Effect of mental fatigue on induced tremor in human knee extensors

In this study, the effects of mental fatigue on mechanically induced tremor at both a low (3–6 Hz) and high (8–12 Hz) frequency were investigated. The two distinct tremor frequencies were evoked using two springs of different stiffness, during 20 s sustained contractions of the knee extensor muscles at 30% maximum voluntary contraction (MVC) before and after 100 min of a mental fatigue task, in 12 healthy (29 ± 3.7 years) participants. Mental fatigue resulted in a 6.9% decrease in MVC and in a 9.4% decrease in the amplitude of the agonist muscle EMG during sustained 30% MVC contractions in the induced high frequency only. Following the mental fatigue task, the coefficient of variation and standard deviation of the force signal decreased at 8–12 Hz induced tremor by 31.7% and 35.2% respectively, but not at 3–6 Hz induced tremor. Similarly, the maximum value and area underneath the peak in the power spectrum of the force signal decreased by 55.5% and 53.1% respectively in the 8–12 Hz range only. In conclusion, mental fatigue decreased mechanically induced 8–12 Hz tremor and had no effect on induced 3–6 Hz tremor. We suggest that the reduction could be attributed to the decreased activation of the agonist muscles.     

Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor

The relationship between Aspergillus niger morphology and citric acid production was investigated in two reactor systems with different configurations, a tubular loop and a stirred tank bioreactor, with operating volumes of 6 and 8 dm³, respectively. Morphology was quantified by image analysis. In each system, morphology, characterized by the parameter P (mean convex perimeter of clumps), and citric acid production, were agitation-dependent and closely linked. Increased agitation caused a reduction of clump sizes and results when both reactors demonstrate that the parameter P should not exceed a threshold value in order to achieve increased productivities. The results obtained from the two reactors were in agreement, both qualitatively and quantitatively. Reducing the fundamentally different mixing conditions of the two bioreactors to the order of the dimensionless mixing parameter relative mixing time (τ_m), results showed that the loop simulated the stirred tank. Also, relationships valid for one system accurately described the results obtained from the other system, demonstrating the validity of the relationship between morphology and productivity for the particular fermentation, regardless of the reactor type. Previous attempts to evaluate the use of loop configurations as scale-up tools and their performance as bioreactors, neglected the morphology of the producer micro-organisms. This study demonstrated the close link between morphology and productivity for citric acid production by A. niger, and identified a morphology parameter that was used successfully to characterize the process performance.     

Histone Deacetylase-3 Mediates Positive Feedback Relationship between Anaphylaxis and Tumor Metastasis

Allergic inflammation has been known to enhance the metastatic potential of tumor cells. The role of histone deacetylase-3 (HDAC3) in allergic skin inflammation was reported. We investigated HDAC3 involvement in the allergic inflammation-promotion of metastatic potential of tumor cells. Passive systemic anaphylaxis (PSA) induced HDAC3 expression and FcϵRI signaling in BALB/c mice. PSA enhanced the tumorigenic and metastatic potential of mouse melanoma cells in HDAC3- and monocyte chemoattractant protein 1-(MCP1)-dependent manner. The PSA-mediated enhancement of metastatic potential involved the induction of HDAC3, MCP1, and CD11b (a macrophage marker) expression in the lung tumor tissues. We examined an interaction between anaphylaxis and tumor growth and metastasis at the molecular level. Conditioned medium from antigen-stimulated bone marrow-derived mouse mast cell cultures induced the expression of HDAC3, MCP1, and CCR2, a receptor for MCP1, in B16F1 mouse melanoma cells and enhanced migration and invasion potential of B16F1 cells. The conditioned medium from B16F10 cultures induced the activation of FcϵRI signaling in lung mast cells in an HDAC3-dependent manner. FcϵRI signaling was observed in lung tumors derived from B16F10 cells. Target scan analysis predicted HDAC3 to be as a target of miR-384, and miR-384 and HDAC3 were found to form a feedback regulatory loop. miR-384, which is decreased by PSA, negatively regulated HDAC3 expression, allergic inflammation, and the positive feedback regulatory loop between anaphylaxis and tumor metastasis. We show the miR-384/HDAC3 feedback loop to be a novel regulator of the positive feedback relationship between anaphylaxis and tumor metastasis.     

A Fundamental Trade-off in Covalent Switching and Its Circumvention by Enzyme Bifunctionality in Glucose Homeostasis

Covalent modification provides a mechanism for modulating molecular state and regulating physiology. A cycle of competing enzymes that add and remove a single modification can act as a molecular switch between “on” and “off” and has been widely studied as a core motif in systems biology. Here, we exploit the recently developed “linear framework” for time scale separation to determine the general principles of such switches. These methods are not limited to Michaelis-Menten assumptions, and our conclusions hold for enzymes whose mechanisms may be arbitrarily complicated. We show that switching efficiency improves with increasing irreversibility of the enzymes and that the on/off transition occurs when the ratio of enzyme levels reaches a value that depends only on the rate constants. Fluctuations in enzyme levels, which habitually occur due to cellular heterogeneity, can cause flipping back and forth between on and off, leading to incoherent mosaic behavior in tissues, that worsens as switching becomes sharper. This trade-off can be circumvented if enzyme levels are correlated. In particular, if the competing catalytic domains are on the same protein but do not influence each other, the resulting bifunctional enzyme can switch sharply while remaining coherent. In the mammalian liver, the switch between glycolysis and gluconeogenesis is regulated by the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). We suggest that bifunctionality of PFK-2/FBPase-2 complements the metabolic zonation of the liver by ensuring coherent switching in response to insulin and glucagon.     

EFFECTS OF DIURETIC HORMONE 31, DROSOKININ,AND ALLATOSTATIN A ON TRANSEPITHELIAL K+ TRANSPORT AND CONTRACTION FREQUENCY IN THE MIDGUT AND HINDGUT OF LARVAL Drosophila melanogaster

Recent studies have identified paracrine and endocrine cells in the midgut of larval Drosophila melanogaster as well as midgut and hindgut receptors for multiple neuropeptides implicated in the control of fluid and ion balance. Although the effects of diuretic factors on fluid secretion by isolated Malpighian tubules of D. melanogaster have been examined extensively, relatively little is known about the effects of such factors on gut peristalsis or ion transport across the gut. We have measured the effects of diuretic hormone 31 (DH31), drosokinin and allatostatin A (AST‐A) on both K⁺ transport and muscle contraction frequency in the isolated gut of larval D. melanogaster. K⁺ absorption across the gut was measured using K⁺‐selective microelectrodes and the scanning ion‐selective electrode technique. Allatostatin A (AST‐A; 1 μM) increased K⁺ absorption across the anterior midgut but reduced K⁺ absorption across the copper cells and large flat cells of the middle midgut. AST‐A strongly inhibited gut contractions in the anterior midgut but had no effect on contractions of the pyloric sphincter induced by proctolin. DH31 (1 μM) increased the contraction frequency in the anterior midgut, but had no effect on K⁺ flux across the anterior, middle, or posterior midgut or across the ileum. Drosokinin (1 μM) did not affect either contraction frequency or K⁺ flux across any of the gut regions examined. Possible functions of AST‐A, DH31, and drosokinin in regulating midgut physiology are discussed.     

Effect of reproductive modes and environmental heterogeneity in the population dynamics of a geographically widespread clonal desert cactus

The dynamics of plant populations in arid environments are largely affected by the unpredictable environmental conditions and are fine-tuned by biotic factors, such as modes of recruitment. A single species must cope with both spatial and temporal heterogeneity that trigger pulses of sexual and clonal establishment throughout its distributional range. We studied two populations of the clonal, purple prickly pear cactus, Opuntia macrocentra, in order to contrast the factors responsible for the population dynamics of a common, widely distributed species. The study sites were located in protected areas that correspond to extreme latitudinal locations for this species within the Chihuahuan Desert. We studied both populations for four consecutive years and determined the demographic consequences of environmental variability and the mode of reproduction using matrix population models, life table response experiments (LTREs), and loop and perturbation analyses. Although both populations seemed fairly stable (population growth rate, λ∼1), different demographic parameters and different life cycle routes were responsible for this stability in each population. In the southernmost population (MBR) LTRE and loop and elasticity analyses showed that stasis is the demographic process with the highest contributions to λ, followed by sexual reproduction, and clonal propagation contributed the least. The northern population (CR) had both higher elasticities and larger contributions of stasis, followed by clonal propagation and sexual recruitment. Loop analysis also showed that individuals in CR have more paths to complete a life cycle than those in MBR. As a consequence, each population differed in life history traits (e.g., size class structure, size at sexual maturity, and reproductive value). Numerical perturbation analyses showed a small effect of the seed bank on the λ of both populations, while the transition from seeds to seedlings had an important effect mainly in the northern population. Clonal propagation (higher survival and higher contributions to vital rates) seems to be more important for maintaining populations over long time periods than sexual reproduction.     

A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.     

Peptides from second extracellular loop of C-C chemokine receptor type 5 (CCR5) inhibit diverse strains of HIV-1

To initiate HIV entry, the HIV envelope protein gp120 must engage its primary receptor CD4 and a coreceptor CCR5 or CXCR4. In the absence of a high resolution structure of a gp120-coreceptor complex, biochemical studies of CCR5 have revealed the importance of its N terminus and second extracellular loop (ECL2) in binding gp120 and mediating viral entry. Using a panel of synthetic CCR5 ECL2-derived peptides, we show that the C-terminal portion of ECL2 (2C, comprising amino acids Cys-178 to Lys-191) inhibit HIV-1 entry of both CCR5- and CXCR4-using isolates at low micromolar concentrations. In functional viral assays, these peptides inhibited HIV-1 entry in a CD4-independent manner. Neutralization assays designed to measure the effects of CCR5 ECL2 peptides when combined with either with the small molecule CD4 mimetic NBD-556, soluble CD4, or the CCR5 N terminus showed additive inhibition for each, indicating that ECL2 binds gp120 at a site distinct from that of N terminus and acts independently of CD4. Using saturation transfer difference NMR, we determined the region of CCR5 ECL2 used for binding gp120, showed that it can bind to gp120 from both R5 and X4 isolates, and demonstrated that the peptide interacts with a CD4-gp120 complex in a similar manner as to gp120 alone. As the CCR5 N terminus-gp120 interactions are dependent on CD4 activation, our data suggest that gp120 has separate binding sites for the CCR5 N terminus and ECL2, the ECL2 binding site is present prior to CD4 engagement, and it is conserved across CCR5- and CXCR4-using strains. These peptides may serve as a starting point for the design of inhibitors with broad spectrum anti-HIV activity.     

Bacillus thuringiensis Cry4Aa insecticidal protein: Functional importance of the intrinsic stability of the unique α4–α5 loop comprising the Pro-rich sequence

The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro¹⁹³Pro¹⁹⁴_Pro¹⁹⁶) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4–α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4–α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro¹⁹³. Further MD simulations revealed that the most critical residue−Pro¹⁹³ for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4–α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.