Cytoplasmic Orientation of the Naphthylphthalamic Acid-Binding Protein in Zucchini Plasma Membrane Vesicles.

Polar transport of the plant hormone auxin is blocked by substances such as N-1-naphthylphthalamic acid (NPA), which inhibit auxin efflux and block polar auxin transport. To understand how auxin transport is regulated in vivo, it is necessary to discern whether auxin transport inhibitors act at the intra- or extracellular side of the plasma membrane. Populations of predominantly in-side-in plasma membrane vesicles were subjected to treatments that reverse the orientation. These treatments, which included osmotic shock, cycles of freezing and thawing, and incubation with 0.05% Brij-58, all increased NPA-binding activity and the accessibility of the binding protein to protease digestion. Marker activities for inside-out vesicles also increased, indicating that these treatments act by altering the membrane orientation. Finally, binding data were analyzed by multiple analyses and indicated that neither the affinity nor abundance of binding sites changed. Kinetic analyses indicated that the change in NPA-binding activity by Brij-58 treatment was due to an increase in the initial rates of both association and dissociation of this ligand. These experiments indicated that the NPA-binding site is on the cytoplasmic face of the plasma membrane in zucchini (Cucurbita pepo L. cv Burpee Fordhook).

Activation of murine epidermal V gamma 5/V delta 1-TCR(+) T cell lines by Glu-Tyr polypeptides.

The physiologic role of gamma delta-T-cell-receptor-bearing cells and the T cell receptor ligands that they recognize is still poorly understood. Previous studies have suggested that one possible antigen for gamma delta-TCR(+) cells is the random copolymer poly-glutamic acid-tyrosine (poly-Glu-Tyr), because poly-Glu-Tyr-reactive gamma delta-TCR(+) hybridoma cells were produced from poly-Glu-Tyr-immunized mice. We have found, however, that clonal V gamma 5/V delta 1-TCR(+) epidermal T cell lines from nonimmune mice also respond to poly-Glu-Tyr by producing cytokines. Other amino acid homopolymers, copolymers, and tripolymers were not stimulatory for the V gamma 5/V delta 1-TCR(+) epidermal T cells, except for poly-glutamic acid-alanine-tyrosine (poly-Glu-Ala-Tyr). Of the poly-Glu-Tyr and poly-Glu-Ala-Tyr polymers, only those that contained Glu and Tyr in an equimolar ratio were stimulatory. The cytokine interleukin-2 was strictly required for the responses to poly-Glu-Ala-Tyr, whereas the responses to poly-Glu-Tyr were merely enhanced with interleukin-2. The response to poly-Glu-Tyr was also enhanced by crosslinking CD28 molecules with plate-bound anti-CD28 crosslinking antibody. This finding suggests that the poly-Glu-Tyr response has a partial dependence on CD28-mediated costimulation, a characteristic of TCR-dependent responses. Consistent with this observation, V gamma 5/V delta 1-TCR-loss variants of the epidermal T cell line could no longer respond to poly-Glu-Tyr. The unpredicted responses of epidermal gamma delta-TCR(+) T cells to poly-Glu-Tyr and poly-Glu-Ala-Tyr demonstrate that the functions of these cells potentially can be triggered by peptidic ligands, probably through a TCR-mediated process.

Age-related differences in NMDA responses in cultured rat hippocampal neurons.

The N-methyl-D-aspartate receptor (NMDAR) is expressed in the cerebral cortex and hippocampus and is important in learning and memory. NMDARs are influenced by aging and implicated in neurodegenerative disorders. We investigated age-related differences in NMDAR ionic currents and intracellular calcium in embryonic (E18), middle-age (9-10 month) and old (26 month) rat hippocampal neurons cultured in serum-free medium for 7-12 days. Responses to 200 microM NMDA with 50 microM glycine were measured using whole cell voltage clamp and fura-2 fluorescence. Embryonic neurons exhibited significantly larger and faster NMDA responses than adults. Old rats had 1.5 fold greater normalized NMDA peak current compared to middle-age rats, while intracellular calcium rose 1.3 fold higher. Differences in regression slopes generated from the integral of NMDA current versus normalized NMDA current indicate age-related differences are not exclusively due to changes in receptor density but likely influenced by changes in receptor function. Corresponding age-related measures of intracellular calcium by fura-2 fluorescence in response to NMDA showed a strong correlation with peak current (r(2)=0.996). Our data support the hypothesis that NMDAR responsiveness is altered during aging with an enhanced NMDA peak current in both old and embryonic neurons compared to middle-age neurons.

Decompression of lumbar spinal stenosis and stabilization with Knodt rods in the elderly patient.

We present a series of 25 elderly patients who exhibited signs and symptoms of neurogenic claudication and who were found to have one or two levels of spinal stenosis. At the time of decompressive surgery, excessive movement was found at the stenotic levels, so a simple stabilization procedure was performed using Knodt rods and a facet fusion. The expectation was that spine fixation would decrease the amount of postoperative back pain, which can be a result of continued abnormal mobility. All of the patients have been followed for 2 or more years. This elderly group of individuals tolerated surgery well, and long-term results were good.

Using a learning contract to successfully discharge an infant on home total parenteral nutrition.

Discharging an infant on home total parenteral nutrition is a complex process involving a variety of health care team providers and family members. Development of a learning contract can bring cohesion and provide a framework for discharge.

Magnetic properties of uncultivated magnetotactic bacteria and their contribution to a stratified estuary iron cycle.

Of the two nanocrystal (magnetosome) compositions biosynthesized by magnetotactic bacteria (MTB), the magnetic properties of magnetite magnetosomes have been extensively studied using widely available cultures, while those of greigite magnetosomes remain poorly known. Here we have collected uncultivated magnetite- and greigite-producing MTB to determine their magnetic coercivity distribution and ferromagnetic resonance (FMR) spectra and to assess the MTB-associated iron flux. We find that compared with magnetite-producing MTB cultures, FMR spectra of uncultivated MTB are characterized by a wider empirical parameter range, thus complicating the use of FMR for fossilized magnetosome (magnetofossil) detection. Furthermore, in stark contrast to putative Neogene greigite magnetofossil records, the coercivity distributions for greigite-producing MTB are fundamentally left-skewed with a lower median. Lastly, a comparison between the MTB-associated iron flux in the investigated estuary and the pyritic-Fe flux in the Black Sea suggests MTB play an important, but heretofore overlooked role in euxinic marine system iron cycle.

Biodegradation of [(14)C] ring-labeled nonylphenol ethoxylate.

Nonylphenol (NP) and the 9-mole ethoxylate of nonylphenol (NPE9) were synthesized with a uniform radioactive (14)C label in the aromatic ring. The [(14)C]NP isomer distribution and [(14)C]NPE9 oligomer distribution closely matched that of commercial NPE9. Biodegradation of [(14)C]NPE9 was examined under conditions simulating a river water environment, and changes in the oligomer distribution and mineralization to (14)CO(2) were monitored for 128 days. Over 40% of the [(14)C]NPE aromatic ring carbon was converted to (14)CO(2) and another 21% was incorporated into the biomass. Primary degradation of NPE (conversion to metabolites other than NP, NPE ethoxylates, and NPE carboxylates) was estimated to be 87-97%. NP was a minor metabolite, accounting for less than 0.4% of the initial NPE. These studies demonstrate that the phenolic ring of NPE is opened, metabolized, and mineralized in the aquatic environment.

The effect of putative diuretic factors on in vivo urine production in the mosquito, Aedes aegypti.

Changes in total urine production were measured in the mosquito, Aedes aegypti, following injection of 5-hydroxytryptamine (5-HT), Culex salinarius diuresin, culekinin depolarizing peptides (CDP-I, II and III) or the A. aegypti leucokinin peptides (ALP-I, II and III). All stimulated total urine production in a dose dependent manner. 5-HT was the least potent in urine production experiments with ED(50) values nearly 100-fold higher than other diuretic agonists. Doses greater than 2x10(-4) &mgr;moles inhibited urine production, suggesting either the occurrence of receptor down regulation, more than one type of 5-HT receptor, or increases in hindgut resorption of urine. The ALPs had relatively low ED(50) values compared to the CDPs suggesting that the endogenous peptides may have higher receptor binding affinities. Injection of mosquitoes with polyclonal antisera raised against either ALP-I or C. salinarius diuresin significantly reduced the response to injections of the respective peptides. The evidence presented above suggests that mosquito leucokinins and the C. salinarius diuresin function in the neuroendocrine regulation of urine production in the mosquito.

Effects of putative diuretic factors on intracellular second messenger levels in the Malpighian tubules of Aedes aegypti.

Intracellular levels of the second messengers, 3',5'-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP(3)) were measured in the Malpighian tubules of Aedes aegypti following the in vitro application of 5-hydroxytryptamine (5-HT) and the putative mosquito diuretic peptides, Culex salinarius diuresin and mosquito leucokinins (culekinin depolarizing peptides (CDPs) I, II, III, A. aegypti leucokinin peptides (ALPs) I, II, III). The C. salinarius diuresin significantly (p<0.05) increased tubule intracellular cAMP concentrations. Treatment of tubules with either 5-HT or CDP-II resulted in significant increases in both intracellular cAMP and IP(3) concentrations. All of the mosquito leucokinins, with the exception of CDP-I, significantly stimulated intracellular IP(3) in isolated tubules. These data suggest that the mosquito leucokinins may function on the Malpighian tubules of A. aegypti by increasing the intracellular Ca(2+) levels through the release of IP(3) sensitive Ca(2+) stores. The physiological relevance of these data to the regulation of mosquito Malpighian tubule function is discussed.

Ambulance sphygmomanometers are frequently inaccurate.

OBJECTIVE: To determine the accuracy and reliability of sphygmomanometers used in a metropolitan emergency medical services (EMS) system. METHODS: As a cross-sectional, convenience sample, 150 sphygmomanometers used by EMS personnel in Milwaukee County, Wisconsin, were evaluated. Each sphygmomanometer was checked for accuracy by connecting the manometer to a new, standard mercury manometer using a "Y" connector. Pressure was checked at readings of 60, 90, 120, and 200 mm Hg. The integrity of the device (leaking) was checked by inflating the cuff around a can to 300 mm Hg and measuring the pressure lost in 1 minute. Devices were determined to be inaccurate if the average of the absolute differences at each pressure deviated by more than 3 mm Hg. The device was determined to be unreliable (leaked) if it lost pressure greater than 15 mm Hg in 1 minute. RESULTS: Twenty-eight percent (41/149) of the devices were inaccurate at 90 mm Hg and 25% (37/149) were inaccurate overall. The overall and 90 mm Hg average deviations were +/- 6.6 and +/- 6.0 mm Hg, respectively. Sixty-three percent (94/150) of the devices were unreliable (leaked). When considering both accuracy and reliability at 90 mm Hg, a total of 73% (109/150) of the devices failed one or both of the criteria. CONCLUSION: This study suggests that an accurate blood pressure measurement may not be reliably obtained with 73% of the sphygmomanometers currently used in the county's EMS system.

Temperature and time interval for culture of postmortem neurons from adult rat cortex.

For a model of neurological disease and ischemia, we extended recent work to culture adult postmortem rat brain neurons. Frontal cortex sections were removed from adult rats immediately following sacrifice and at different postmortem intervals and with the brain at either 22 degrees C or 4 degrees C. Brain could be stored four times longer at 4 degrees C between sacrifice and neuronal disaggregation to achieve the same 20% recovery of live cells from those plated compared to 22 degrees C. Each milligram of rat frontal cortex was estimated by the optical disector method to contain 160,000 neurons. When cells were isolated as rapidly as possible, 9% of the neurons originally present in the brain were viable. Various postmortem intervals from 2 to 24 hr resulted in a reduction from 6% to 3% of the cells originally present. After 5 days in culture, viable neurons were 23-42% of those isolated. Neuron-like cells that survived represented 40-75% of the viable cells, or 0.5-2.75% of those originally estimated to be present in the brain. Electrophysiology experiments show that cells isolated 0 and 24 hr postmortem had neuronal electrical properties, including an average resting membrane potential of -48 mV, voltage-sensitive currents, and action potentials. Neuron-like cells were immunoreactive for neuron-specific enolase, neurofilament 200, glutamate, MAP2, and tau after 2 weeks in culture. These experiments show that neuron-like cells can be reliably cultured from adult rat cortex up to 6 hr postmortem when stored at 22 degrees C and up to 24 hr postmortem when stored at 4 degrees C. These findings should encourage donation of human postmortem brain neurons for studies on ischemia, adult pharmacology, and neurological disease.

The role of arginine vasotocin and prostaglandin F2 alpha on oviposition and luteolysis in the common snapping turtle Chelydra serpentina.

The administration of arginine vasotocin (AVT) to gravid snapping turtles with steroidogenically active corpora lutea and high plasma progesterone concentration (1480 +/- 155 pg/ml) did not trigger oviposition, whereas 12 days after ovulation when luteolysis occurred and plasma progesterone concentration was low (570 +/- 78 pg/ml), treatment with AVT caused oviposition. Controls with high plasma progesterone concentration (1605 +/- 185 pg/ml) oviposited 15-23 days after ovulation when plasma progesterone concentration dropped to 201 +/- 35 pg/ml. Deluteinization-induced oviposition was initiated 15 hr after surgery and was completed by 30 hr. Oviposition of complete clutches occurred and was correlated with a significant drop in plasma progesterone. Sham-operated turtles did not exhibit oviposition and no significant change in progesterone concentration was observed. A single injection of prostaglandin F2 alpha (PGF) in recently ovulated turtles induced early luteolysis and a significant decrease in plasma progesterone concentration after 24-30 hr. A single administration of PGF caused the disappearance of steroidogenic features such as the smooth endoplasmic reticulum and mitochondria with tubular cristae 48 hr later. Also PGF triggered the invasion of a hyaline-like material from the luteal theca into the luteal cell mass which eventually induced luteolysis. The role of AVT, PGF, and progesterone in relation to egg retention and oviposition is discussed.

Regionally specific modulation of brain apolipoprotein E in the mouse during the estrous cycle and by exogenous 17beta estradiol.

Studies have suggested that 17beta estradiol (E2) can modify apolipoprotein E (apoE) expression. The current study determined if apoE protein varied in different regions of the mouse brain as a function of the estrous cycle and if E2 could increase apoE protein expression. In this study apoE concentration was lowest on estrus in the hippocampus, cingulate cortex and frontal cortex. In contrast, apoE concentration was highest on estrus in the olfactory bulb and cerebellum. There were no differences in the striatal apoE expression throughout the estrous cycle. Exogenous E2 significantly raised tissue levels of apoE in the olfactory bulb and cerebellum at 5 days after treatment. There was a slight, but nonsignificant increase in cortical expression of apoE and no change in striatum. Immunocytochemical localization studies found estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in cortical neurons and glia. In the cerebellum and olfactory bulb, ERbeta was seen primarily in glia. ERalpha was not observed in the cerebellum and was rare in the olfactory bulb. Neither ERalpha nor ERbeta was seen in the striatum. Our data show regional differences in the production of apoE throughout the estrous cycle. In addition, exogenous E2 has regionally specific effects on apoE expression. Regional variability in apoE production appears to vary as a function of the estrogen receptor subtype.

Response of murine gamma delta T cells to the synthetic polypeptide poly-Glu50Tyr50.

Random heterocopolymers of glutamic acid and tyrosine (pEY) evoke strong, genetically controlled immune responses in certain mouse strains. We found that pE50Y50 also stimulated polyclonal proliferation of normal gamma delta, but not alpha beta, T cells. Proliferation of gamma delta T cells did not require prior immunization with this Ag nor the presence of alpha beta T cells, but was enhanced by IL-2. The gamma delta T cell response proceeded in the absence of accessory cells, MHC class II, beta 2-microglobulin, or TAP-1, suggesting that Ag presentation by MHC class I/II molecules and peptide processing are not required. Among normal splenocytes, as with gamma delta T cell hybridomas, the response was strongest with V gamma 1+ gamma delta T cells, and in comparison with related polypeptides, pE50Y50 provided the strongest stimulus for these cells. TCR gene transfer into a TCR-deficient alpha beta T cell showed that besides the TCR, no other components unique to gamma delta T cells are needed. Furthermore, interactions between only the T cells and pE50Y50 were sufficient to bring about the response. Thus, pE50Y50 elicited a response distinct from those of T cells to processed/presented peptides or superantigens, consistent with a mechanism of Ig-like ligand recognition of gamma delta T cells. Direct stimulation by ligands resembling pE50Y50 may thus selectively evoke contributions of gamma delta T cells to the host response.

Early preferential stimulation of gamma delta T cells by TNF-alpha.

Although recent findings indicate that gamma delta T cells influence both early innate and Ag-specific adaptive host responses, it has remained unclear what triggers gamma delta T cell reactivity. Investigating very early T cell activation in mouse and human models of bacterial infection, we measured CD69 expression as an indicator of early cellular activation. Both murine alpha beta and gamma delta T cells responded polyclonally to systemic bacterial infections, and to LPS. However, gamma delta T cells responded more strongly to the bacteria and to LPS. In vitro LPS-stimulated human T cells showed a similar differential response pattern. We identified TNF-alpha as mediator of the early differential T cell activation, and of differential proliferative responses. The stronger response of gamma delta T cells to TNF-alpha was correlated with higher inducible expression levels of TNF-Rp75. Among unstimulated splenocytes, more gamma delta T cells than alpha beta T cells expressed CD44 at high levels. The data suggest that TNF-Rp75 determines the differential T cell reactivity, and that most gamma delta T cells in the normal spleen are present in a presensitized state. As TNF-alpha stimulates activated T cells, it may early preferentially connect gamma delta T cell functions with those of cells that produce this cytokine, including activated innate effector cells and Ag-stimulated T lymphocytes.