. We found 5 new host records for E. inyoni, 3 for E. vagrantis, and 3 for I. brevicauda. The 2 additional oocyst morphotypes, both from the tundra shrew (Sorex tundrensis), are putative new species. Sporulated oocysts of Eimeria beringiacea n. sp. are subspheroidal, 17.7 x 15.6 microm (14-24 x 13-20 microm) with a length (L)/width (W) ratio of 1.1 (1.0-1.4); these lack a micropyle (M), an oocyst residuum (OR), and a polar granule (PG). Sporocysts are ellipsoidal, 10.3 x 6.1 microm (7-14 x 4-8 microm), with a L/W ratio of 1.7 (1.3-2.3) and have a Stieda body (SB), Substieda body (SSB), and sporocyst residuum (SR). Oocysts of Eimeria tundraensis n. sp. are spheroidal to subspheroidal, 24.8 x 23.5 microm (23-26 x 22-25 microm), with a L/W ratio of 1.1 (1.0-1.2); these lack a M and OR, but a single PG is present. Sporocysts are elongate ellipsoidal, 15.4 x 8.3 microm (13-17 x 7-9 microm), with a L/W ratio of 1.9 (1.4-2.1) and have a SB, SSB, and SR.     

The protection against gentamicin nephrotoxicity in the streptozotocin-induced diabetic rat is not related to gender.

Since gender can influence the renal toxicity of a drug in a given species, the present study was undertaken to evaluate the role of sex in the protection against gentamicin (G)-induced nephrotoxicity afforded by diabetes mellitus (DM) in the rat. We have compared the effects of administration of G (40 mg/kg/day, for 14 days) on male and female DM Sprague-Dawley rats. Non-diabetic animals of both sexes receiving identical doses of G served as controls. At the end of the experiment on day 14, both female (F) and male (M) control groups had similar and marked evidence of nephrotoxicity: elevation of plasma creatinine (F 1.7 +/- 0.7; M 2.8 +/- 0.6 mg/dl), decrease in endogenous 24-h creatinine clearance (Ccr) (F0.3 +/- 0.1; M 0.2 +/- 0.1 ml/min/100 g BW), and histological evidence of severe acute tubular necrosis. In marked contrast, the DM rats showed no functional or morphological evidence of renal damage throughout the study regardless of their gender (day 14: plasma creatinine: F 0.2 +/- 0.03; M 0.2 +/- 0.02; Ccr: F 1.2 +/- 0.1; M 1.6 +/- 0.1 ml/min/100 g BW), and they also accumulated less G in their kidney cortex than the C rats. The male controls exhibited higher renal cortex accumulation of G than the female controls (p < 0.05), whereas the opposite occurred in the DM groups (p < 0.01). Because the validity of using Ccr for the evaluation of GFR changes in experimental nephrotoxicity has been questioned, we have compared, in a separate experiment, three different methods of estimation of GFR (simultaneous short clearances of inulin and Ccr, and 24-h Ccr) in conscious female Sprague-Dawley rats undergoing the same treatment with G described above. At no time during the study did the method used for estimation of the GFR influence the results. We conclude that male and female Sprague-Dawley rats with diabetes are functionally and morphologically equally protected against G. Furthermore, no gender-related differences in the magnitude of G-induced nephrotoxicity was demonstrated in the non-diabetic control animals.     

IP3-Gated Channels and their Occurrence Relative to CNG Channels in the Soma and Dendritic Knob of Rat Olfactory Receptor Neurons

Olfactory receptor neurons respond to odorants with G protein-mediated increases in the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) and/or inositol-1,4,5-trisphosphate (IP3). This study provides evidence that both second messengers can directly activate distinct ion channels in excised inside-out patches from the dendritic knob and soma membrane of rat olfactory receptor neurons (ORNs). The IP3-gated channels in the dendritic knob and soma membranes could be classified into two types, with conductances of 40 +/- 7 pS (n = 5) and 14 +/- 3 pS (n = 4), with the former having longer open dwell times. Estimated values of the densities of both channels from the same inside-out membrane patches were very much smaller for IP3-gated than for CNG channels. For example, in the dendritic knob membrane there were about 1000 CNG channels x microm(-2) compared to about 85 IP3-gated channels x microm(-2). Furthermore, only about 36% of the dendritic knob patches responded to IP3, whereas 83% of the same patches responded to cAMP. In the soma, both channel densities were lower, with the CNG channel density again being larger ( approximately 57 channels x microm(-2)) than that of the IP3-gated channels ( approximately 13 channels x microm(-2)), with again a much smaller fraction of patches responding to IP3 than to cAMP. These results were consistent with other evidence suggesting that the cAMP-pathway dominates the IP3 pathway in mammalian olfactory transduction.     

Myogenic and vasoconstrictor responsiveness of skeletal muscle arterioles is diminished by hindlimb unloading.

The purpose of the present study was to determine whether hindlimb unloading of rats alters vasoconstrictor and myogenic responsiveness of skeletal muscle arterioles. After either 2 wk of hindlimb unloading (HU) or cage control (C), second-order arterioles were isolated from the white portion of gastrocnemius (WG; C: n = 9, HU: n = 10) or soleus (Sol; C: n = 9, HU: n = 10) muscles and cannulated with two micropipettes connected to reservoir systems for in vitro study. Intraluminal pressure was set at 60 cmH2O. The arterioles were exposed to step changes in intraluminal pressure ranging from 20 to 140 cmH2O to determine myogenic responsiveness and to KCl (10-100 mM) and norepinephrine (10(-9)-10(-4) M) to determine vasoconstrictor responsiveness. Although maximal diameter of WG arterioles was not different between C (185 +/- 12 microm) and HU (191 +/- 14 microm) rats, WG arterioles from HU rats developed less spontaneous tone (C: 33 +/- 5%, HU 20 +/-3%), were unable to maintain myogenic tone at pressures from 140 to 100 cmH2O, and were less sensitive to the vasoconstrictor effects of KCl and norepinephrine (as indicated by a higher agonist concentration that produced 50% of maximal vasoconstrictor response). In contrast, maximal diameter of Sol arterioles from HU rats (117 +/- 12 microm) was smaller than that in C rats (148 +/- 14 microm). However, the development of spontaneous tone (C: 30 +/- 4%, HU: 36 +/- 5%), myogenic activity, and the responsiveness to vasoconstrictor agonists were not different between Sol arterioles from C and HU rats. These results indicate that hindlimb unloading diminishes the myogenic autoregulatory and contractile responsiveness of arterioles from muscle composed of type IIB fibers and suggest that the compromised ability to elevate vascular resistance after exposure to microgravity may be related to these vascular alterations. In addition, hindlimb unloading appears to induce vascular remodeling of arterioles from muscle composed of type I fibers, as indicated by the decrease in maximal diameter of arterioles from Sol muscle.     

Structure-activity studies of the inhibition of FabI,the enoyl reductase from Escherichia coli,by triclosan: kinetic analysis of mutant FabIs

Triclosan, a common antibacterial additive used in consumer products, is an inhibitor of FabI, the enoyl reductase enzyme from type II bacterial fatty acid biosynthesis. In agreement with previous studies [Ward, W. H., Holdgate, G. A., Rowsell, S., McLean, E. G., Pauptit, R. A., Clayton, E., Nichols, W. W., Colls, J. G., Minshull, C. A., Jude, D. A., Mistry, A., Timms, D., Camble, R., Hales, N. J., Britton, C. J., and Taylor, I. W. (1999) Biochemistry 38, 12514-12525], we report here that triclosan is a slow, reversible, tight binding inhibitor of the FabI from Escherichia coli. Triclosan binds preferentially to the E.NAD(+) form of the wild-type enzyme with a K(1) value of 23 pM. In agreement with genetic selection experiments [McMurry, L. M., Oethinger, M., and Levy, S. B. (1998) Nature 394, 531-532], the affinity of triclosan for the FabI mutants G93V, M159T, and F203L is substantially reduced, binding preferentially to the E.NAD(+) forms of G93V, M159T, and F203L with K(1) values of 0.2 microM, 4 nM, and 0.9 nM, respectively. Triclosan binding to the E.NADH form of F203L can also be detected and is defined by a K(2) value of 51 nM. We have also characterized the Y156F and A197M mutants to compare and contrast the binding of triclosan to InhA, the homologous enoyl reductase from Mycobacterium tuberculosis. As observed for InhA, Y156F FabI has a decreased affinity for triclosan and the inhibitor binds to both E.NAD(+) and E.NADH forms of the enzyme with K(1) and K(2) values of 3 and 30 nM, respectively. The replacement of A197 with Met has no impact on triclosan affinity, indicating that differences in the sequence of the conserved active site loop cannot explain the 10000-fold difference in affinities of FabI and InhA for triclosan.     

Rabbit skeletal muscle myosin light chain kinase. The calmodulin binding domain as a potential active site-directed inhibitory domain

A synthetic peptide modeled after the calmodulin (CaM)-binding domain of rabbit skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys5-Lys-Asn-Phe-Ile-Ala10-Val-Ser-Ala-Ala-+ ++Asn15-Arg-Phe-Glycyl amide (M5), inhibited the CaM-independent chymotryptic fragment of the enzyme, C35 (Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. G. (1985) J. Biol. Chem. 260, 11275-11285), with a Ki of 3.2 +/- 2.1 microM. Inhibition was competitive with respect to the peptide substrate Lys-Lys-Arg-Ala-Ala5-Arg-Ala-Thr-Ser-Asn10-Val-Phe-Ala and was of the noncompetitive linear mixed type with respect to ATP. M5 and homologues with a serine residue substituted at positions 9, 13, or 14 were phosphorylated with the following order of preference: M5(Ser9) greater than M5(Ser13) much greater than M5(Ser14) greater than M5. The order of preference observed agreed with that predicted by comparison of the sequence of these peptides with the phosphorylation sites of myosin P-light chains. Both inhibition of C35 by M5 and phosphorylation of M5 and its serine-substituted homologues were severely curtailed by the addition of a stoichiometric excess of CaM over peptide. Thus, synthetic peptides modeled after the CaM-binding domain of skeletal muscle myosin light chain kinase can function as calmodulin-regulated active site-directed inhibitors of the enzyme.