Simultaneous detection of different glycosidase activities by 19F NMR spectroscopy

A fast method for the simultaneous detection of different glycosidolytic activities in commercially available enzyme preparations and crude culture filtrates was found in using, as substrate, a mixture of different glycosyl fluorides and 19F NMR spectroscopy as a screening technique. Accompanying studies regarding the hydrolytic stability of these fluorides in various buffer systems, as well as conditions of their long-term storage, were carried out. A simple procedure for the preparation of beta-D-mannopyranosyl fluoride in gram quantities is given.     

Characterisation of the high-molecular weight fructan isolated from garlic (Allium sativum L.)

A high molecular weight fructan was isolated from garlic and the structure determined by enzymatic, chemical and spectroscopic (NMR) methods. It was found that the garlic fructan belongs to the neokestose family. It has a (2 --> 1)-linked beta-D-Fruf backbone with (2 --> 6)-linked beta-D-Fruf side chains. A structural model was postulated for a degree of polymerisation of about 58. This model was substantiated using an endo-inulinase purified from Aspergillus ficuum and by 1H and 13C NMR spectroscopy.     

Characterization and Evolution of mariner Elements from Closely Related Species of Fruit Flies (Diptera: Tephritidae)

Mariner elements were amplified using the polymerase chain reaction from two species of tephritid flies, Ceratitis rosa and Trirhithrum coffeae. The sequences were ∼1.3 kb in length. None of these elements appeared to be functional, as in every case the open reading frame (ORF) was disrupted by the presence of frameshifts or stop codons. These elements, Crmar1 and Tcmar1, are very similar to the Ccmar1 element previously amplified from the closely related tephritid species C. capitata and are members of the mellifera subfamily of mariner elements. The phylogeny and pattern of divergence of these elements were examined in relation to the phylogeny of the host species. It is highly probable that the elements were present in the ancestral lineage prior to the divergence of the three species. The copy numbers of the elements within each species are very different, ranging from about 10 in T. coffeae to 5,000 in C. rosa. The possible mechanisms which determine the copy number of an element in the host genome are discussed. Received: 25 April 1997 / Accepted: 31 July 1997     

Regulation of dendritic excitability by activity-dependent trafficking of the A-type K+ channel subunit Kv4.2 in hippocampal neurons

Voltage-gated A-type K+ channel Kv4.2 subunits are highly expressed in the dendrites of hippocampal CA1 neurons. However, little is known about the subcellular distribution and trafficking of Kv4.2-containing channels. Here we provide evidence for activity-dependent trafficking of Kv4.2 in hippocampal spines and dendrites. Live imaging and electrophysiological recordings showed that Kv4.2 internalization is induced rapidly upon glutamate receptor stimulation. Kv4.2 internalization was clathrin mediated and required NMDA receptor activation and Ca2+ influx. In dissociated hippocampal neurons, mEPSC amplitude depended on functional Kv4.2 expression level and was enhanced by stimuli that induced Kv4.2 internalization. Long-term potentiation (LTP) induced by brief glycine application resulted in synaptic insertion of GluR1-containing AMPA receptors along with Kv4.2 internalization. We also found evidence of Kv4.2 internalization upon synaptically evoked LTP in CA1 neurons of hippocampal slice cultures. These results present an additional mechanism for synaptic integration and plasticity through the activity-dependent regulation of Kv4.2 channel surface expression.     

Feeding Ecology and Regurgitation–Reingestion Behavior of the Critically Endangered Indri indri in the Maromizaha Protected Area,Eastern Madagascar

International Journal of Primatology - Deforestation around the world is a major threat to primates. Understanding primate species’ habitat and dietary requirements is critical in creating...     

DPP6 establishes the A-type K(+) current gradient critical for the regulation of dendritic excitability in CA1 hippocampal neurons

Subthreshold-activating A-type K(+) currents are essential for the proper functioning of the brain, where they act to delay excitation and regulate firing frequency. In CA1 hippocampal pyramidal neuron dendrites, the density of A-type K(+) current increases with distance from the soma, playing an important role in synaptic integration and plasticity. The mechanism underlying this gradient has, however, remained elusive. Here, dendritic recordings from mice lacking the Kv4 transmembrane auxiliary subunit DPP6 revealed that this protein is critical for generating the A-current gradient. Loss of DPP6 led to a decrease in A-type current, specifically in distal dendrites. Decreased current density was accompanied by a depolarizing shift in the voltage dependence of channel activation. Together these changes resulted in hyperexcitable dendrites with enhanced dendritic AP back-propagation, calcium electrogenesis, and induction of synaptic long-term potentiation. Despite enhanced dendritic excitability, firing behavior evoked by somatic current injection was mainly unaffected in DPP6-KO recordings, indicating compartmentalized regulation of neuronal excitability.     

Iron uptake mediated by binding of H-ferritin to the TIM-2 receptor in mouse cells

Ferritin binds specifically and saturably to a variety of cell types, and recently several ferritin receptors have been cloned. TIM-2 is a specific receptor for H ferritin (HFt) in the mouse. TIM-2 is a member of the T cell immunoglobulin and mucin domain containing (TIM) protein family and plays an important role in immunity. The expression of TIM-2 outside of the immune system indicates that this receptor may have broader roles. We tested whether ferritin binding to TIM-2 can serve as an iron delivery mechanism. TIM-2 was transfected into normal (TCMK-1) mouse kidney cells, where it was appropriately expressed on the cell surface. HFt was labeled with (55)Fe and (55)Fe-HFt was incubated with TIM-2 positive cells or controls. (55)Fe-HFt uptake was observed only in TIM-2 positive cells. HFt uptake was also seen in A20 B cells, which express endogenous TIM-2. TIM-2 levels were not increased by iron chelation. Uptake of (55)Fe-HFt was specific and temperature-dependent. HFt taken up by TIM-2 positive cells transited through the endosome and eventually entered a lysosomal compartment, distinguishing the HFt pathway from that of transferrin, the classical vehicle for cellular iron delivery. Iron delivered following binding of HFt to TIM-2 entered the cytosol and became metabolically available, resulting in increased levels of endogenous intracellular ferritin. We conclude that TIM-2 can function as an iron uptake pathway.     

Variable effects of climate change on six species of North American birds

Many recent studies have shown that birds are advancing their laying date in response to long-term increases in spring temperatures. These studies have been conducted primarily in Europe and at local scales. If climate change is a large-scale phenomenon, then we should see responses at larger scales and in other regions. We examined the effects of long-term temperature change on the laying dates and clutch sizes of six ecologically diverse species of North American birds using 50 years of nest record data. As predicted, laying dates for most (four of six) species were earlier when spring temperatures were warmer. Over the long-term, laying dates advanced over time for two species (red-winged blackbirds, Agelaius phoeniceus and eastern bluebirds, Sialia sialis). Laying date of song sparrows (Melospiza melodia) also advanced with increasing temperature when the analysis was restricted to eastern populations. Neither laying date nor clutch sizes changed significantly over time in the remaining species (American coot, Fulica americana, killdeer, Charadrius vociferous, and American robin, Turdus migratorius), an unsurprising result given the lack of increase in temperatures over time at nest locations of these species. This study indicates that the relationship between climate change and breeding in birds is variable within and among species. In large-scale analyses of North American birds, four of seven species have shown advances in laying dates with increasing temperature (including song sparrows in the east). These variable responses within and among species highlight the need for more detailed studies across large spatial scales.     

Thrombopoietin complements G(i)- but not G(q)-dependent pathways for integrin {alpha}(IIb){beta}(3) activation and platelet aggregation

Binding of thrombopoietin (TPO) to the cMpl receptor on human platelets potentiates aggregation induced by a number of agonists, including ADP. In this work, we found that TPO was able to restore ADP-induced platelet aggregation upon blockade of the G(q)-coupled P2Y1 purinergic receptor but not upon inhibition of the G(i)-coupled P2Y12 receptor. Moreover, TPO triggered platelet aggregation upon co-stimulation of G(z) by epinephrine but not upon co-stimulation of G(q) by the thromboxane analogue U46619. Platelet aggregation induced by TPO and G(i) stimulation was biphasic, and cyclooxygenase inhibitors prevented the second but not the first phase. In contrast to ADP, TPO was unable to induce integrin alpha(IIb)beta(3) activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin alpha(IIb)beta(3) was blocked by antagonists of the G(q)-coupled P2Y1 receptor but was completely restored by the simultaneous co-stimulation of cMpl receptor by TPO. Inside-out activation of integrin alpha(IIb)beta(3) induced by TPO and G(i) stimulation occurred independently of thromboxane A(2) production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and G(i) activation of integrin alpha(IIb)beta(3) was suppressed by wortmannin and Ly294002, suggesting a critical regulation by phosphatidylinositol 3-kinase. We found that TPO did not activate phospholipase C in human platelets and was unable to restore ADP-induced phospholipase C activation upon blockade of the G(q)-coupled P2Y1 receptor. TPO induced a rapid and sustained activation of the small GTPase Rap1B through a pathway dependent on phosphatidylinositol 3-kinase. In ADP-stimulated platelets, Rap1B activation was reduced, although not abolished, upon blockade of the P2Y1 receptor. However, accumulation of GTP-bound Rap1B in platelets activated by co-stimulation of cMpl and P2Y12 receptor was identical to that induced by the simultaneous ligation of P2Y1 and P2Y12 receptor by ADP. These results indicate that TPO can integrate G(i), but not G(q), stimulation and can efficiently support integrin alpha(IIb)beta(3) activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.     

Kinetic study of the antiport mechanism of an Escherichia coli zinc transporter, ZitB

ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (K(m)) of 104.9 +/- 5.4 microm and 90.1 +/- 3.7 microm, respectively. Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+. H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a Km of 19.9 nm for H+. Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H(+) gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+. The exchange stoichiometry of metal ion for proton is likely to be 1:1.