History dependence of human muscle-fiber conduction velocity during voluntary isometric contractions

The conduction velocity (CV) of a muscle fiber is affected by the fiber's discharge history going back ～1 s. We investigated this dependence by measuring CV fluctuations during voluntary isometric contractions of the human brachioradialis muscle. We recorded electromyogram (EMG) signals simultaneously from multiple intramuscular electrodes, identified potentials belonging to the same motor unit using EMG decomposition, and estimated the CV of each discharge from the interpotential interval. In 12 of 14 subjects, CV increased by ～10% during the first second after recruitment and then fluctuated by about ±2% in a way that mirrored the fluctuations in the instantaneous firing rate. The CV profile could be precisely described in terms of the discharge history by a simple mathematical model. In the other two subjects, and one subject retested after cooling the arm, the CV fluctuations were inversely correlated with instantaneous firing rate. In all subjects, CV was additionally affected by very short interdischarge intervals (<25 ms): it was increased in doublets at recruitment, but decreased in doublets during continuous firing and after short interdischarge intervals in doubly innervated fibers. CV also exhibited a slow trend of about -0.05%/s that did not depend on the immediate discharge history. We suggest that measurements of CV fluctuations during voluntary contractions, or during stimulation protocols that involve longer and more complex stimulation patterns than are currently being used, may provide a sensitive approach for estimating the dynamic characteristics of ion channels in the human muscle-fiber membrane.     

Nomenclature changes in African Eumolpinae with reference to type specimens preserved in the Muséum national d’Histoire naturelle,Paris (Coleoptera: Chrysomelidae)

Based on examination of type specimens preserved in the Muséum national d’Histoire naturelle, Paris, 50 new combinations in African Eumolpinae species described by Lefèvre and Pic in the genera Angoleumolpus Pic, 1938, Cheiriphyle Jacoby, 1901, Eryxia Baly, 1865, Eurydemus Chapuis, 1874, Microeurydemus Pic, 1938, Microsyagrus Pic, 1952, Pagria Lefèvre, Rhembastus Harold, 1877, Syagrus Chapuis, 1874 and Thysbina Weise, 1902 are proposed. The following new names are introduced to correct homonymies: Afroeurydemus fortesculptus nom. nov. for Microsyagrus cribricollis Pic, 1952, Afroeurydemus parvomaculatus nom. nov. for Syagrus quadrimaculatus Pic, 1940, Afroeurydemus selmani nom. nov. for A. signatus Selman, 1972, Microsyagrus punctaticollis nom. nov. for M. trinotatus Pic, 1952, Obelistes bryanti nom. nov. for O. nigrovittatus Bryant, 1952, and Pathius pici nom. nov. for Microeurydemus pallidus Pic, 1952. The following synonyms are established: Angoleumolpus Pic, 1938 n. syn. of Obelistes Lefèvre, 1885, Thysbina gabonica Pic, 1952 n. syn. of Ennodius murrayi (Chapuis, 1874), and Timentes flavipes Selman, 1965 n. syn. of Timentes camerunensis (Pic, 1953). Lectotypes are designated where appropriate.     

Chimeric piggyBac transposases for genomic targeting in human cells

Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.     

Modulation of transforming growth factor-beta 1 stimulated elastin and collagen production and proliferation in porcine vascular smooth muscle cells and skin fibroblasts by basic fibroblast growth factor,transforming growth factor-α, and insulin-like growth factor-I

During tissue repair and development, matrix accumulation is modulated as multiple signals impinge on target cells. We have investigated the effects of combinations of the mitogenic cytokines, basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGF-α), and insulin-like growth factor-1 (IGF-1) with transforming growth factor-beta 1 (TGF-β1) with respect to the production of two matrix components, elastin and type I collagen. Using specific enzyme-linked immunoassays for detection of secreted precursors in both vascular smooth muscle cells and skin fibroblasts from the domestic pig, production of these two fibrous proteins was shown to be strongly stimulated by TGF-β1. In the smooth muscle cell, both bFGF and TGF-α were potent antagonists of TGF-β1-mediated matrix production, whereas IGF-1 was only weakly additive with respect to elastin production. Antagonism was also evident to a lesser extent in skin fibroblasts. Reduced responsiveness to TGF-β1 did not appear to be due to a switch to a proliferative state, since TGF-β1 itself acted as a mitogen in confluent SMC, and TGF-α was only a weak mitogen in confluent fibroblast cultures. Although a predominant effect of TGF-β is matrix accumulation, these findings suggest that this property will be significantly modified by the cytokine context. © 1993 Wiley-Liss, Inc.     

Artificial and engineered chromosomes: developments and prospects for gene therapy

At the gene therapy session of the ICCXV Chromosome Conference (2004), recent advances in the construction of engineered chromosomes and de novo human artificial chromosomes were presented. The long-term aims of these studies are to develop vectors as tools for studying genome and chromosome function and for delivering genes into cells for therapeutic applications. There are two primary advantages of chromosome-based vector systems over most conventional vectors for gene delivery. First, the transferred DNA can be stably maintained without the risks associated with insertion, and second, large DNA segments encompassing genes and their regulatory elements can be introduced, leading to more reliable transgene expression. There is clearly a need for safe and effective gene transfer vectors to correct genetic defects. Among the topics discussed at the gene therapy session and the main focus of this review are requirements for de novo human artificial chromosome formation, assembly of chromatin on de novo human artificial chromosomes, advances in vector construction, and chromosome transfer to cells and animals.     

Deficiency in mouse Y chromosome long arm gene complement is associated with sperm DNA damage

Background  

Mice with severe non-PAR Y chromosome long arm (NPYq) deficiencies are infertile in vivo and in vitro. We have previously shown that sperm from these males, although having grossly malformed heads, were able to fertilize oocytes via intracytoplasmic sperm injection (ICSI) and yield live offspring. However, in continuing ICSI trials we noted a reduced efficiency when cryopreserved sperm were used and with epididymal sperm as compared to testicular sperm. In the present study we tested if NPYq deficiency is associated with sperm DNA damage - a known cause of poor ICSI success.     

Specific vulnerability of mouse spinal cord motoneurons to membrane depolarization

Intracellular calcium (Ca²⁺) concentration determines neuronal dependence on neurotrophic factors (NTFs) and susceptibility to cell death. Ca²⁺ overload induces neuronal death and the consequences are thought to be a probable cause of motoneuron (MN) degeneration in neurodegenerative diseases. In the present study, we show that membrane depolarization with elevated extracellular potassium (K⁺) was toxic to cultured embryonic mouse spinal cord MNs even in the presence of NTFs. Membrane depolarization induced an intracellular Ca²⁺ increase. Depolarization-induced toxicity and increased intracellular Ca²⁺ were blocked by treatment with antagonists to some of the voltage-gated Ca²⁺ channels (VGCCs), indicating that Ca²⁺ influx through these channels contributed to the toxic effect of depolarization. Ca²⁺ activates the calpains, cysteine proteases that degrade a variety of substrates, causing cell death. We investigated the functional involvement of calpain using a calpain inhibitor and calpain gene silencing. Pre-treatment of MNs with calpeptin (a cell-permeable calpain inhibitor) rescued MNs survival; calpain RNA interference had the same protective effect, indicating that endogenous calpain contributes to the cell death caused by membrane depolarization. These findings suggest that MNs are especially vulnerable to extracellular K⁺ concentration, which induces cell death by causing both intracellular Ca²⁺ increase and calpain activation.     

Turing-Hopf instability in biochemical reaction networks arising from pairs of subnetworks

A three dimensional nutrient-plant-herbivore model was proposed and conditions for boundedness, positive invariance, existence and stability of different equilibrium points, Hopf-bifurcation and global stability were obtained. We performed numerical simulations to observe the simultaneous effect of the top-down and the bottom-up mechanism on the system. It was found that nutrient enrichment destroyed the coexistence steady state of the system. This nutrient enrichment could be due to high nutrient input rate or high nutrient recycling rate. In both cases the system showed instability. Moreover, these results were independent of the grazing pressure and the predation functional form.     

Production of S-adenosyl- L-methionine by a mutant strain of Kluyveromyces lactis

S-Adenosyl-l-methionine (AdoMet) was produced by a mutant strain Kluyveromyces lactis AM-65 grown on whey. A full factorial design method of three factors – (NH₄)₂SO₄ (factor x ₁), corn steep liquor (factor x ₂) and l-methionine (factor x ₃) on three levels – was used to determine the optimal medium conditions for the production of AdoMet. A time course shake-flask experiment in optimal whey medium (x ₁=3.1 g l^–1, x ₂=12.7 g l^–1, x ₃=4.6 g l^–1) was also carried out and the results confirmed the results of the factorial design and subsequent quadratic modelling and optimization of AdoMet production which reached 90 mg g^–1 cell dry wt.