A comprehensive study on the role of the Yersinia pestis virulence markers in an animal model of pneumonic plague.

We determined the role of Yersinia pestis virulence markers in an animal model of pneumonic plague. Eleven strains of Y. pestis were characterized using PCR assays to detect the presence of known virulence genes both encoded by the three plasmids as well as chromosomal markers. The virulence of all Y. pestis strains was compared in a mouse model for pneumonic plague. The presence of all known virulence genes correlated completely with virulence in the Balb/c mouse model. Strains which lacked HmsF initially exhibited visible signs of disease whereas all other strains (except wild-type strains) did not exhibit any disease signs. Forty-eight hours post-infection, mice which had received HmsF(-) strains regained body mass and were able to control infection; those infected with strains possessing a full complement of virulence genes suffered from fatal disease. The bacterial loads observed in the lung and other tissues reflected the observed clinical signs as did the cytokine changes measured in these animals. We can conclude that all known virulence genes are required for the establishment of pneumonic plague in mammalian animal models, the role of HmsF being of particular importance in disease progression.

Impact velocities of the teeth after a sudden unloading at various initial bite forces, degrees of mouth opening, and distances of travel.

A potentially dangerous situation arises when an individual bites on hard and brittle food which suddenly breaks, since the impact velocity of the lower teeth onto the upper teeth after the food is broken can be high and may cause dental damage. The present experiments were designed to study the magnitude of the impact velocity after a sudden unloading at various initial bite forces, degrees of mouth opening, and distances of travel. Subjects were asked to perform a static biting task during which the resistance to the bite was suddenly removed. The upward mandible movement was arrested after a certain distance. The velocity of the lower teeth at impact was calculated just before the mandible came to a standstill in combinations of 4 different bite forces (100, 80, 60, and 40 N), 4 different initial degrees of mouth opening (33.5, 30.5, 27.5, and 24.5 mm), and 3 different distances of travel of the mandible (4.5, 3.0, and 1.5 mm). We found that the bite force rapidly declined after the unloading, resulting in a small impact velocity of the lower front teeth. This impact velocity largely depended on the magnitude of the initial bite force and the distance traveled; it was barely sensitive to variations in degree of initial mouth opening. The maximal velocity of the lower teeth was 0.43 m/s (at an initial bite force of 100 N). This maximum was reached after a distance of travel of about 4 mm in 12 ms. The data suggest that the rapid decline in bite force coupled with a limitation of impact velocity is due to the force-velocity properties of the active jaw muscles and is not caused by neural control.

The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins.

We describe the cloning and characterisation of the BiP gene homologues of the filamentous fungi Aspergillus niger and Aspergillus awamori. The BiP genes of these black Aspergilli encode an identical protein of 672 amino acids, which has a high homology with the BiP protein from Saccharomyces cerevisiae and contains a putative signal sequence of 38 amino acids. The DNA sequences of the Aspergillus BiP genes diverge in particular in the three intronic sequences and the 5'- and 3'- noncoding regions. Sequences resembling Heat Shock Elements (HSE) and Unfolded Protein Response (UPR) elements, as found in the yeast KAR2 promoter, are present in the 5' non-transcribed regions of both genes. The expression of the A. niger bipA gene is increased by heat shock and tunicamycin treatment.

Contribution of the digastric muscles to the control of bite force in man.

The contribution of the (co-contracting) digastric muscles to the rapid decline in bite-force magnitude after unloading of a static bite was investigated by asking participants to perform two different biting tasks with sudden unloading, and correlating the degree of co-contraction of the digastrics (as derived from their electromyograms) with the impact force, the impact velocity (as measured after a travel distance of 5 mm), and the residual force when the jaw system was in static conditions again after the impact. Co-contraction of the digastrics was varied by asking participants to perform the biting task while controlling bite force (force-controlled experiments) or jaw position (position-controlled experiments). In half of the experiments, participants co-contracted their digastrics more strongly in the position-controlled than the force-controlled experiments. However, there was no clear relation between the level of co-contraction and the magnitude of the impact force, the impact velocity and the residual force. The results imply that co-contraction of the digastric muscles is not sufficient to explain the reduction in bite force and the low impact velocity after an unexpected jaw-closing movement. Two other possible mechanisms that reduce forces in an unloaded jaw system are: (1) force velocity properties of the activated jaw muscles in conjunction with creep of the aponeurotic sheets of the jaw muscles, resulting in a slow partial recovery of the biting force after impact: (2) force length properties of jaw-opening muscles, an activity not recorded here.

Influence of visual feedback on human isometric bite-force tremor.

In contrast to recent reports, during an isometric short forceful bite, visual feedback had a significant influence on the force tremor spectrum. The value of a 'half-value frequency', being the frequency f1/2 at which, with increasing frequency, the amplitude of the spectrum for the first time drops to half its initial value, was used as an indicator for the spectral behavior. Under visual feedback, the amplitude contribution to the force spectrum in the 3-5 Hz frequency range was much larger than after deprivation of visual feedback. Elevations in the frequency range between 3 and 5 Hz in the force spectrum are interpreted as an expression of a visual feedback loop with a tau between 100 and 200 ms. This is supported by the visual/oral reaction times recorded, which were between 110 and 190 ms.

Secretion of heterologous proteins by Aspergillus niger: production of active human interleukin-6 in a protease-deficient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein.

To develop improved methods for heterologous protein production in Aspergillus niger, we studied the secretion of human interleukin-6 (hIL6). Since in vitro experiments with culture medium revealed that hIL6 was rapidly degraded, several protease-deficient strains of A. niger were isolated and tested for reduced degradation of hIL6 compared with the wild-type strain. The mutant strain giving the least degradative effect on hIL6 (designated AB1.13) was transformed with several hIL6-expression plasmids. Initially, hIL6 was expressed using various signal sequences fused to the sequence of mature hIL6. The resulting transformants did not produce detectable amounts of hIL6, despite high transcription levels in one transformant. We hypothesized that hIL6 was not efficiently processed during passage along the secretion pathway. Therefore, hIL6 was expressed as a fusion protein with glucoamylase, a protein which is efficiently secreted by A. niger and expression of which can easily be measured enzymatically. To obtain mature hIL6, a sequence encoding the KEX2 cleavage-site (Lys-Arg) was inserted between glucoamylase and hIL6 sequences. Mature active hIL6 was found to be secreted in the extracellular medium. Using this combined approach of transforming a protease-deficient strain with a fusion construct containing the KEX2 site, up to 15 mg l-1 active hIL6 was obtained in shake-flask culture. A fusion construct without the KEX2 site resulted in substantially higher production of the fusion protein, but hIL6 was not active in the fused form. These results indicate that A. niger contains a protease with similar specificity as the KEX2 protease from yeast.

An electromyographic study of whether the digastric muscles are controlled by jaw-closing proprioceptors in man.

Whether in the oral system the digastric muscles (which lack muscle spindles) are under the control of proprioceptive information from the masseter muscles (which contain muscle spindles) was investigated by analysing whether and how the masseters and digastrics showed coordinated behaviour during a static, forceful bite. Subjects were asked to maintain a 100-N force for 15 s with and without visual guidance; bite force exerted, and masseter and digastric electromyograms (EMGs) were recorded. Under visual guidance all subjects co-contracted their digastric muscles during the isometric bite. They held the force for a short time, followed by periods with fluctuations (peak-to-peak force amplitude about 15-20 N). Fluctuations in bite force correlated with the masseter EMGs, the maximum in the correlogram occurring at about -50 ms with the force lagging the masseter. In 75% of the subjects a significant periodic component in the masseter and in the force spectra was found at about 4 Hz. This was also seen in the amplitude spectra of the forces, which showed in 80% of the subjects a significant elevation between 7-10 Hz as well. No correlation between the digastric EMGs and the bite forces, and between the EMGs of masseter and digastric could be detected. Spectra of digastric EMGs showed no prominent maxima. When subjects were deprived of visual feedback, maxima at -50 ms in the cross-correlation functions of the masseters and the forces were reduced considerably; periodicities of +/- 250 ms disappeared.(ABSTRACT TRUNCATED AT 250 WORDS)

Digastric muscle response as a function of knowledge of the task to be performed.

Whether the motor programme executed by the digastric muscles during a forceful bite is modified according to a subject's expectation that the resistance between the teeth will change was investigated. There were two experimental conditions: (1) tracking a ramp (drawn on an oscilloscope screen) by biting (isometrically) on a force transducer and holding it at 120 N, and (2) tracking the same ramp with a sudden unloading at 100 N. There were two groups of experiments: (1) control experiments in which subjects underwent a sudden and unexpected unloading of the jaw, and (2) experiments in which subjects were previously informed whether or not there was to be an unloading. In all experiments the subjects co-contracted their digastric muscles during the bite as compared to the state at rest. The subjects' responses fell into the three different types: (i) those who varied the level of tonic digastric activity only as a function of the experimental condition, (ii) those who co-contracted the digastric muscles at the same time as the masseter muscles, and (iii) those who changed the contraction pattern of the digastric muscles as a function of the experimental condition. If modulation of the digastric muscles occurred this is a 'feedforward' strategy mainly based on immediate past performance.

Bite-force endurance in patients with temporomandibular joint osteoarthrosis and internal derangement.

The aim of this study was to investigate the potential clinical relevance of testing bite force endurance in patients with articular temporomandibular disorders. The endurance of a 50 N bite force was measured in 51 patients with painful temporomandibular joint disorders. The results were compared to those of a control group of 20 subjects. The force exerted was sustained until this task could not be continued because of intolerable pain or fatigue. The endurance test was repeated following therapy. Testing bite force endurance could be reliably carried out (paired t-test not significant, product-moment correlation coefficient 0.87). The mean endurance time in the patient group was significantly different from that of the control group (t = 7.43, df = 69, P < 0.01). The 95% confidence intervals for patients and controls did not show any overlap. No difference in endurance time between diagnostic subgroups could be detected (F = 1.30, df = 4,46, P < 0.28). Following treatment, all patients showed a significant increase of endurance time (t = 8.09, df = 50, P < 0.01) and reported a decrease in post-test pain. The mean difference between pre- and post-treatment endurance was 60s. Subjects of the control group stopped the biting effort predominantly because of muscle fatigue. By contrast, the main reason of the patients to cease the effort was TMJ pain. The results of this study indicate that the discriminatory power of the test is sufficient to justify its utility as a complementary tool in assessing the functional capacity of the masticatory system.

Perception of forces exerted by jaw and thumb.

By comparing the results of force matching between the jaw and the thumbs, whether subjects have any knowledge about the magnitude of exerted forces irrespective of the motor system was studied. Subjects were asked to match isometric forces of their own choice exerted by flexion of one of the thumbs with the jaw, and the other way around. The results were compared with control experiments in which subjects matched forces exerted by flexion of one of the thumbs with the other thumb and vice versa. None of the subjects was able to match correctly in all experimental conditions. All subjects displayed inconsistent matching behaviour, showing a mixture of correct matching and mismatching. This holds both for absolute matches (in N), and for matches relative to the maximal forces. The results show that knowledge about the magnitude of exerted forces is different for the jaw and the thumbs. Sensations about isometric forces exerted by the jaw or the thumbs are different within each subject and from subject to subject.

Clinical significance of bite force reproduction ability.

The objective of this study was to evaluate the clinical relevance of measurement of bite force reproduction ability. This parameter was measured at reference force levels of 2, 10, and 50 N in a group of patients with articular and nonarticular temporomandibular disorders and in a control group. The ability to reproduce the reference forces was measured at four equidistant occasions. All subjects poorly and imprecisely reproduced the reference force levels. A trend in the matches or in their imprecision could not be found (P greater than 0.05). Bite force reproduction ability did not differ between the patient group and the control group (P greater than 0.05). It was concluded that measurement of bite force reproduction ability does not provide a useful clinical assessment tool.

Psychophysical investigations into the tenderness of meat.

Tenderness of five veal meat cuts was determined by two groups of subjects: a panel of skilled butchers and a consumers' panel. The butchers estimated tenderness with and without assistance of visual information. For the consumers a procedure to measure the oral sensation by a forced choice method of successive comparison was developed. It was shown that: imprecision of the opinions of the consumers was greater than that of the butchers; use of additional visual information did not affect the butchers' precision; and the butchers' views of tenderness had a poor relationship to the consumers' oral perception of this quality.

A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae.

A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. oryzae strain deficient in orotidine-5'-phosphate decarboxylase (pyrG) and the vector pAO4-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular pAO4-2 resulted in the appearance of Pyr+ transformants at a frequency of up to 20 per micrograms of DNA, whereas with linear pAO4-2 up to 200 transformants per micrograms DNA were obtained. In 75% of the Pyr+ transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pAO4-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding beta-galactosidase, and uidA, encoding beta-glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.

Fusion proteins with multiple copies of the major antigenic determinant of foot-and-mouth disease virus protect both the natural host and laboratory animals.

Proteins consisting of one, two or four copies of the amino acid sequence 137 to 162, which contains the major immunogenic site of VP1 of foot-and-mouth disease virus, attached to the N-terminus of beta-galactosidase have been expressed in Escherichia coli cells. In guinea-pigs the protein containing one copy (P71) of the viral determinant elicited only low levels of neutralizing antibody whereas protective levels were elicited by the proteins containing two (P72) or four (P74) copies of the determinant. Single inoculations of the P72 and P74 proteins containing as little as 2 micrograms or 0.8 micrograms of peptide respectively were sufficient to protect all the animals against challenge infection. Moreover, the equivalent of 40 micrograms of peptide in P74 protected pigs against challenge infection after one inoculation.

The control of bite-force in relation to jaw position and load in man.

Whether bite-force is controlled independently of mouth opening, and jaw position is controlled independently of the imposed load of the mandible, was examined. Subjects were asked to match, at 15 and 30 mm inter-incisor distance, reference forces of 2, 10 and 50 N exerted at 15 and 30 mm mouth opening. They were able to grade bite-force, as matches made of the different forces hardly overlapped. However, at a 5 per cent level of significance in 90 per cent of the cases, the matches were dependent on jaw position. The imprecision of the 2 N matches was about 40 per cent of the reference force; those of the 10 and 50 N matches were about 25 per cent. Thus a mechanism controlling bite-force with any degree of precision is absent in the jaw system.

A hierarchy of neural control of mastication in the rat.

In rats anaesthetized with ketamine, rhythmic jaw-opening and jaw-closing movements were induced by palatal stimulation. The two masseter muscles (jaw-closing) and the four digastric muscles (jaw-opening) were fitted with electrodes, which could be used either for electrical stimulation or for recording electromyographic responses. Electrical stimulation of the masseters in the phase when the digastrics were the contracting muscles, caused responses in the digastrics. The amplitude of these responses was dependent on whether the stimulated masseters were active or not. The responses in digastric persisted when contraction of the masseters during stimulation was prevented by dantrolene sodium but they disappeared when the masseteric nerves were blocked with xylocaine. The responses in digastric are thus reflexes from stimulating afferent fibres in the masseteric nerves. Likewise, electrical stimulation of the four digastrics in the phase when the masseters were contracting, caused responses in the masseters. The amplitude of these responses, however, was independent of the state of activity of the stimulated digastrics. Furthermore, the responses in masseter disappeared when contraction of the digastrics was prevented by dantrolene sodium; but they persisted when the digastric nerves were blocked with xylocaine, provided the digastrics continued to twitch to the electric stimuli. The responses in masseter are thus reflexes in masseter caused by mechanical stretch transmitted from the digastric twitches. In the rhythmic preparation, prevention of contraction of the masseters of digastrics by dantrolene sodium or xylocaine leaves the overall frequency and amplitude of the evoked rhythmic activity unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of fusion proteins containing antigenic determinants of foot-and-mouth disease virus.

Part of the genome of foot-and-mouth disease virus (FMDV) type 01,BFS, including the sequence encoding the capsid polypeptide VP1, was cloned in Escherichia coli following a new cloning strategy. The clone containing the VP1 sequence was used for the construction of two expression plasmids encoding VP1 fusion proteins. Subsequently, substantial amounts of the two VP1-beta-galactosidase fusion proteins, containing either one (amino acid region 140-160) or two (amino acid regions 140-160 and 200-213) antigenic determinants of the virus, were synthesized by E. coli bacteria. The protein containing the amino acid region 140-160 of VP1 fused to beta-galactosidase efficiently induced antibodies in rabbits specifically reacting with FMDV type 01,BFS. The same protein was also capable of eliciting neutralizing antibodies. The fusion protein containing both antigenic determinants did not efficiently induce antibodies reacting with FMDV.

Synthesis of fusion proteins with multiple copies of an antigenic determinant of foot-and-mouth disease virus.

A series of four expression plasmids coding for fusion proteins containing foot-and-mouth disease virus (FMDV) sequences was constructed. The fusion proteins contain a large part of beta-galactosidase from Escherichia coli preceded (N-terminal) by 1, 2, 4 or 8 repeats of the antigenic determinant of FMDV consisting of amino acids 137-162 of the capsid polypeptide VP1. All four fusion proteins were efficiently produced in E. coli host bacteria. Immunization of rabbits resulted in FMDV-specific, neutralizing antibodies, the response being dependent on the number of repeats. With enzyme-linked immunosorbent-assay techniques it was shown that the FMDV antigenic determinants are exposed on the surface of the fusion proteins under non-denaturing conditions.