Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores

When injected into the cytoplasm of Vero cells, nucleoplasmin rapidly concentrates in a narrow layer around the nuclear envelope and then accumulates within the nucleus. Transport into the nucleus can be reversibly arrested at the perinuclear stage by metabolic inhibitors or by chilling. Nucleoplasmin-coated colloidal gold particles concentrate around the nuclear envelope of Vero cells or Xenopus oocytes, and by electron microscopy of oocytes appear to be associated with fibrils attached to nuclear pore complexes. Perinuclear accumulation is not observed for the nonmigrating nucleoplasmin core fragment or nonnuclear proteins. We propose two steps in nuclear migration of proteins: rapid binding around the nuclear envelope, possibly to pore-associated fibrils, followed by slower, energy-dependent translocation through nuclear pores.     

Selective perturbation of the myosin recovery stroke by point mutations at the base of the lever arm affects ATP hydrolysis and phosphate release

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys(84) and Arg(704), we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release.     

Uptake of sulfate but not phosphate by Mycobacterium tuberculosis is slower than that for Mycobacterium smegmatis

Knowledge of the metabolic pathways used by Mycobacterium tuberculosis during infection is important for understanding its nutrient requirements and host adaptation. However, uptake, the first step in the utilization of nutrients, is poorly understood for many essential nutrients, such as inorganic anions. Here, we show that M. tuberculosis utilizes nitrate as the sole nitrogen source, albeit at lower efficiency than asparagine, glutamate, and arginine. The growth of the porin triple mutant M. smegmatis ML16 in media with limiting amounts of nitrate and sulfate as sole nitrogen and sulfur sources, respectively, was delayed compared to that of the wild-type strain. The uptake of sulfate was 40-fold slower than that of the wild-type strain, indicating that the efficient uptake of these anions is dependent on porins. The uptake by M. tuberculosis of sulfate and phosphate was approximately 40- and 10-fold slower than that of M. smegmatis, respectively, which is consistent with the slower growth of M. tuberculosis. However, the uptake of these anions by M. tuberculosis is orders of magnitude faster than diffusion through lipid membranes, indicating that unknown outer membrane proteins are required to facilitate this process.     

The rate of tension recovery in cardiac muscle correlates with the relative residual tension prevailing after restretch

Isolated cardiac muscles generate tension more quickly at higher levels of Ca(2+) activation. We investigated the molecular mechanisms underlying this effect in permeabilized rat myocardial preparations by measuring the rate of tension recovery following brief shortening/restretch perturbations. Separate series of experiments used Ca(2+)-activating solutions with different pH values (pH 6.75, 7.00, and 7.25) and different phosphate (P(i)) concentrations (0, 2.5, and 5.0 mM added P(i)) to modulate the recovery kinetics. Subsequent analysis showed that the rate of tension recovery correlated (P < 0.001) with the relative residual tension, that is, the minimum tension measured immediately after restretch normalized to the steady-state isometric tension for the experimental condition. This new finding suggests that the rate at which cardiac muscles develop force increases with the proportion of cross bridges bound to the thin filament and is strong evidence of cooperative contractile activation.     

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

Reduction in primary production followed by rapid recovery of plant biomass in response to repeated mid-season droughts in a semiarid shrubland

The frequency and severity of extreme weather events, including droughts, are expected to increase due to the climate change. Climate manipulation field experiments are widely used tools to study the response of key parameters like primary production to the treatments. Our study aimed to detect the effect of drought on the aboveground biomass and primary production both during the treatments as well as during the whole growing seasons in semiarid vegetation. We estimated aboveground green biomass of vascular plants in a Pannonian sand forest-steppe ecosystem in Hungary. We applied non-destructive field remote sensing method in control and drought treatments. Drought treatment was carried out by precipitation exclusion in May and June, and was repeated in each year from 2002. We measured NDVI before the drought treatment, right after the treatment, and at the end of the summer in 2011 and 2013. We found that the yearly biomass peaks, measured in control plots after the treatment periods, were decreased or absent in drought treatment plots, and consequently, the aboveground net primary production was smaller than in the control plots. At the same time, we did not find general drought effects on all biomass data. The studied ecosystem proved resilient, as the biomass in the drought-treated plots recovered by the next drought treatment. We conclude that the effect of drought treatment can be overestimated with only one measurement at the time of the peak biomass, while multiple within-year measurements better describe the response of biomass.     

Time-resolved measurements of phosphate release by cycling cross-bridges in portal vein smooth muscle.

The rate of release of inorganic phosphate (Pi) from cycling cross-bridges in rabbit portal-anterior mesenteric vein smooth muscle was determined by following the fluorescence of the Pi-reporter, MDCC-PBP (Brune, M., J. L. Hunter, S. A. Howell, S. R. Martin, T. L. Hazlett, J. E. T. Corrie, and M. R. Webb. 1998. Biochemistry. 37:10370-10380). Cross-bridge cycling was initiated by photolytic release of ATP from caged-ATP in Triton-permeabilized smooth muscles in rigor. When the regulatory myosin light chains (MLC20) had been thiophosphorylated, the rate of Pi release was biphasic with an initial rate of 80 microM s-1 and amplitude 108 microM, decreasing to 13.7 microM s-1. These rates correspond to fast and slow turnovers of 1.8 s-1 and 0.3 s-1, assuming 84% thiophosphorylation of 52 microM myosin heads. Activation by Ca2+-dependent phosphorylation subsequent to ATP release resulted in slower Pi release, paralleling the rate of contraction that was also slower than after thiophosphorylation, and was also biphasic: 51 microM s-1 and 13.2 microM s-1. These rates suggest that the activity of myosin light chain kinase and phosphatase ("pseudo-ATPase") contributes <20% of the ATP usage during cross-bridge cycling. The extracellular "ecto-nucleotidase" activity was reduced eightfold by permeabilization, conditions in which the ecto-ADPase was 17% of the ecto-ATPase. Nevertheless, the remaining ecto-ATPase activity reduced the precision of the estimate of cross-bridge ATPase. We conclude that the transition from fast to slow ATPase rates reflects the properties and forces directly acting on cross-bridges, rather than the result of a time-dependent decrease in activation (MLC20 phosphorylation) occurring in intact smooth muscle. The mechanisms of slowing may include the effect of positive strain on cross-bridges, inhibition of the cycling rate by high affinity Mg-ADP binding, and associated state hydrolysis.     

The deficit of the isometric tetanic tension redeveloped after a release of frog muscle at a constant velocity

Frog sartorius muscles tetanized isometrically were released at a constant velocity from lengths lL to lS (delta l = lL -lS; Ls greater than lO). The tension PS redeveloped after the release was lower than the isometric tension PS at LS, and higher than the isometric tension PL at lL. The tension deficit D is defined as the difference PS-PS. The timing of the release during the tetanus did not influence D. D/PO was proportional to delta l/lO. The proportionality constant k was equal to 1.35 +/- 0.19 (n = 8) when the velocity of release was 2.5 mm/s. When the muscles were released the same delta l, D was found to be an exponential decreasing function of the velocity. The tension deficit was also found in experiments performed in the region lS less than lO. The proportionality constant k was smaller, but the influence of the velocity of the release on D was not modified. When the velocity of the release was changed during the release, D changed accordingly, showing that the effects of delta l and V are multiplicative. These facts suggest a working hypothesis based on the concept that the actin filaments which enter the overlap region during a release are strained by the tetanic stress and therefore unable to make normal cross-bridges.     

Selective deafferentation of hand cutaneous territory is followed by changes in fibre type distribution of a forearm muscle in the horse

Based on previous observations that capsaicin can selectively damage group III and IV afferents and induce muscle fibre transformation, we hypothesized that eliminating, by means of capsaicin, the group III and IV afferents of a peripheral territory it could lead to a fibre transformation in a muscle involved in the flexor reflexes of the same peripheral territory. Therefore, capsaicin was injected into the palmar nerves of the forelimb of the horse to investigate if eliminating group III and IV afferents from the hand of the horse a muscle fibre transition would occur in the flexor carpi radialis (FCR) muscle, which is involved in the flexor reflexes of the finger itself. 120 days after capsaicin injection, type I slow fibres increased and type IIA fast fibres decreased. We presume that the long lasting deafferentation of the ergo-nociceptive fibres causes a plastic remodelling in the central nervous system and indirectly influences the motoneuron excitability via short or long loop-pathways enhancing their tonic discharge.     

Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers.

To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers.     

Overexpression of sTnC polypeptide in muscle cells is controlled by its rapid degradation

Neutrophil apoptosis is a constitutive process that can be enhanced or delayed by signals induced by various stimuli. We investigated the role of phospholipase D (PLD) in neutrophil apoptosis. The apoptotic rate of neutrophils was found to be increased by 1-butanol and decreased by the exogenous addition of PLD. Moreover, the delay of apoptosis by apoptosis-delaying stimuli such as granulocyte/macrophage colony-stimulating factor or lipopolysaccharide (LPS) was also blocked by 1-butanol. Unstimulated PLD activity in cultured cells for 20 h was higher than that in freshly isolated cells and further increased in cultured cells with LPS. These results suggest that PLD is involved in the up-regulation of neutrophil survival.