A versatile assay for total cellulase activity using U-[14C]-labelled bacterial cellulose

Summary A versatile and sensitive assay for total cellulase activity and not just carboxymethylcellulase activity, is described. The method is equally suitable for studying the kinetics of solubilization of cellulose by growing cells or isolated enzyme fractions.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

3-Phenylpropanoic Acid Improves the Affinity of Ruminococcus albus for Cellulose in Continuous Culture

A continuous-culture device, adapted for use with solid substrates, was used to evaluate the effects of 3-phenylpropanoic acid (PPA) upon the ability of the South African strain Ruminococcus albus Ce63 to ferment cellulose. Steady states of fermentation were established with a dilution rate of 0.17 h⁻¹, and the extent and volumetric rates of cellulose fermentation were determined over four consecutive days. When the growth medium contained no additions (control), 25 μM phenylacetate alone, 25 μM PPA alone, or 25 μM each of phenylacetate and PPA, the extent of cellulose hydrolysis was determined to be 41.1, 35.7, 90.2, and 86.9%, respectively, and the volumetric rate of cellulose hydrolysis was 103.0, 97.9, 215.5, and 230.4 mg liter⁻¹ h⁻¹, respectively. To evaluate the effect of PPA availability on affinity for cellulose, the values for dilution rate and extent of cellulose hydrolysis were used in combination with values for maximum specific growth rate determined from previous studies of growth rates and kinetics of cellulose hydrolysis. The findings support the contention that PPA maintains a competitive advantage for R. albus when grown in a dynamic, fiber-rich environment.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

H5N1 whole-virus vaccine induces neutralizing antibodies in humans which are protective in a mouse passive transfer model

Background

Vero cell culture-derived whole-virus H5N1 vaccines have been extensively tested in clinical trials and consistently demonstrated to be safe and immunogenic; however, clinical efficacy is difficult to evaluate in the absence of wide-spread human disease. A lethal mouse model has been utilized which allows investigation of the protective efficacy of active vaccination or passive transfer of vaccine induced sera following lethal H5N1 challenge.

Methods

We used passive transfer of immune sera to investigate antibody-mediated protection elicited by a Vero cell-derived, non-adjuvanted inactivated whole-virus H5N1 vaccine. Mice were injected intravenously with H5N1 vaccine-induced rodent or human immune sera and subsequently challenged with a lethal dose of wild-type H5N1 virus.

Results

Passive transfer of H5N1 vaccine-induced mouse, guinea pig and human immune sera provided dose-dependent protection of recipient mice against lethal challenge with wild-type H5N1 virus. Protective dose fifty values for serum H5N1 neutralizing antibody titers were calculated to be ≤1∶11 for all immune sera, independently of source species.

Conclusions

These data underpin the confidence that the Vero cell culture-derived, whole-virus H5N1 vaccine will be effective in a pandemic situation and support the use of neutralizing serum antibody titers as a correlate of protection for H5N1 vaccines.     

Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model

In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)₃-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines.     

Modelling, simulation and optimisation of a human vertical jump.

This paper describes an efficient biomechanical model of the human lower limb with the aim of simulating a real human jump movement consisting of an upword propulsion, a flying and a landing phase. A multiphase optimal control technique is used to solve the muscle force sharing problem. To understand how intermuscular control coordinates limb muscle excitations, the human body is reduced to a single lower limb consisting of three rigid bodies. The biomechanical system is activated by nine muscle-tendon actuators representing the basic properties of muscles during force generation. For the calculation of the minimal muscle excitations of the jump movement, the trajectory of the hip joint is given as a rheonomic constraint and the contact forces (ground reaction forces) are determined by force plates. Based on the designed musculoskeletal model and on the differential equations of the multibody system, muscle excitations and muscle forces necessary for a vertical jump movement are calculated. The validity of the system is assessed comparing the calculated muscle excitations with the registered surface electromyogramm (EMG) of the muscles. The achieved results indicate a close relationship between the predicted and the measured parameters.