Oxidative regulation of fatty acid-induced tau polymerization

Alzheimer's disease (AD) is characterized by the presence of amyloid-positive senile plaques and tau-positive neurofibrillary tangles. Aside from these two pathological hallmarks, a growing body of evidence indicates that the amount of oxidative alteration of vulnerable molecules such as proteins, DNA, and fatty acids is elevated in the brains of AD patients. It has been hypothesized that the elevated amounts of protein oxidation could lead directly to the formation of neurofibrillary tangles through a cysteine-dependent mechanism. We have tested this hypothesis in an in vitro system in which tau assembly is induced by fatty acids. Using sulfhydryl protective agents and site-directed mutagenesis, we found that cysteine-dependent oxidation of the tau molecule is not required for its polymerization and may even be inhibitory. However, by adjusting the oxidative environment of the polymerization reaction through the addition of a strong antioxidant or through the addition of an oxidizing system consisting of iron, adenosine diphosphate, and ascorbate, we found that oxidation does play a major role in our in vitro paradigm. The results indicated that fatty acid oxidation, the amount of which is found to be elevated in AD patients, can facilitate the polymerization of tau. However, "overoxidation" of the fatty acids can inhibit the process. Therefore, we postulate that specific fatty acid oxidative products could provide a direct link between oxidative stress mechanisms and the formation of neurofibrillary tangles in AD.     

A bactericidal monoclonal antibody elicits a change in its antigen, OspB of Borrelia burgdorferi, that can be detected by limited proteolysis

mAb CB2, directed against outer surface protein B (OspB), causes bacteriolysis of Borrelia burgdorferi in the absence of complement. How this happens is unknown. We examined the effect of mAb binding on OspB tertiary structure by using limited proteolysis to probe changes in protein conformation. Truncated OspB (tOspB) that lacked N-terminal lipid was cleaved by four enzymes: trypsin, endoproteinase Arg-C, endoproteinase Asp-N, and endoproteinase Glu-C. CB2 affected the cleavage by trypsin and Arg-C, but not by AspN or Glu-C. None of the enzymes cleaved CB2 under these conditions. Both trypsin and Arg-C cleaved tOspB near the N-terminus; CB2 slowed the rate of cleavage, but did not affect the identity of the sites cleaved. Irrelevant mAb had no effect, indicating that the effect was specific. CB2 was active against tOspB of strain B31, but not against tOspB of strain BEP4, to which it does not bind, suggesting that binding was required to elicit the effect on cleavage. With trypsin, CB2 showed a maximal effect at 8 mol of tOspB to 1 mol of mAb. At this ratio, not enough CB2 was present to bind all the tOspB; therefore, either CB2 shows turnover or CB2 acts by binding tOspB and effecting a change in this tOspB such that it, in turn, propagates the effect in other molecules of tOspB. Regardless of the mechanism, these data show that CB2 elicits a change in tOspB that can be measured by its reduced susceptibility to protease cleavage.     

The gene encoding TBC1D1 with homology to the tre-2/USP6 oncogene, BUB2, and cdc16 maps to mouse chromosome 5 and human chromosome 4

TBC1D1 is the founding member of a family of related proteins with homology to tre-2/UPS6, BUB2, and cdc16 and containing the tbc box motif of 180-220 amino acids. This protein family is thought to have a role in differentiation and in regulating cell growth. We set out to map the TBC1D1 gene in mouse and human. Segregation analysis of a TBC1D1 RFLP in two independent mouse RI (recombinant inbred) lines reveals that mouse Tbc1d1 is closely linked to Pgm1 on chromosome 5. The human TBC1D1 gene was assigned to human chromosome 4p15.1-->4q21 using Southern blot analyses of genomic DNAs from rodent-human somatic cell lines. A human-specific genomic fragment was observed in the somatic cell lines containing human chromosome 4 or the 4p15.1-->4q21 region of the chromosome. TBC1D1 maps to the region containing the ortholog of mouse Pgm1 adding another locus to this long region of conserved synteny between mouse and man.     

Interstitial pH, K(+), lactate, and phosphate determined with MSNA during exercise in humans

The purpose of the present study was to use the microdialysis technique to simultaneously measure the interstitial concentrations of several putative stimulators of the exercise pressor reflex during 5 min of intermittent static quadriceps exercise in humans (n = 7). Exercise resulted in approximately a threefold (P < 0.05) increase in muscle sympathetic nerve activity (MSNA) and 13 +/- 3 beats/min (P < 0.05) and 20 +/- 2 mmHg (P < 0.05) increases in heart rate and blood pressure, respectively. During recovery, all reflex responses quickly returned to baseline. Interstitial lactate levels were increased (P < 0.05) from rest (1.1 +/- 0.1 mM) to exercise (1. 6 +/- 0.2 mM) and were further increased (P < 0.05) during recovery (2.0 +/- 0.2 mM). Dialysate phosphate concentrations were 0.55 +/- 0. 04, 0.71 +/- 0.05, and 0.48 +/- 0.03 mM during rest, exercise, and recovery, respectively, and were significantly elevated during exercise. At the onset of exercise, dialysate K(+) levels rose rapidly above resting values (4.2 +/- 0.1 meq/l) and continued to increase during the exercise bout. After 5 min of contractions, dialysate K(+) levels had peaked with an increase (P < 0.05) of 0.6 +/- 0.1 meq/l and subsequently decreased during recovery, not being different from rest after 3 min. In contrast, H(+) concentrations rapidly decreased (P < 0.05) from resting levels (69.4 +/- 3.7 nM) during quadriceps exercise and continued to decrease with a mean decline (P < 0.05) of 16.7 +/- 3.8 nM being achieved after 5 min. During recovery, H(+) concentrations rapidly increased and were not significantly different from baseline after 1 min. This study represents the first time that skeletal muscle interstitial pH, K(+), lactate, and phosphate have been measured in conjunction with MSNA, heart rate, and blood pressure during intermittent static quadriceps exercise in humans. These data suggest that interstitial K(+) and phosphate, but not lactate and H(+), may contribute to the stimulation of the exercise pressor reflex.     

Limb congestion enhances the synchronization of sympathetic outflow with muscle contraction

In this report, we examined if the synchronization of muscle sympathetic nerve activity (MSNA) with muscle contraction is enhanced by limb congestion. To explore this relationship, we applied signal-averaging techniques to the MSNA signal obtained during short bouts of forearm contraction (2-s contraction/3-s rest cycle) at 40% maximal voluntary contraction for 5 min. We performed this analysis before and after forearm venous congestion; an intervention that augments the autonomic response to sustained static muscle contractions via a local effect on muscle afferents. There was an increased percentage of the MSNA noted during second 2 of the 5-s contraction/rest cycles. The percentage of total MSNA seen during this particular second increased from minute 1 to 5 of contraction and was increased further by limb congestion (control minute 1 = 25.6 +/- 2.0%, minute 5 = 32.8 +/- 2.2%; limb congestion minute 1 = 29.3 +/- 2.1%, minute 5 = 37.8 +/- 3.9%; exercise main effect <0.005; limb congestion main effect P = 0.054). These changes in the distribution of signal-averaged MSNA were seen despite the fact that the mean number of sympathetic discharges did not increase over baseline. We conclude that synchronization of contraction and MSNA is seen during short repetitive bouts of handgrip. The sensitizing effect of contraction time and limb congestion are apparently due to feedback from muscle afferents within the exercising muscle.     

Effects of hypoxia, anoxia, and endogenous ethanol on thermoregulation in goldfish, Carassius auratus

Effects of hypoxia, anoxia, and endogenous ethanol (EtOH) on selected temperature (T(sel)) and activity in goldfish were evaluated. Blood and brain EtOH concentrations ([EtOH]) and brain oxygen partial pressure (PO(2)) were quantified at crucial ambient oxygen pressures. Below a threshold value near 31 Torr, T(sel) decreased as a function of environmental PO(2). T(sel) of 15 degrees C-acclimated fish was approximately 10 degrees C at the onset of anoxia and changed little over 2 h. Activity showed a similar response pattern. Brain [EtOH] was significantly elevated above control levels after 1 h anoxia. In normoxic water, T(sel) remained different in previously anoxic and normoxic control fish for approximately 20 min. Blood [EtOH] of previously anoxic fish remained significantly elevated ([EtOH] >4.0 micromol/g blood), and activity was significantly depressed at 20 min. Brain PO(2) reached normal levels in <3 min. We conclude that [EtOH] (brain or blood) and brain PO(2) are not proximal causes of either behavioral anapyrexia (hypothermia) or inactivity in goldfish exposed to oxygen-depleted environments.     

Mechanism of high susceptibility of iron-overloaded mouse to Vibrio vulnificus infection

Vibrio vulnificus produces fulminant septicemia in humans with underlying conditions, particularly those with diseases that elevate the iron level. The effect of a high iron level on the virulence of V. vulnificus was therefore investigated in mice treated with iron dextran. The mice loaded with iron became highly susceptible to V. vulnificus infection, the LD50 (50% lethal dose) decreased five logs when infected per peritoneum. However, when infected via the oral route, the LD50 was affected little unless the mouse was treated with an additional drug such as cyclophosphamide or D-galactosamine. Mice with or without iron-overloading died when the bacterial concentration in the blood reached 10(5) cfu/ml or above. Iron increased the growth rate of the bacteria, both inside and outside of the animal, quickly reaching a lethal concentration in the iron-overloaded mouse. V. vulnificus, grown with or without the addition of iron, showed strong cytotoxicity on the isolated cells or within the animal at high bacterial concentration. Iron overload stimulated the production of tumor necrosis factor alpha (TNF-alpha), a major factor of septic shock, in mice upon infection with the bacteria, probably caused by the endotoxin; however, the neutrophils, whose migration is effected by TNF-alpha, appeared to be less active. Taken together, the major virulence factor of V. vulnificus appeared to be the accelerated growth of bacteria to quickly reach the lethal level and the lower activity of immune cells including neutrophil as a result of iron-overloading. These two effects manifest other virulence factors, the host's as well as bacterial. Such factors, other than TNF-alpha stimulated by the endotoxin, enhanced cytotoxicity, which kills the host cells including the host's immune cells.     

Critical phases of the brain development

Various effects were shown to act alter the proceeding of histogenetic processes in the embryonic neural tissue. Studies in mice revealed that, depending on the embryogenesis stage when a lack of serotonin occurred, the lack of this monoamine entails either death of the embryo or a teratogenic effect, or underdevelopment of brain structures. In the rat model of a shorten hypoxia, a disorder of neural tissue histogenesis in early stages of development, was found, as well as a reduction of its sensitivity to lack of oxygen by the end of embryogenesis.     

Changes in the neuronal activity in the dorsolateral pontine region caused by electrical stimulation of the brainstem regions inhibiting movement and the muscle tone

Activity of 44 mesencephalic locomotor area's (MLR) units and 38 pontine inhibitory area's (PIA) units was recorded during stimulation of the giganto-cellular reticular nucleus and oral pontine reticular nucleus inducing the hindlimb muscle tone inhibition in decerebrated rats. The muscle tone suppression was always accompanied by a decrease in the MLR and an increase in the PIA unit discharges. Stimulation of the brainstem inhibitory area seems to activate reticulospinal inhibitory system and suppress some MLR units relating to locomotion and muscle tone.