Hsc70 Rapidly Engages Tau after Microtubule Destabilization

The microtubule-associated protein Tau plays a crucial role in regulating the dynamic stability of microtubules during neuronal development and synaptic transmission. In a group of neurodegenerative diseases, such as Alzheimer disease and other tauopathies, conformational changes in Tau are associated with the initial stages of disease pathology. Folding of Tau into the MC1 conformation, where the amino acids at residues 7–9 interact with residues 312–342, is one of the earliest pathological alterations of Tau in Alzheimer disease. The mechanism of this conformational change in Tau and the subsequent effect on function and association to microtubules is largely unknown. Recent work by our group and others suggests that members of the Hsp70 family play a significant role in Tau regulation. Our new findings suggest that heat shock cognate (Hsc) 70 facilitates Tau-mediated microtubule polymerization. The association of Hsc70 with Tau was rapidly enhanced following treatment with microtubule-destabilizing agents. The fate of Tau released from the microtubule was found to be dependent on ATPase activity of Hsc70. Microtubule destabilization also rapidly increased the MC1 folded conformation of Tau. An in vitro assay suggests that Hsc70 facilitates formation of MC1 Tau. However, in a hyperphosphorylating environment, the formation of MC1 was abrogated, but Hsc70 binding to Tau was enhanced. Thus, under normal circumstances, MC1 formation may be a protective conformation facilitated by Hsc70. However, in a diseased environment, Hsc70 may preserve Tau in a more unstructured state, perhaps facilitating its pathogenicity.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Host‐associated differentiation in a pecan and water hickory Aphidomorpha community

Host‐associated differentiation (HAD) is the formation of genetically distinct, host‐associated populations created and maintained by ecologically mediated reproductive isolation. HAD potentially accounts for species origins in parasites, including herbivorous insects. Although case studies testing the occurrence of HAD are accumulating, it is still unclear how common HAD is and which specific ecological traits explain its occurrence. To address these issues, studies are needed that include negative results (i.e., instances in which parasite populations do not exhibit HAD) and test for HAD across communities (i.e., several parasite species on the same set of host species). In this study, HAD was tested in a community of six species of Aphidomorpha (Hemiptera) that share a host‐plant pair: pecan [Carya illinoinensis (Wangenh.) K.Koch] and water hickory [Carya aquatica (F.Michx) Nutt., both Juglandaceae] trees. All six species are parthenogenetic and three species are endophagous, traits that can exacerbate host‐specific selection. AFLP markers were employed to detect the presence of genetically distinct, host‐associated populations for each insect species. Strict HAD (i.e., the occurrence of genetically distinct pecan‐associated and water hickory‐associated genotypes) was found in Phylloxera notabilis Pergande (Phylloxeridae), Phylloxera devastatrix Pergande, and Monelliopsis pecanis Bissel (Aphididae). Monellia caryella Fitch (Aphididae) displayed a pattern of partial HAD (i.e., the occurrence of only a genetically distinct pecan‐associated genotype). No HAD was found in Melanocallis caryaefoliae Davis (Aphididae) or Phylloxera texana Stoetzel. The pattern of HAD occurrence in the pecan and water hickory Aphidomorpha community indicated that neither parthenogenesis nor endophagy sufficiently explain the occurrence of HAD in this system.     

Internalization, recycling, and redistribution of vasopressin receptors in rat hepatocytes

Three hours after isolation, cultured hepatocytes have approximately 150,000 surface vasopressin receptors/cell, and these exhibit a Kd for 125I-vasopressin of 6 nM based on calculation of Koff/Kon, or a Kd of 9.5 nM based on Scatchard plot analysis. After the binding of 125I-vasopressin to its receptor on the hepatocyte surface, this complex is internalized with a t1/2 of 3-6 min. Following this internalization, the number of vasopressin receptors on the cell surface is restored both in vitro and in the isolated perfused liver with a t1/2 of 8-10 min. This restoration is blocked in vitro by incubation of the hepatocytes at 18 degrees C, but not by cycloheximide, suggesting that internalized vasopressin receptors recycle back to the cell surface. Prolonged incubation of hepatocytes with vasopressin results in the loss of greater than 75% of the vasopressin surface binding at concentrations of vasopressin approximately equivalent to its Kd. The binding of vasopressin to cultured hepatocytes 3-5 h after isolation resembles binding to the isolated perfused whole liver with respect to receptor dynamics. During culture for 48 h, however, we observe a progressive loss of hepatocyte surface vasopressin receptors. Concomitant with this reduction in surface receptors with time in culture, there appears to be a marked elevation in intracellular receptors.     

Purification and characterization of the rat liver vasopressin (V1) receptor

Utilizing a proteoliposome reconstitution system, we have purified the rat liver V1 vasopressin receptor to near homogeneity. The receptor was purified approximately 21,000-fold from rat liver membranes, using differential detergent solubilization, size exclusion gel filtration, lectin affinity, and ion-exchange chromatography. The purified receptor exhibits a Kd of 6 nM, when, prior to solubilization, the membranes were exposed to 1 microM vasopressin. This resulted in the association of a pertussis toxin-insensitive guanine nucleotide-binding protein with the receptor during most of the purification procedure. In the absence of this association, the receptor had a Kd of approximately 30 nM. Association of the receptor with a G-protein was confirmed by the ability of vasopressin to stimulate the hydrolysis of [gamma-32P]GTP. The specific activity of the vasopressin-stimulated hydrolysis was 25 nmol/min/mg, approximately 8,000-fold higher than values obtained with crude reconstituted receptor preparations. Cross-linking of 125I-vasopressin to a partially purified preparation of receptor demonstrated that the receptor had a molecular weight of approximately 68,000 under reducing conditions, and 58,000 under nonreducing conditions. The purification procedure may prove useful in purifying a number of small peptide hormone receptors (e.g. bradykinin, angiotensin II) and perhaps their associated G-proteins as well.     

Human neutrophil migration is enhanced by beta-endorphin

Using a serum-free chemotaxis-under-agarose assay, we measured the effect of beta-endorphin on directed migration of human neutrophils toward 10(-7) M N-formyl-methionyl-leucyl-phenylalanine (FMLP). Neutrophils were pre-incubated with a range of beta-endorphin concentrations. beta-endorphin enhanced migration of neutrophils toward FMLP. This effect was maximal at 10(-9) M beta-endorphin. Naloxone inhibited the beta-endorphin effect, suggesting that enhanced migration is mediated via an opiate receptor.     

Response of cytochrome a,a3 to carbon monoxide in canine hearts with prior infarcts

The effects of moderate levels of carbon monoxide (CO) on the oxidation-reduction state of cytochrome a,a3 (cyt a,a3) were examined in the hearts of twelve dogs with a prior myocardial infarction. Exposure to ten minutes CO produced a carboxyhemoglobin (CO-Hb) level of 9.4%, a level experienced by heavy smokers. Accompanying the exposure to CO, cyt a,a3 became more reduced; 17.4% +/- 4.7%. Exposure to CO was accompanied by an increase of 33% +/- 4% in the rate of cyt a,a3 reduction following occlusion of the left circumflex coronary artery and a decrease of 24% +/- 8% in the rate of cyt a,a3 oxidation with release. There was also a decrease in the magnitude of cyt a,a3 reduction from 86% +/- 9% to 70% +/- 11%. These results indicate that moderate levels of CO trap cyt a,a3 in the reduced state which impairs the ability of the heart to recover from transient ischemic episodes.