Development of the mouse mandibles and clavicles in the absence of skeletal myogenesis.

In this report we employed double-knock-out mouse embryos and fetuses (designated as Myf5-/-: MyoD-/- that completely lacked striated musculature to study bone development in the absence of mechanical stimuli from the musculature and to distinguish between the effects that static loading and weight-bearing exhibit on embryonic development of skeletal system. We concentrated on development of the mandibles (= dentary) and clavicles because their formation is characterized by intramembranous and endochondral ossification via formation of secondary cartilage that is dependent on mechanical stimuli from the adjacent musculature. We employed morphometry and morphology at different embryonic stages and compared bone development in double-mutant and control embryos and fetuses. Our findings can be summarized as follows: a) the examined mutant bones had significantly altered shape and size that we described morphometrically, b) the effects of muscle absence varied depending on the bone (clavicles being more dependent than mandibles) and even within the same bone (e.g., the mandible), and c) we further supported the notion that, from the evolutionary point of view, mammalian clavicles arise under different influences from those that initiate the furcula (wishbone) in birds. Together, our data show that the development of secondary cartilage, and in turn the development of the final shape and size of the bones, is strongly influenced by mechanical cues from the skeletal musculature.

Effects of clofazimine on potassium uptake by a Trk-deletion mutant of Mycobacterium tuberculosis.

OBJECTIVES: This study was designed to investigate the effects of the membrane-active, anti-mycobacterial agent, clofazimine, on potassium (K+)-uptake by a mutant of Mycobacterium tuberculosis (MTB), in which the Trk system, the major K+ transporter of this microbial pathogen, had been selectively inactivated. METHODS: The ceoB and ceoC genes of MTB, which encode the TrkA proteins, CeoB and CeoC, were deleted by homologous recombination, and the double-knockout mutant and wild-type strains compared with respect to K+ uptake and growth in the presence and absence of clofazimine (0.015-2.5 mg/L) using radioassay procedures. RESULTS: Surprisingly, the magnitudes of K+ uptake and rate of growth of the ceoBC-knockout mutant were significantly (P < 0.05) greater than those of the wild-type strain, due, presumably, to induction of a back-up transporter. Exposure of both the wild-type strain and ceoBC-knockout mutant of MTB to clofazimine was accompanied by dose-related decreases in K+ uptake, as well as growth, which were of comparable magnitude for both strains. CONCLUSIONS: These observations demonstrate that the major K+ transporter of MTB, Trk, as well as an uncharacterized inducible back-up system, is equally sensitive to the inhibitory actions of clofazimine.

Global expression profiling of strains harbouring null mutations reveals that the five rpf-like genes of Mycobacterium tuberculosis show functional redundancy.

SETTING: Aged, dormant cultures of Mycobacterium tuberculosis can be resuscitated by a secreted, proteinaceous growth factor from Micrococcus luteus, known as resuscitation-promoting factor (Rpf). M. tuberculosis contains five rpf-like genes, rpfA (Rv0867c), rpfB (Rv1009), rpfC (Rv1884c), rpfD (Rv2389c) and rpfE (Rv2450c), that bear significant similarity to Mi. luteus rpf, suggesting that these too may play a role in growth and/or reactivation from a quiescent state. OBJECTIVE AND DESIGN: Unmarked deletion mutants of each of the five rpf-like genes of M. tuberculosis H37Rv were constructed and their phenotypes and global gene expression profiles were assessed. RESULTS AND CONCLUSIONS: Deletion of any one of the rpf-like genes did not affect growth or survival of M. tuberculosis in liquid culture, but some alterations in colony-forming ability and colonial morphology were observed. Global gene expression profiling suggested that loss of rpfC affected the expression of the largest number of genes and there was a significant overlap in the differential gene expression profile of the rpfC mutant with those of the rpfB, rpfD and rpfE mutants. The expression profile of the rpfA mutant was notably less similar, but inverse associations with genes affected in the other mutants were observed. These results, together with those obtained by real-time, quantitative RT-PCR, suggest that the rpf-like genes serve wholly or partially overlapping functions in M. tuberculosis.

A genome screen of systemic lupus erythematosus using affected-relative-pair linkage analysis with covariates demonstrates genetic heterogeneity.

Systemic lupus erythematosus (SLE) appears to be the consequence of complex genetics and of only partly understood environmental contributions. Previous work by ourselves and by others has established genetic effects on 1q, 2q, 4p, 6p, and 16p using SLE as the phenotype. However, individual SLE affecteds are extraordinarily different from one another by clinical and laboratory measures. This variation may have a genetic basis; if so, it is advantageous to incorporate measures of between-family clinical variability as covariates in a genetic linkage analysis of affected relative pairs (ARPs) to allow for locus heterogeneity. This approach was applied to genome scan marker data from 160 pedigrees multiplex for SLE and containing 202 ARPs. Because the number of potential covariates was large, we used both ad hoc methods and formal principal components analysis to construct four composite covariates using the SLE classification criteria plus age of onset, ethnicity, and sex. Linkage analysis without covariates has detected evidence for linkage at 1q22-24, 2q37, 4p16, 12p12-11, and 17p13. Linkage analysis with these covariates uncovered linkage at 13p11, 17q11-25, and 20q12 and greatly improved evidence for linkage at 1q22-24, 2q37, 12p12-11, and 17p13. Follow-up analysis identified the original variables contributing to locus heterogeneity in each of these locations. In conclusion, allowing for locus heterogeneity through the incorporation of covariates in linkage analysis is a useful way to dissect the genetic contributions to SLE and uncover new genetic effects.

Biocontrol of the sugarcane borer Eldana saccharina by expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA genes in sugarcane-associated bacteria.

The cry1Ac7 gene of Bacillus thuringiensis strain 234, showing activity against the sugarcane borer Eldana saccharina, was cloned under the control of the tac promoter. The fusion was introduced into the broad-host-range plasmid pKT240 and the integration vector pJFF350 and without the tac promoter into the broad-host-range plasmids pML122 and pKmM0. These plasmids were introduced into a Pseudomonas fluorescens strain isolated from the phylloplane of sugarcane and the endophytic bacterium Herbaspirillum seropedicae found in sugarcane. The ptac-cry1Ac7 construct was introduced into the chromosome of P. fluorescens using the integration vector pJFF350 carrying the artificial interposon Omegon-Km. Western blot analysis showed that the expression levels of the integrated cry1Ac7 gene were much higher under the control of the tac promoter than under the control of its endogenous promoter. It was also determined that multicopy expression in P. fluorescens and H. seropedicae of ptac-cry1Ac7 carried on pKT240 caused plasmid instability with no detectable protein expression. In H. seropedicae, more Cry1Ac7 toxin was produced when the gene was cloned under the control of the Nm(r) promoter on pML122 than in the opposite orientation and bioassays showed that the former resulted in higher mortality of E. saccharina larvae than the latter. P. fluorescens 14::ptac-tox resulted in higher mortality of larvae than did P. fluorescens 14::tox. An increased toxic effect was observed when P. fluorescens 14::ptac-tox was combined with P. fluorescens carrying the Serratia marcescens chitinase gene chiA, under the control of the tac promoter, integrated into the chromosome.

Introduction of the Serratia marcescens chiA gene into an endophytic Pseudomonas fluorescens for the biocontrol of phytopathogenic fungi.

An endophytic strain of Pseudomonas fluorescens was isolated from micropropagated apple plantlets and introduced into beans (Phaseolus vulgaris) via their root tips. It was shown to be present as an endophyte in the roots at a level of 1.2 x 10(5) CFU/g fresh weight. The gene coding for the major chitinase of Serratia marcescens, chiA, was cloned under the control of the tac promoter into the broad-host-range plasmid pKT240 and the integration vector pJFF350. Pseudomonas fluorescens carrying tacchiA either on the plasmid or integrated into the chromosome is an effective biocontrol agent of the phytopathogenic fungus Rhizoctonia solani on bean seedlings under plant growth chamber conditions.

Disc lesions and the mechanics of the intervertebral joint complex.

STUDY DESIGN: Correlations between tears in the disc and the mechanics of both the intervertebral joint and vertebral body bone were analyzed. OBJECTIVES: To examine the effect of disc degeneration on the mechanics of spinal motion segments. SUMMARY OF BACKGROUND DATA: Degeneration of the intervertebral disc results in changes to the mechanics of the spine. The actual effect of tear type and size on the mechanics of the intervertebral joint is unknown. METHODS: Thirty spinal specimens (median age, 68 years) were divided into T12-L1, L2-L3, and L4-L5 motion segments. Mechanical tests recorded stiffness in flexion, extension, and torsion. Disc morphology was ascertained by taking three transverse sections of the disc and mapping and measuring the concentric tears, radial tears, and rim lesions. The severity of each tear type within each disc then was quantified. Bone cubes from the adjacent vertebral bodies were tested in compression to determine the elastic moduli and tested to failure in the longitudinal direction. RESULTS: Groups with tears were older and had reduced bone elastic moduli than groups without tears. Extension stiffness for the intact joint tended to increase with increasing tear severity. A decrease in torsional stiffness was present with increased severity of rim lesions at both L2-L3 and L4-L5. CONCLUSIONS: Tears in the intervertebral disc are reflected in a reduction in vertebral bone elastic modulus and in changes in the mechanics of the intervertebral joints in flexion, extension, and torsion.

Staphylococcus aureus nuclease is a useful secretion reporter for mycobacteria.

A secretion reporter system based on Staphylococcus aureus nuclease (nuc) was developed for use in mycobacteria. Fusion of secretion signals to the reporter cloned in a shuttle vector, pBPnuc1, resulted in halo formation around colonies of Mycobacterium smegmatis and Mycobacterium tuberculosis grown on DNase agar plates containing Methyl Green indicator dye. This in-situ detection system was used to identify secreted proteins by screening a pBPnuc1::H37Rv nuc gene fusion library in M. smegmatis. The clones identified in this screen all formed colony halos when present in M. tuberculosis grown on indicator media. The proteins corresponded to DesA2, a stearoyl-acyl carrier protein desaturase, PepA, a putative serine protease and the Apa antigen, which is the ATP-binding subunit of an ABC transport system. Of these proteins, only PepA and Apa contained recognizable leader peptides.

Failure strengths of suture versus biodegradable arrow for meniscal repair: an in vitro study.

Advances in our understanding of meniscal function and consequences of menisectomy have spawned meniscal repair techniques that yield success rates approaching 90% in properly selected patients. Biodegradable implants have been fashioned for meniscal fixation to simplify the technique and minimize neurovascular complications. We performed the current study to determine the in vitro biomechanical behavior of the BIOFIX Meniscal Arrow, a polylactic acid tack developed for meniscal repair. Eight pairs of menisci were harvested from cadaveric knees kept frozen before testing. Peripheral vertical tears were created in the posterior horn of all menisci, and each was subsequently repaired using a vertical loop suture of 2-0 Ethibond and a Meniscal arrow. Ultimate load to failure of each method was determined on a Hounsfield H25KM Universal Testing machine. The mean failure load for the suture group was 58.3 N compared with the Arrow group mean of 29.6 N (P < .001). All sutures failed by rupture at the knot but did not pull through the meniscus. All but one of the arrows failed by pulling out of the meniscus. The Arrows also permitted gapping at the repair site at considerably lesser loads than the sutures subject to strain. The concept of a biodegradable tack is appealing. Vertical loop sutures should be the standard by which their biomechanical performance is judged. We suggest modifications to the Arrow design that could enhance the fixation strength of this implant.