Activity-dependent endocytic sorting of kainate receptors to recycling or degradation pathways

Kainate receptors (KARs) play important roles in the modulation of neurotransmission and plasticity, but the mechanisms that regulate their surface expression and endocytic sorting remain largely unknown. Here, we show that in cultured hippocampal neurons the surface expression of GluR6-containing KARs is dynamically regulated. Furthermore, internalized KARs are sorted into recycling or degradative pathways depending on the endocytotic stimulus. Kainate activation causes a Ca2+- and PKA-independent but PKC-dependent internalization of KARs that are targeted to lysosomes for degradation. In contrast, NMDAR activation evokes a Ca2+-, PKA- and PKC-dependent endocytosis of KARs to early endosomes with subsequent reinsertion back into the plasma membrane. These results demonstrate that GluR6-containing KARs are subject to activity-dependent endocytic sorting, a process that provides a mechanism for both rapid and chronic changes in the number of functional receptors.     

A liquid chromatographic-tandem mass spectrometric method for the determination of two selective thymidylate synthase inhibitors, BGC945 and BGC638, in mouse plasma

A LC-tandem mass spectrometry method to quantify the quinazoline-based thymidylate synthase inhibitors BGC945 and BGC638 in mouse plasma was developed. BGC945 and BGC638 were extracted from mouse plasma using protein precipitation with acetonitrile. Chromatography was performed on a Fluophase RP 5 microm, 100 mmx2.0mm i.d. column using a gradient of ammonium acetate and acetonitrile as a mobile phase with a flow rate of 0.2 mLmin(-1). The injection volume for each sample was 20 microL with a total run time of 7.5 min. This method was validated in the range 25-4000 nM (r2=0.99). The analytical assay performance showed that the method was accurate (mean intra- and inter-day assay R.E. were below 12% and 11%, respectively), reproducible (mean intra- and inter-day R.S.D. were less than 13% and 5% for all quality control levels, respectively) and sensitive (lower limit of quantification was 25 nM) in the range studied. This validated method has been used to define the first pharmacokinetic report of BGC945 and BGC638 in mice.     

GROWTH AND PHOTOSYNTHESIS OF ULVA ROTUNDATA (CHLOROPHYTA) AS A FUNCTION OF TEMPERATURE AND SQUARE WAVE IRRADIANCE IN INDOOR CULTURE1

Temperature and photon flux density (PFD) vary independently in estuaries, e.g. high PFD may occur at any temperature, so it is necessary to consider synergistic effects of these factors on algal growth. Because natural PFD is highly variable and daylength changes confound seasonal temperature cycles, it is easier to interpret factorial experiments in controlled laboratory conditions. Clonal Ulva rotundata Blid. (Chlorophyta) has been studied extensively in outdoor culture. In this study it was maintained indoors under square wave photoperiods at five PFDs and three temperatures. Growth rate, photqsynthetic light response (P-I) curves, and photosystem II chlorophyll fluorescence properties were measured at the growth temperature following acclimation. Interactions between PFD and growth temperature were strongly indicated in all physiological parameters measured. Greatest PFD response occurred at the highest temperature, and the largest temperature response occurred at the highest PFD. Light-saturated photosynthesis (P_m) dark respiration (R_d), and light-limited quantum yield (Φ_m) were sufficient to describe acclimation status. The light-saturation parameter (I_k) was redundant and potentially misleading. Although U. rotundata exhibits a great amplitude of photoacclimation, it apparently has little capacity for temperature acclimation compared to the kelp, Laminaria saccharina, for which published data indicate similar photosynthetic rates over a broad range of growth temperatures. Diurnal variation of P_m and R_d at a growth PFD of ～ 1700 ± 200 μmol photons · m^?2· s^?1 was similar to the pattern observed previously in outdoor culture, suggesting endogenous control of these parameters. Quantum yield and the ratio of variable to maximum chlorophyll fluorescence (F_v/F_m), which were depressed in midday sunlight exceeding ～ 1500 μmol photons · m^?2· s^?1, were relatively invariant through the day in indoor culture, indicating that these parameters are controlled primarily by instantaneous PFD. Growth and fluorescence data are also presented for some other macroalgae for comparative purposes.     

Phylogenetic analysis of Dunaliella (Chlorophyta) emphasizing new benthic and supralittoral isolates from Great Salt Lake

Dunaliella, a commercially important chlorophyte, is globally distributed in saline habitats. Morphological species have not been definitively reconciled with phylogenetic analyses. Considerable genetic diversity continues to be discovered in new isolates, especially from soil and benthic habitats. Twenty‐nine new isolates from Great Salt Lake, Utah, many from benthic or supralittoral habitats, were phylogenetically analyzed using ITS1+5.8S+ITS2 in comparison to a broad sampling of available sequences. A few new isolates align in one branch of a bifurcated monophyletic Dunaliella salina clade and several cluster within monophyletic D. viridis. Several others align with relatively few unnamed strains from other locations, comprising a diverse clade that may represent two or more new species. The overall Dunaliella clade is relatively robust, but the nearest outgroups are ambiguously placed with extremely long branches. About half of the isolates, all from benthic or supralittoral habitats, have been persistently sarcinoid in liquid media since isolation. This trait is spread across the Dunaliella phylogeny. The morphology of two sarcinoid strains was documented with light microscopy, revealing an extensive glycocalyx. Clumping behavior of unicellular and sarcinoid strains was unaffected by presence or absence of Mg²⁺ or Ca²⁺, addition of lectin‐inhibiting monosaccharides, or water‐soluble factors from morphologically opposite strains. Results from this investigation have significantly expanded our current understanding of Dunaliella diversity, but it seems likely that much remains to be discovered with additional sampling.     

A Model for Studying the Hemostatic Consumption or Destruction of Platelets

A fundamental issue in understanding homeostasis of the hematopoietic system is to what extent intrinsic and extrinsic factors regulate cell fate. We recently revisited this issue for the case of blood platelets and concluded that platelet life span is largely regulated by internal factors, in contrast to the long-held view that accumulated damage from the environment triggers clearance. However, it is known that in humans there is an ongoing fixed requirement for platelets to maintain hemostasis and prevent bleeding; hence a proportion of platelets may be consumed in such processes before the end of their natural life span. Whether it is possible to detect this random loss of platelets in normal individuals at steady-state is unknown. To address this question, we have developed a mathematical model that independently incorporates age-independent random loss and age-dependent natural senescent clearance. By fitting to population survival curves, we illustrate the application of the model in quantifying the fixed requirement for platelets to maintain hemostasis in mice, and discuss the relationship with previous work in humans. Our results suggest a higher requirement for platelets in mice than in humans, however experimental uncertainty in the data limits our ability to constrain this quantity. We then explored the relationship between experimental uncertainty and parameter constraint using simulated data. We conclude that in order to provide useful constraint on the random loss fraction the standard error in the mean of the data must be reduced substantially, either through improving experimental uncertainty or increasing the number of experimental replicates to impractical levels. Finally we find that parameter constraint is improved at higher values of the random loss fraction; thus the model find utility in situations where the random loss fraction is expected to be high, for example during active bleeding or some types of thrombocytopenia.     

Hypothyroidism increases serotonin turnover and sympathetic activity in the adult rat.

Adult, male Sprague-Dawley rats underwent surgical thyroidectomy (Tx) or sham surgery. In all three experiments from which data are reported, a 3-week recovery period was allowed. In experiments I and II, baseline measurements of colonic temperature (Tc) and urinary norepinephrine excretion (NE) were obtained, and both variables were monitored daily for the duration of the studies. After baseline measurements, half of each surgical group was given either triiodothyronine (T3) or vehicle injections subcutaneously; in experiment I replacements continued for 1.5 days, while in experiment II T3 replacement continued for 3.5 days. Rats were decapitated at the end of each experiment and serotonin (5-HT) turnover was measured in brainstem. Serotonin turnover in rostral and caudal brainstem was increased with Tx (p less than 0.05). Increased turnover in caudal brainstem was normalized by T3 only in experiment II. Similarly, decreased Tc and elevated NE with Tx were normalized in experiment II but not in experiment I. In experiment III, NE measurements normalized on a creatinine excretion basis indicated that increased NE is evident with Tx, irrespective of normalization procedure. Significant correlations between 5-HT in caudal brainstem and metabolic correlates of sympathetic function, concurrent normalization of NE and 5-HT in caudal brainstem, plus work from other laboratories describing sympathoexcitatory serotonergic neurons located in the caudal brainstem suggest that the central and peripheral changes in the hypothyroid rat are causally related.     

Fungi Identify the Geographic Origin of Dust Samples

There is a long history of archaeologists and forensic scientists using pollen found in a dust sample to identify its geographic origin or history. Such palynological approaches have important limitations as they require time-consuming identification of pollen grains, a priori knowledge of plant species distributions, and a sufficient diversity of pollen types to permit spatial or temporal identification. We demonstrate an alternative approach based on DNA sequencing analyses of the fungal diversity found in dust samples. Using nearly 1,000 dust samples collected from across the continental U.S., our analyses identify up to 40,000 fungal taxa from these samples, many of which exhibit a high degree of geographic endemism. We develop a statistical learning algorithm via discriminant analysis that exploits this geographic endemicity in the fungal diversity to correctly identify samples to within a few hundred kilometers of their geographic origin with high probability. In addition, our statistical approach provides a measure of certainty for each prediction, in contrast with current palynology methods that are almost always based on expert opinion and devoid of statistical inference. Fungal taxa found in dust samples can therefore be used to identify the origin of that dust and, more importantly, we can quantify our degree of certainty that a sample originated in a particular place. This work opens up a new approach to forensic biology that could be used by scientists to identify the origin of dust or soil samples found on objects, clothing, or archaeological artifacts.     

Interaction of monoclonal antibodies with alpha-bungarotoxin and (-)-nicotine binding sites in goldfish brain. Identification of putative nicotinic acetylcholine receptor subtypes

Monoclonal antibodies raised against the nicotinic acetylcholine receptor of Electrophorus electricus electroplaque have been used as probes to characterize putative nicotinic acetylcholine receptors in goldfish brain. One monoclonal antibody (mAb), mAb 47, recognized a protein which binds both (-)-[3H]nicotine and 125I-alpha-bungarotoxin with high affinity. Another monoclonal antibody (mAb 172) recognized a protein which binds (-)-[3H]nicotine but not 125I-alpha-bungarotoxin. Both antibodies precipitated a protein(s) (biosynthetically labeled with [35S]methionine) in the absence, but not in the presence, of excess purified nicotinic acetylcholine receptor from Torpedo nobiliana. The dilution of mAb 47 that precipitated half of the maximum amount of 125I-alpha-bungarotoxin binding protein was the same as that which precipitated half of the maximum amount of (-)-[3H]nicotine binding activity. When used in combination, the two antibodies precipitated more (-)-[3H]nicotine radioactivity than either antibody alone. The (-)-[3H]nicotine and 125I-alpha-bungarotoxin binding component-mAb complexes were characterized by sucrose density centrifugation. In the presence of either mAb 172 or 47, the (-)-[3H] nicotine binding component migrated further into the gradient, but only mAb 47 shifted the 125I-alpha-bungarotoxin peak. Incubation of solubilized brain extract with alpha-bungarotoxin-coupled Sepharose reduced the amount of (-)-[3H]nicotine radioactivity precipitated by mAb 47 but not by mAb 172. These data suggest that the antibodies may recognize distinct subtypes of (-)-nicotine binding sites in goldfish brain, one subtype which binds both 125I-alpha-bungarotoxin and (-)-[3H]nicotine and a second subtype which binds only (-)-[3H] nicotine.     

Chromatin condensation involving lamellar strands in spermiogenesis of Goniobasis proxima

Young spermatids of the prosobranch mollusc Goniobasis have spherical nuclei with diffuse centers and condensation of the chromatin only at their peripheries. At later stages, this condensation becomes uniform for the entire nucleus, and has a lamellar appearance in sectioned material. In specimens prepared using freeze-etch techniques, these lamellae are resolvable into chromatin strands, each of which has a helical substructure, with a period of ca. 100 Å and a pitch angle of 35°. The strands are ca. 130–140 Å in diameter (comparable to measurements made on micrographs of sectioned material). Most of the strands are oriented in the long axis of the elongating nucleus, but a few are oblique, suggesting that they might serve some sort of binding function. In the mature spermatozoon the chromatin is very compact and dense, and all evidence of the lamellar or stranded appearance is absent, in both sectioned and freeze-etch preparations.     

Elephant (Loxodonta africana) impact on trees used by nesting vultures and raptors in South Africa

Negative influences on the establishment and persistence of large trees used by tree‐nesting birds as nesting sites represent a potential threat to vultures and raptors. We monitored large trees and their surrounding vegetation and analysed whether trees with nesting sites are at risk due to elephant impact. Trees with nests did not differ in elephant impact from control trees without nests, and the survival rates of trees with nests and the actual nests within the trees showed that nests decreased at a faster rate than the trees themselves. Elephant damage did not affect the persistence of nests over the 5‐year monitoring period. However, the presence of insects and fungus on large trees was negatively related to tree survival, thereby indicating that elephant impact could indirectly facilitate insect and fungus attack and shorten the lifespan of a tree.