Bacillus licheniformis as an object for the propagaton of heterologous genetic material from bacilli

Bacillus licheniformis was transformed with plasmids pUB110 and pJJ10 (pUB110 - pBR322) isolated from Bac. subtilis and Escherichia coli, respectively. It was revealed that the structure and genetic properties of the plasmids did not change during the transformation process. pJJ101 (pJJ10-rib) DNA isolated from E. coli and containing helper pJJ10 plasmid was used, as a recipient. It was shown that pJJ101 rib markers were "rescued" by the resident plasmid during transformation of Bac. licheniformis (pJJ10). Plasmid pLP1 containing ribB, ribD, Kmr genes and the pUB110 replicator, was isolated from the transformants. pLP1 plasmid might be considered as a detected derivative of the parental pJJ101 plasmid. The deletion is presented by 3,9 MD segment that contains the pBR322 replicator. pLP1 DNA is capable of transforming plasmidless strains of Bac. licheniformis and Bac. subtilis.     

In vitro analysis of kinematics and elastostatics of the human rib cage during thoracic spinal movement for the validation of numerical models

Neither kinematic nor stiffness properties of the rib cage during thoracic spinal motion were investigated in previous studies, while being essential for the accurate validation of numerical models of the whole thorax. The aim of this in vitro study therefore was to quantify the kinematics and elastostatics of the human rib cage under defined boundary conditions. Eight fresh frozen human thoracic spine specimens (C7-L1, median age 55 years, ranging from 40 to 60 years) including entire rib cages were loaded quasi-statically in flexion/extension, lateral bending, and axial rotation using pure moments of 5 Nm. Relative motions of ribs, thoracic vertebrae, and sternal structures as well as strains on the ribs were measured using optical motion tracking of 150 reflective markers per specimen, while specimens were loaded displacement-controlled with a constant rate of 1°/s for 3.5 cycles. The third full cycle was used to determine relative angles and strains at full loading of the spine for all motion directions. Largest relative angles were found in the main loading directions with only small motions at the mid-thoracic levels. Highest strains of the intercostal spaces were detected in the anterior section of the lowest fourth of the rib cage, showing compressions and elongations of more than 10% in all spinal motion planes. Elastostatic rib deformation was generally less than 1%. Rib-sternum relative motions exhibited complex motion patterns, overall showing relative angles below 2°. The results indicate that rib cage structures are not macroscopically deformed during spinal motion, but exhibit characteristic reproducible kinematics patterns.     

Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81

The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied. The mutants were isolated from a genetic line of Pichia guilliermondii. Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium. An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium. The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class. The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency. Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81. Thus, riboflavin biosynthesis in P. guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81. The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes. The hybrids of rib81 mutants with natural nonflavinogenic strain P. guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium. Apparently, the strain NF1453-1 contains an unaltered gene RIB81.     

Synergistic effect of rib80 and hit mutations on riboflavin biosynthesis and iron transport in the yeast Pichia guilliermondii

Mutant strains of the yeast Pichia guilliermondii, carrying both rib80 and hit mutations in a haploid genome, were derived from previously obtained strains with defective rib80 or hit genes, exerting negative control of the riboflavin biosynthesis and iron transport in Pichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of the rib80 and hit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of 55Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

枯草芽孢杆菌核黄素操纵子与ribC基因的修饰与遗传效应

[目的]研究核黄素操纵子(rib)组成型高表达,以及ribC基因低水平表达对枯草芽孢杆菌过量合成核黄素的影响.[方法]在染色体原位修饰启动子,用mRNA稳定子替换mRNA前导区,使rib操纵子组成型高表达;修饰ribC基因的启动子,降低ribC基因的表达水平.采用qRT-PCR方法,表征基因的相对表达水平;通过摇瓶发酵,测定重组菌的生物量和核黄素产量,表征相关基因修饰所表现的遗传效应.[结果]用gsiB mRNA稳定子替换核黄素操纵子的mRNA前导区,使其相对表达水平提高了约1 500倍.ribC基因启动子-35区的首个碱基由“T”突变为“C”,使ribC基因的表达水平下降了97％以上.得到的重组菌株LX34在补加蔗糖20 g/L的LB培养基上摇瓶发酵36 h,可积累核黄素2.1 g/L,同时生物量没有明显下降.[结论]使用gsiB mRNA稳定子,能够有效地提高目标基因或操纵子的表达水平;启动子-35区首个碱基的点突变,能够有效降低ribC基因的表达水平;rib操纵子过量表达和ribC基因低水平表达,使细胞能够过量合成并积累核黄素.     

Coupling between rib cage and abdominal compartments of the relaxed chest wall

The volume displacements of the rib cage and abdomen of relaxed seated subjects were measured as functions of pleural pressure with the chest wall expanded by airway pressure and with the chest wall distorted by an external force applied to the rib cage. From the measured displacements for the two independent loads, the three compliances that describe the mechanical properties of the relaxed chest wall modeled as a linear elastic system with two degrees of freedom were obtained. The cross compliance that describes the coupling between the rib cage and abdomen was found to be small and positive, 0.01-0.02 1/cmH2O. The displacement of the rib cage by the external force was consistent with the displacement predicted by use of standard methods for calculating the mechanical advantage of the force.     

Vertebral and rib anatomy in Caperea marginata: Implications for evolutionary patterning of the mammalian vertebral column

The pygmy right whale, Caperea marginata, is a rare mysticete cetacean with an unusual suite of axial skeletal characters. Distally expanded first ribs, a long thorax with broadly overlapping vertebral transverse processes, plate‐like posterior ribs, and a short tail contrast with other cetaceans and suggest unique developmental patterning. Twenty‐four individuals of diverse ontogenetic age were available for analysis. Multiple, variable examples of incomplete rib fusion in dependent calves indicate that the first rib of adults is an ontogenetic fusion product of ribs 1 and 2. The composite rib articulates by way of its anterior (Rib1) component to the sternum and by way of its posterior (Rib2) component with thoracic vertebra 2. Thoracic vertebra 1 lacks rib articulations. When rib fusion is taken into account, the most frequent column formulas are C7T18L1Cd16–17 = 42–43 and C7T17L1Cd16–18 = 41–43. Thoracic and lumbar series are not reciprocal in count, arguing against their developmental linkage. Instead, parallel reduction in both lumbar and caudal counts supports the existence of neocete patterning in Caperea, as in all other living cetaceans. Ontogenetic vertebral column elongation is most marked in the posterior thorax, lumbos, and anterior tail. Vertebrae in these column regions are excellent predictors of total body length.     

Thixotropy of rib cage respiratory muscles in normal subjects.

In this study, we searched for signs of thixotropic behavior in human rib cage respiratory muscles. If rib cage respiratory muscles possess thixotropic properties similar to those seen in other skeletal muscles in animals and humans, we expect resting rib cage circumference would be temporarily changed after deep rib cage inflations or deflations and that these aftereffects would be particularly pronounced in trials that combine conditioning deep inflations or deflations with forceful isometric contractions of the respiratory muscles. We used induction plethysmography to obtain a continuous relative measure of rib cage circumference changes during quiet breathing in 12 healthy subjects. Rib cage position at the end of the expiratory phase (EEP) was used as an index of resting rib cage circumference. Comparisons were made between EEP values of five spontaneous breaths immediately before and after six types of conditioning maneuvers: deep inspiration (DI); deep expiration (DE); DI combined with forceful effort to inspire (FII) or expire (FEI); and DE combined with forceful effort to inspire (FIE) or expire (FEE), both with temporary airway occlusion. The aftereffects of the conditioning maneuvers on EEP values were consistent with the supposition that human respiratory muscles possess thixotropic properties. EEP values were significantly enhanced after all conditioning maneuvers involving DI, and the aftereffects were particularly pronounced in the FII and FEI trials. In contrast, EEP values were reduced after DE maneuvers. The aftereffects were statistically significant for the FEE and FIE, but not DE, trials. It is suggested that respiratory muscle thixotropy may contribute to the pulmonary hyperinflation seen in patients with chronic obstructive pulmonary disease.     

Effects of the rib cage on thoracic spine flexibility.

Besides protecting the internal organs of the thorax, the rib cage is the site of numerous muscle attachments. It also decreases the overall flexibility of the thoracic spine. This study developed finite element (FE) models of the thoracic spine with and without the rib cage, and the effects of the rib cage on thoracic spine flexibility were determined. The numerical models were validated by comparing the maximum rotation of the models for several loading cases with experimental data in the literature. After adapting the material properties for the discs and ligaments, the calculated maximum rotations differed from the measured median values by less than 1 degrees without the rib cage and by less than 2.5 degrees with it. The rib cage decreased the mean flexibility of the thoracic spine by 23% to 47%, depending on the loading plane. Assuming the ribs to be rigid beams required a corresponding reduction of ligament stiffnesses in order to achieve the same agreement of the maximum rotations with the measured median values. Interconnecting the FE thoracic spine model plus rib cage with the existing detailed FE lumbar spine model improves the simulation of force directions of muscles attached to the rib cage or thoracolumbar spine. In addition, such a model is suitable for determining the effects of lumbar spine implants on spinal balance.     

The first rib of hominoids

Homo sapiens is unique among extant hominoids in displaying a univertebral articular pattern for the first rib; that is, the head of the first rib articulates only with the body of the first thoracic vertebra. All other hominoids, indeed virtually all other mammals, display a bivertebral pattern; that is, the head of the first rib articulates with the bodies of both the seventh cervical and the first thoracic vertebrae, as well as the intervening disk. Two fossil hominid partial first ribs, A.L. 288-lax and A.L. 333-118, show that the univertebral pattern was fully established in the hominid lineage by the appearance of Australopithecus afarensis. Four hypotheses, based in functional anatomy, can be postulated for the evolution of the univertebral pattern: (1), it increases the volume (via increased length) of the neck, which could, in turn, compensate for the functional loss of the laryngeal sac systems in hominid vocalization; (2), it is a consequence of the more barrel-shaped thorax in hominids; (3), it is a consequence of functional modifications in the hominid shoulder girdle; and/or (4), it is a consequence of modifications in hominid first rib motion while breathing in an upright stance. Fossil evidence supports all but the first hypothesis, and most strongly supports the third. However, evidence for the first hypothesis does suggest that the evolution of descent of the upper respiratory system in the hominid lineage may have been permitted by the presence of the univertebral pattern, while the reverse is probably not true. Furthermore, fossil evidence for the third hypothesis shows that, by the appearance of A. afarensis, the hominid upper limb had been freed from locomotor constraints, which concomitantly confirms full adaptation to upright posture. Thus, because of their potential relationship with upright posture, the two remaining hypotheses (i.e., "thoracic shape" and "first rib movement during breathing") also have support from the fossil evidence.     

Functional characteristics of plasmids of Shigella sonnei 47 strains

The phenotypic characteristics of Shigella sonnei strain 47 containing 7 plasmids of low molecular weight and 2 plasmids 60-100 Md large have been studied. The strains of Escherichia coli containing the single plasmids or plasmid groups from Shigella sonnei have been obtained by transformation and conjugation. The comparison of phenotypes of the obtained strains has helped to find the plasmid location of the determinants for streptomycin resistance (P7), genes for colicinogenicity and colicin immunity (P5), the enzymes of host cell specificity system Sso47I (P6), Sso47II (P4), and the genes for the conjugative DNA transfer (P9). Escherichia coli strains producing individual restriction enzymes SsoI and SsoII have been isolated.     

Action of the diaphragm on the rib cage inferred from a force-balance analysis

Displacements of the rib cage are determined by the intrinsic passive properties of the rib cage, rib cage musculature, pleural and abdominal pressures, and the diaphragm. The diaphragm's mechanical actions on the rib cage are inferred from a force-balance analysis in which the diaphragm is seen to cause expansion of the rib cage by pulling cephalad at its insertions on the lower ribs (insertional component) and by raising intra-abdominal pressure, which pushes outward on the diaphragm's zone of apposition to the rib cage (appositional component). Goldman and Mead suggested that the diaphragm, acting alone, could drive both the rib cage and abdomen on their passive characteristics. The force-balance analysis shows that the diaphragm's inspiratory action on the rib cage is less than predicted by Goldman and Mead, but that in the special circumstances of their experiment (low lung volumes), the appositional component is large and the rib cage can be driven close to its passive characteristics. The force-balance analysis is consistent with recent observations by other investigations and is incompatible with the model proposed by Macklem and colleagues and with the Goldman-Mead hypothesis. Experiments on three subjects produced data consistent with the force-balance analysis, showing that the inspiratory action of the diaphragm on the rib cage is greatest at low lung volumes.     

Overexpression of genes involved in vesicular trafficking to the vacuole defends against lethal effects of oxidative damage.

Anticancer bleomycins and structurally-related analogs are oxidative agents that mimic ionizing radiation in many of their cellular effects. The current study was designed to better understand this class of radiomimetic and oxidative drugs, and how cells defend against them to become resistant. Based on some of the properties conferred by the blm5-1 mutation of Saccharomyces cerevisiae, a multi-step cloning strategy was developed to search for genes that protect cells against oxidative damage and lethal effects of bleomycin treatments. The strategy employed blm5-1 mutant strains to search for genes that rescued the drug hypersensitivities conferred by the mutation, and utilized the inability of homozygous blm5-1 mutant diploid strains to grow at elevated temperatures. This approach identified the VPS3, VPS8 and PEP7 genes that function in vesicular trafficking between the endosome and the yeast vacuole via the carboxypeptidase Y (CpY) pathway. Mutant blm5-1 strains possess several phenotypic characteristics consistent with CpY mutants, including reduced mitotic growth rates and sporulative abilities. However, blm5-1 strains were not found to be defective in the transport of CpY into the vacuole. We suggest that the ability of the VPS3, VPS8 and PEP7 genes to rescue lethal effects of oxidative damage resulted from the overexpression of these genes.     

Fracture toughness of manatee rib and bovine femur using a chevron-notched beam test

Bone is an anisotropic material with a hierarchical structure consisting of organic matrix, minerals and water. Fracture toughness (K(C)) has been shown to be a good index to assess the mechanical performance of bone. A chevron-notched (CN) beam test, a standard fracture mechanics test successfully applied to many other materials, was used to determine the transverse-direction fracture toughness in manatee rib and bovine femur cortical bone. Although human and bovine bone has been well studied, there is virtually no information on the toughness of manatee rib bone. As a biological material, manatee rib is interesting for study in that it is a highly mineralized bone. Three major advantages of the CN specimen test are: (1) it is easier to reach plane strain condition; (2) there is no fatigue-precracking needed; and (3) it is relatively easy to produce stable crack propagation before catastrophic fracture. The fracture toughness values of manatee rib and bovine femur were measured to be 4.5 +/- 0.5 MPa m(1/2) and 5.8 +/- 0.5 MPa m(1/2), respectively. Based on the microstructures shown in SEM images, two features that contributed to the greater fracture toughness of bovine femur were identified as greater osteon density and lesser porosity.     

Analysis of human chest wall motion using a two-compartment rib cage model.

We present a model of chest wall mechanics that extends the model described previously by Macklem et al. (J. Appl. Physiol. 55: 547-557, 1983) and incorporates a two-compartment rib cage. We divide the rib cage into that apposed to the lung (RCpul) and that apposed to the diaphragm (RCab). We apply this model to determine rib cage distortability, the mechanical coupling between RCpul and RCab, the contribution of the rib cage muscles to the pressure change during spontaneous inspiration (Prcm), and the insertional component of transdiaphragmatic pressure in humans. We define distortability as the relationship between distortion and transdiaphragmatic pressure (Pdi) and mechanical coupling as the relationship between rib cage distortion and the pressure acting to restore the rib cage to its relaxed configuration (Plink), as assessed during bilateral transcutaneous phrenic nerve stimulation. Prcm was calculated at end inspiration as the component of the pressure displacing RCpul not accounted for by Plink or pleural pressure. Prcm and Plink were approximately equal during quiet breathing, contributing 3.7 and 3.3 cmH2O on average during breaths associated with a change in Pdi of 3.9 cmH2O. The insertional component of Pdi was measured as the pressure acting on RCab not accounted for by the change in abdominal pressure during an inspiration without rib cage distortion and was 40 +/- 12% (SD) of total Pdi. We conclude that there is substantial resistance of the human rib cage to distortion, that, along with rib cage muscles, contributes importantly to the fall in pleural pressure over the costal surface of the lung.     

利用美国SMV株系初步鉴定中国大豆鉴别寄主的抗性基因

参照美国的大豆花叶病毒(SMV)株系鉴别系统,利用G1、G2、G3、G5和G7 5个株系对24个中国鉴别寄主进行接种鉴定,初步对中国SMV大豆鉴别寄主的抗性基因进行了推断.结果表明:'Davis'、'文丰5号'、'齐黄1号'、'齐黄10号'、'徐豆1号'、'徐豆2号'、'吉林26'(PI 612720A,B)、'铁丰25'和'诱变30'的表现型与'York'相同,可能携带相同的抗性基因Rsv1-y;'合丰25'可能携带一个新的Rsv1-n基因;'吉林26'(PI 597411A)和'1138-2'(PI 592914)可能携带Rsv3基因;'吉林26'(PI 597411B)、'早18'、'早熟18'、'吉林21'、'鲁豆4号'抗所有鉴定的SMV株系,可能携带Rsv1-h、Rsv4、Rsv1+3、Rsv1+4或Rsv3+4基因.利用SMV接种鉴定的反应型是对大豆抗性基因进行初步筛选的有效方法,本研究结果对构建中国SMV株系通用鉴别系统有一定借鉴意义.     

Investigation of virulence genes in clinical isolates of Yersinia enterocolitica

In this study, we aimed to investigate the distribution of virulence genes in clinical isolates of pathogenic Yersinia enterocolitica. Two thousand six hundred stool samples were collected from 2600 patients with diarrhea, and were tested using the culture method and real-time PCR. Then, all isolates of pathogenic Y. enterocolitica cultured from the culture method were examined for virulence genes (inv, ail, ystA, ystB, ystC, yadA, virF) by PCR and for the presence of plasmid by four phenotypic tests. As a result, 160 pathogenic strains were successfully detected by the culture method, including bio/serotype 1A/unknown (4), 1B/unknown (8), 2/O:9 (39), 2/unknown (7), 3/O:3 (22), 3/unknown (6), 4/O:3 (55), 4/unknown (10) and 5/unknown (9). The positive rate of virulence genes tested in 160 isolates was inv (100%), ail (94%), ystA (93%), ystB (7.5%), ystC (5%), yadA (89%) and virF (82%) while the phenotypic test included autoagglutination (87%), binding of crystal violet (89%), calcium-dependent growth (74%) and Congo red absorption (78%), respectively. Finally, we found that not all pathogenic Y. enterocolitica necessarily carry all traditional virulence genes in both chromosomes and plasmids to cause illness. Perhaps, some of them, lacking some traditional virulence genes, contain other unknown virulence markers that interact with each other and play an important role in the diverse pathogenesis of pathogenic Y. enterocolitica.     

Augmentation of the atrophic mandible with a vascularized rib graft

A patient with a 29-year history of denture wearing had her mandible augmented with a vascularized rib graft. The rib was contoured to fit the arch of the mandible and vascularized by means of the facial arteries to the intercostal vessels. The nutrient artery was not included in the vascularized bone graft. Cortical cancellous bone chips were packed around the rib to augment the buccal surface of the rib graft. Periosteal perfusion and vascularity of the transferred rib were well documented at 1 week, 1 month, and 6 months postoperatively by radionuclide scintigraphy. It is now 24 months after surgery and the patient began wearing a denture 2 months after the rib transfer. She underwent a split-thickness skin graft vestibuloplasty and floor-of-the-mouth lowering 12 months following the rib transfer with improvement in her denture-wearing capabilities, which has remained constant for 10 months. Vertical resorption of the graft at 3 months was 10 percent, at 6 months 15 percent, and it has stabilized at 25 percent.     

Computed tomography, histological and ultrasonic measurements of adolescent scoliotic rib hump geometrical and material properties

In Adolescent Idiopathic Scoliosis (AIS), numerical models can enhance orthopaedic or surgical treatments and provide reliable insights into the mechanism of progression. Computational methods require knowledge of relevant parameters, such as the specific geometrical or material properties of the AIS rib, about which there is currently a lack of information. The aim of our study was to determine the geometrical and material properties (Young's modulus [E] and Poisson's ratio [ν]) for AIS rib bones. Twelve ribs extracted during gibbectomy on 15 and 17 year old girls were tested using computed tomography (CT) scanner, histology and ultrasonic scanner. The mean porosity (± standard deviation (SD)) is 1.35 (±0.52)% and the mean (±SD) bone mineral density is 2188 (±19)mmHA/cc. The cortical part of the AIS rib hump is found to be thicker than physiological values in the literature. To mimic the rib hump for an AIS girl, our results suggest that ribs should be modeled as hollow circular cylinders with a 10.40 (±1.02)mm external radius and 7.56mm (±0.75) internal radius, and material properties with a mean E of 14.9GPa (±2.6) and a mean ν of 0.26 (±0.08).     

Comparative genome analysis of Campylobacter jejuni using whole genome DNA microarrays

Whole genome DNA microarrays were constructed and used to investigate genomic diversity in 18 Campylobacter jejuni strains from diverse sources. New algorithms were developed that dynamically determine the boundary between the conserved and variable genes. Seven hypervariable plasticity regions (PR) were identified in the genome (PR1 to PR7) containing 136 genes (50%) of the variable gene pool. When comparisons were made with the sequenced strain NCTC11168, the number of absent or divergent genes ranged from 2.6% (40 genes) to 10.2% (163) and in total 16.3% (269) of the genes were variable. PR1 contains genes important in the utilisation of alternative electron acceptors for respiration and may confer a selective advantage to strains in restricted oxygen environments. PR2, 3 and 7 contain many outer membrane and periplasmic proteins and hypothetical proteins of unknown function that might be linked to phenotypic variation and adaptation to different ecological niches. PR4, 5 and 6 contain genes involved in the production and modification of antigenic surface structures.