Modelling motor units in 3D: influence on muscle contraction and joint force via a proof of concept simulation

Functional heterogeneity is a skeletal muscle’s ability to generate diverse force vectors through localised motor unit (MU) recruitment. Existing 3D macroscopic continuum-mechanical finite element (FE) muscle models neglect MU anatomy and recruit muscle volume simultaneously, making them unsuitable for studying functional heterogeneity. Here, we develop a method to incorporate MU anatomy and information in 3D models. Virtual fibres in the muscle are grouped into MUs via a novel “virtual innervation” technique, which can control the units’ size, shape, position, and overlap. The discrete MU anatomy is then mapped to the FE mesh via statistical averaging, resulting in a volumetric MU distribution. Mesh dependency is investigated using a 2D idealised model and revealed that the amount of MU overlap is inversely proportional to mesh dependency. Simultaneous recruitment of a MU’s volume implies that action potentials (AP) propagate instantaneously. A 3D idealised model is used to verify this assumption, revealing that neglecting AP propagation results in a slightly less-steady force, advanced in time by approximately 20 ms, at the tendons. Lastly, the method is applied to a 3D, anatomically realistic model of the masticatory system to demonstrate the functional heterogeneity of masseter muscles in producing bite force. We found that the MU anatomy significantly affected bite force direction compared to bite force magnitude. MU position was much more efficacious in bringing about bite force changes than MU overlap. These results highlight the relevance of MU anatomy to muscle function and joint force, particularly for muscles with complex neuromuscular architecture.

     

Inducible transport of citrate in Lactobacillus rhamnosus ATCC 7469

Lactobacillus rhamnosus ATCC 7469 exhibited diauxie when grown in a medium containing both glucose and citrate as energy source. Glucose was used as the primary energy source during the glucose-citrate diauxie. Uptake of citrate was carried out by an inducible citrate transport system. The induction of citrate uptake system was repressed in the presence of glucose. This repression was reversible and mediated by cAMP.     

Introduction and expression of foreign DNA in isolated spinach chloroplasts by electroporation

An electroporation-mediated method for the study of foreign gene expression within chloroplasts has been developed. The chloroplast expression vector pHD203-GUS, which consists of coding regions for β-glucuronidase (GUS) and chloramphenicol acetyltransferase (CAT) separated by a double psbA promoter fragment from pea (in opposite orientation) was electroporated into spinach chloroplasts and the transient gene expression was examined. Conditions for the expression of the reporter genes have been optimized. Both CAT and GUS activities were detected in chloroplasts electroporated with pHD203-GUS, but not with nuclear expression vector pBI221 or negative control pUC18. No GUS activity was detected when pHD203-GUS was electroporated into spinach protoplasts. Dot immunoblot analysis using anti-GUS antibody confirmed the existence of GUS protein in chloroplasts electroporated with chloroplast-specific vector but not the negative controls, excluding the possibilities of endogenous GUS or bacterial contamination. The expression of GUS protein in treated chloroplasts was further confirmed by Western blot analysis.     

The NADH-binding subunit of respiratory chain complex I is nuclear-encoded in plants and identified only in mitochondria

In higher plants, genes for subunits of respiratory chain complex I (NADH:ubiquinone oxidoreductase) have so far been identified solely in organellar genomes. At least nine subunits are encoded by the mitochondrial DNA and 11 homologues by the plastid DNA. One of the 'key' components of complex I is the subunit binding the substrate NADH. The corresponding gene for the mitochondrial subunit has now been cloned and identified in the nuclear genome from potato ( Solanum tuberosum ). The mature protein consists of 457 amino acids and is preceded by a mitochondrial targeting sequence of 30 amino acids. The protein is evolutionarily related to the NADH-binding subunits of complex I from other eukaryotes and is well conserved in the structural domains predicted for binding the substrate NADH, the FMN and one iron-sulphur cluster. Expression examined in different potato tissues by Northern blot analysis shows the highest steady-state mRNA levels in flowers.
Precursor proteins translated in vitro from the cDNA are imported into isolated potato mitochondria in a ΔΨ-dependent manner. The processed translation product has an apparent molecular mass of 55 kDa, identical to the mature protein present in the purified plant mitochondrial complex I. However, the in-vitro translated protein is not imported into isolated chloroplasts. To further investigate whether the complex I-like enzyme in chloroplasts contains an analogous subunit for binding of NAD(P)H, different plastid protein fractions were tested with a polyclonal antiserum directed against the bovine 51 kDa NADH-binding subunit. In none of the different thylakoid or stroma protein fractions analysed were specific crossreactive polypeptides detected. These results are discussed particularly with respect to the structure of a potential complex I in chloroplasts and the nature of its acceptor site.     

Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA-specific antiserum. PIA was shown to be a linear β-1,6-linked glucosaminoglycan composed of at least 130 2-deoxy-2-amino-D-glucopyrano-syl residues of which 80–85% are N-acetylated, the rest being non-N-acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the IcaABC-carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co-transcribed from the mapped ica A promoter. Ica A contains four potential transmembrane helices, indicative of a membrane location. The deduced Ica A sequence shows similarity to those of polysaccharide-polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N-acetylglucosaminyltransferase involved in lipo-chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that Ica A has N-acetylglucosaminyltransferase activity in the formation     

Light-dependent and tissue-specific expression of the H-protein of the glycine decarboxylase complex.

Glycine decarboxylase is a mitochondrial enzyme complex, which is the site of photorespiratory CO2 and NH3 release. Although the proteins that constitute the complex are located within the mitochondria, because of their intimate association with photosynthesis their expression is controlled by light. Comparisons of the kinetics of mRNA accumulation between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the H-protein of glycine decarboxylase during the greening of etiolated Arabidopsis thaliana suggest that their expression is controlled in parallel. A genomic clone for the H-protein (gdcH) was isolated from Arabidopsis and sequenced. The upstream region from -856 to +62 was fused to the beta-glucuronidase (GUS) reporter gene, and this construct was transformed into tobacco. This 5' upstream regulatory region appears to control GUS expression in a manner very similar to that of the endogenous H-protein gene. Constructs with deletions in the 5' upstream region were transformed into tobacco. These deletions revealed that light-dependent and tissue-specific expression was largely controlled by a 259-bp region between -376 and -117 bp. This region contains several putative GT boxes with the GGTTAA consensus core sequence. Once these strong light-dependent elements were removed, a second level of control was revealed. In constructs in which the gdcH 5' regulatory region was shortened to -117 bp or less, there was more GUS activity in the roots than in the leaves, and in dark-grown plants than in light-grown plants. This suggests that more proximal control elements may be responsible for the constitutive low levels of gene expression noted in all nonphotosynthetic tissues.     

Proteinase Inhibitor II Gene Expression Induced by Electrical Stimulation and Control of Photosynthetic Activity in Tomato Plants

Mechanical damage and heat stimulation were used to activateproteinase inhibitor II (Pin2) gene expression in tomato plantsin both treated (local induction) and non-treated tissues (systemicinduction). Both stimuli have been shown to generate electricalsignals, leading to a systemic activation of gene expression.Treatment of tomato leaves with electrical current resultedin the accumulation of Pin2 mRNA in the local and systemic leaves.Additionally, all treatments inducing Pin2 gene activity gaverise to a significant alteration of stomatal aperture. However,heat stimulation provoked a different response in the stomatalparameters than mechanical wounding or electric treatment. Bothmechanical damage and electrical stimulation activated two characteristictime constants in the gas exchange relaxation kinetics. Conversely,heat stimulation resulted in only one major time constant. Theresults clearly show that direct current application to tomatoleaves initiates Pin2 mRNA accumulation locally and systemically.In addition, they suggest the participation of a second slowelectrical/hydraulic component in the wound response mechanismof tomato plants and a possible alternative pathway regulatingheat-induced Pin2 gene expression. (Received February 13, 1995; Accepted April 14, 1995)     

The rhodium(III) trisacetonitrile complexes: a re-investigation of the synthesis of fac- and mer- [RhCl3(CH3CN)3], their characterization by 2D NMR spectroscopy and the X-ray crystal structure of mer-[RhCl3(CH3CN)3] · CH3CN

It is shown that the reaction of RhCl₃·3H₂O with acetonitrile normally produces mixtures of mer- and fac-[RhCl₃(CH₃CN)₃] (1a and 1b, respectively). The IR and ¹H NMR spectra of these isomers were re-investigated. Their two-dimensional (¹⁰³Rh,¹H) NMR spectra were also recorded. Equilibrium and exchange studies of 1a and 1b in CD₃C were performed. It was found that in 1a the exchange rate of the nitrile molecule trans to Cl is much faster than those of mutually trans nitriles. Also the nitrile molecules in 1b underwent fast exchange in CD₃CN; however, their rate was slightly faster than that of the more labile CH₃CN in 1a. The X-ray crystal structure of mer-[RhCl₃(CH₃CN)₃]·CH₃CN (1c) was determined. Crystal data: triclinic space group

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Distribution and quantification of alpha1-integrin subunit in rat organs

Summary The ₁₁-integrin is known to be a receptor for collagen and laminin mediating cell-matrix interactions. A monoclonal antibody, 33.4, which specifically inhibits the ₁-integrin-mediated in vitro cell-collagen binding of rat hepatocytes and hepatoma-derived A-cells (Löster et al., 1994), was used to purify by immunoaffinity chromatography the ₁-integrin subunit from rat liver in large quantities for inducing a polyclonal antiserum. In immunoblot analysis on membrane extracts of several rat organs this polyclonal antiserum recognized only a 190 kDa-band, suggesting that it is highly specific for the ₁-integrin subunit. A sandwich-ELISA with monoclonal antibody 33.4 and the polyclonal antiserum against the ₁-integrin subunit, respectively, enabled the quantitative expression pattern of the ₁-integrin subunit to be studied in different rat organs. With the exceptions of brain (not detectable) and muscle (low concentration), the ₁-integrin subunit was detectable in almost all organs of the digestive, respiratory and urogenital system as well as in lymphatic organs. The highest relative concentrations of ₁-integrin subunit were found in uterus, lung and spleen, whereas in seminal vesicle, stomach, parotid gland, epididymis, kidney and liver only modest concentrations were evident. The organ distribution and localization of ₁-integrin subunit were studied by immunohistochemistry with monoclonal and polyclonal antibodies. Immunoreactivity was present in the plasma membranes of all smooth muscle cells, vascular endothelial cells of many organs and fibrocyte-fibroblast sheaths in the heart and kidney. Since these cells are in close contact with collagen-containing basal membranes as well as reticular fibrils, strong evidence exists that in rat tissues the ₁-integrin subunit is expressed at sites where collagen is present and might be involved in vivo in cell—ollagen binding.Dedicated to Professor Peter Sitte on the occasion of his 65th birthday