Cocaine and its derivatives blunt neutrophil functions without influencing phosphorylation of a 47-kilodalton component of the reduced nicotinamide-adenine dinucleotide phosphate oxidase

Cocaine and its derivatives blunted responses of neutrophils (cell/cell aggregation, up-regulation of the receptor for C3bi (CR3, CD11b/CD18), generation of superoxide anion (O2-) and degranulation to various stimuli. The order of potency of these agents was the same as that for local anesthesia: tetracaine greater than bupivacaine greater than cocaine greater than lidocaine. Neutrophil aggregation elicited by the chemoattractant FMLP (10(-7) M) was inhibited by cocaine (10 mM) to 13.6 +/- 6% of control (p less than 0.002); the IC50 was approximately 4 mM. Cocaine and the other local anesthetics not only inhibited the upregulation of CR3 and O2- generation, but also blocked degranulation of cytochalasin B-treated cells. Cocaine (10 mM) reduced beta-glucuronidase and lysozyme secretion to 4.3 +/- 0.7 and 13 +/- 2.2% controls, respectively; its IC50 was 4 mM. Local anesthetics added after ligand/receptor engagement (FMLP) interrupted aggregation and halted generation of O2-. Moreover, local anesthetics rapidly inhibited aggregation, O2- generation, and degranulation elicited by PMA (1 microgram/ml) or the Ca ionophore A23187 (10 microM): the effects of cocaine could therefore not be attributed to unique actions at the FMLP receptor. Peak levels of intracellular Ca2+ ([Ca]i) at 5 to 10 s, and levels of [Ca]i 120 s after FMLP in Fura 2-loaded cells were significantly lower in cells treated with lidocaine, findings that could be explained by enhanced 45Ca2+ efflux from neutrophils. In cells loaded with bis(carboxyethyl)carboxyfluorescine (pH indicator) local anesthetics failed to affect the initial FMLP-induced (0 to 15 s) drop of pHi but inhibited the later (120 s) realkalinization of the cytosol (lidocaine, bupivacaine). Most remarkably, autoradiographs of SDS gels prepared from stimulated, 32P-labeled neutrophils treated with local anesthetics showed no difference from resting cells, either with respect to patterns of phosphorylation and dephosphorylation or their kinetics. Labeling of a 47-kDa protein, a component of the reduced nicotinamide-adenine dinucleotide phosphate-oxidase system, was unchanged. The effects of local anesthetics, which blunt neutrophil responses without affecting protein phosphorylation, suggest that protein phosphorylation is an insufficient signal for neutrophil activation. Inasmuch as cocaine and its derivatives affect cell functions at sites distal to activation of protein kinase C, these agents should prove useful in uncoupling protein phosphorylation from functional responses.     

Intermitochondrial junctions in the extraocular muscle of the rat

Summary Intermitochondrial junctions with a spacing of 17–21 nm were observed in the superior rectus muscle of a rat. Periodic rounded densities are aligned midway between the apposed outer mitochondrial membranes at some of these junctions. Such densities have a diameter of about 8–10 nm and a center-to-center spacing of about 26–30 nm. These junctions occur in cases where one mitochondrial profile is enclosed within another or where two profiles are interlocked so that their combined overall form has a smoothly contoured profile. Intermitochondrial junctions seem not to have been previously described in muscle, but have been reported in other kinds of tissues. In agreement with those previous reports, the presently observed intermitochondrial junctions usually involve mitochondria that display atypical features indicative of tissue abnormality or stress. Such junctions were never observed in normal extraocular muscle.This study was supported by Grants EY-00309, EY-01297 and EY-01842 from the National Eye Institute (USPHS) and in part by a grant from the Eye Surgery Fund, Inc. and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc.     

Identification of Kel1p,a Kelch Domain-containing Protein Involved in Cell Fusion and Morphology in Saccharomyces cerevisiae

We showed previously that protein kinase C, which is required to maintain cell integrity, negatively regulates cell fusion (Philips, J., and I. Herskowitz. 1997. J. Cell Biol. 138:961–974). To identify additional genes involved in cell fusion, we looked for genes whose overexpression relieved the defect caused by activated alleles of Pkc1p. This strategy led to the identification of a novel gene, KEL1, which encodes a protein composed of two domains, one containing six kelch repeats, a motif initially described in the Drosophila protein Kelch (Xue, F., and L. Cooley. 1993. Cell. 72:681– 693), and another domain predicted to form coiled coils. Overexpression of KEL1 also suppressed the defect in cell fusion of spa2Δ and fps1Δ mutants. KEL2, which corresponds to ORF YGR238c, encodes a protein highly similar to Kel1p. Its overexpression also suppressed the mating defect associated with activated Pkc1p. Mutants lacking KEL1 exhibited a moderate defect in cell fusion that was exacerbated by activated alleles of Pkc1p or loss of FUS1, FUS2, or FPS1, but not by loss of SPA2. kel1Δ mutants form cells that are elongated and heterogeneous in shape, indicating that Kel1p is also required for proper morphology during vegetative growth. In contrast, kel2Δ mutants were not impaired in cell fusion or morphology. Both Kel1p and Kel2p localized to the site where cell fusion occurs during mating and to regions of polarized growth during vegetative growth. Coimmunoprecipitation and two-hybrid analyses indicated that Kel1p and Kel2p physically interact. We conclude that Kel1p has a role in cell morphogenesis and cell fusion and may antagonize the Pkc1p pathway.     

In vivo release of endogenous dopamine from rat caudate nucleus by beta-phenylethylamine and alpha,alpha,-dideutero-beta-phenylethylamine

The release of endogenous dopamine (DA) has been measured in the rat striatum following the intracardial administration of various doses of beta-phenylethylamine (PEA) or alpha,alpha-dideutero-beta-phenylethylamine (deuterated PEA). The release was significantly increased for a period of approximately 15 minutes by a dose of 25 mg/kg PEA. Both the dose required to stimulate DA release and the duration of the effect were in good agreement with previously reported behavioral and locomotor effects of administered PEA. When the animals were given 25 mg/kg of deuterated PEA, the increase in DA release was both longer lasting and significantly greater in magnitude than that observed in response to the non-deuterated amine. The results of these experiments provide direct evidence that DA release is stimulated by amounts of PEA known to cause behavioral effects and locomotor activity in rats, and suggest that these effects are likely to be mediated, at least in part, by DA.     

Urinary growth hormone excretion in 657 healthy children and adults: normal values, inter- and intraindividual variations.

Urinary growth hormone (u-GH) excretion was measured in 547 healthy children and 110 adults by ELISA with a detection limit of 1.1 ng/l u-GH after prior concentration of the urine samples (20- to 30-fold). u-GH excretion values were significantly dependent on the pubertal stage (p less than 0.0001) with maximum values in Tanner stage 3 for girls and 4 for boys. This corresponded to a peak in u-GH excretion between 11.5-14.5 years in girls and 12.5-16 years in boys. Additionally, u-GH excretion in adults was significantly higher than in prepubertal children (p less than 0.001). The day/night ratio of u-GH excretion (pg/h) was significantly higher in females than in males (p less than 0.01). In Tanner stages 1-4, u-GH excretion during the day was lower than that at night, whereas the opposite was true in late puberty and in adult women. The interindividual variation of u-GH excretion within the same Tanner stage was considerable and approximately double the intraindividual variation. The day-to-day variation could be further reduced by collection of three consecutive urine samples. The variations were larger if night samples instead of 24-hour samples were considered. The expression of u-GH excretion in nanograms per gram creatinine did not diminish the observed variation and blunted the pubertal increase in u-GH excretion. In conclusion, (1) u-GH excretion depends significantly on age, sex and pubertal maturation as does the day/night ratio of u-GH excretion. (2) The interindividual variation in u-GH excretion is considerable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Litter decomposition across multiple spatial scales in stream networks

Spatial scale is a critical consideration for understanding ecological patterns and controls of ecological processes, yet very little is known about how rates of fundamental ecosystem processes vary across spatial scales. We assessed litter decomposition in stream networks whose inherent hierarchical nature makes them a suitable model system to evaluate variation in decay rates across multiple spatial scales. Our hypotheses were (1) that increasing spatial extent adds significant variability at each hierarchical level, and (2) that stream size is an important source of variability among streams. To test these hypotheses we let litter decompose in four riffles in each of twelve 3rd-order streams evenly distributed across four 4th-order watersheds, and in a second experiment determined variation in decomposition rate along a stream-size gradient ranging from orders 1 to 4. Differences in decay rates between coarse-mesh and fine-mesh litter bags accounted for much of the overall variability in the data sets, and were remarkably consistent across spatial scales and stream sizes. In particular, variation across watersheds was minor. Differences among streams and among riffles were statistically significant, though relatively small, leaving most of the total variance (51%) statistically unexplained. This result suggests that variability was generated mainly within riffles, decreasing successively with increasing scale. A broad range of physical and chemical attributes measured at the study sites explained little of the variance in decomposition rate. This, together with the strong mesh-size effect and greater variability among coarse-mesh bags, suggests that detritivores account, at least partly, for the unexplained variance. These findings contrast with the widespread perception that variability of ecosystem characteristics, including process rates, invariably increases (1) with spatial extent and (2), in stream networks, when analyses encompass headwaters of various size. An important practical implication is that natural variability need not compromise litter decomposition assays as a means of assessing functional ecosystem integrity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytosolic Ras supports eye development in Drosophila

Ras proteins associate with cellular membranes as a consequence of a series of posttranslational modifications of a C-terminal CAAX sequence that include prenylation and are thought to be required for biological activity. In Drosophila melanogaster, Ras1 is required for eye development. We found that Drosophila Ras1 is inefficiently prenylated as a consequence of a lysine in the A(1) position of its CAAX sequence such that a significant pool remains soluble in the cytosol. We used mosaic analysis with a repressible cell marker (MARCM) to assess if various Ras1 transgenes could restore photoreceptor fate to eye disc cells that are null for Ras1. Surprisingly, we found that whereas Ras1 with an enhanced efficiency of membrane targeting could not rescue the Ras1 null phenotype, Ras1 that was not at all membrane targeted by virtue of a mutation of the CAAX cysteine was able to fully rescue eye development. In addition, constitutively active Ras1(12V,C186S) not targeted to membranes produced a hypermorphic phenotype and stimulated mitogen-activated protein kinase (MAPK) signaling in S2 cells. We conclude that the membrane association of Drosophila Ras1 is not required for eye development.