In vivo crystal formation in Escherichia coli of an over-expressed soluble form of penicillin-binding protein 5

Accumulation of either native membrane-bound or soluble variants of PBP5 over-expressed in the cytoplasm was investigated by electron microscopy of ultra-thin sections. One of the soluble forms of PBP5 (PBP5s353) formed well-ordered crystals inside the cells. Cells sectioned perpendicular to their long axis showed a diamond-shaped crystal whereas cells cut parallel to their long axis contained a long, narrow crystal. In both sectioning directions an ordered ultrastructure was visible as shown by optical diffraction. Computer processing was used to enhance the crystal images. From this the unit cell parameters were calculated as a = 7.6 nm, b = 4 nm, c = 4.2 nm, gamma = 75 degrees. The calculated unit-cell volume of 120 nm3 is large enough to contain one protein molecule.     

Enhanced Ig production by human peripheral lymphocytes induced by aggregated C1q

Because B cells express receptors for C1q, we have investigated the role of C1q in the stimulation of B cells. When B cells were cultured in the presence of C1q that had been frozen, T cells, and suboptimal concentrations of PWM, there was a dose-dependent enhancement of IgM, IgG, and IgA by the B cells. No significant enhancement of Ig production by B cells was seen in the absence of T cells or PWM. The contribution of T cells or PWM could be replaced by supernatants of PMA and Con A-activated PBMC (T cell growth factor). C1q that had been frozen, in contrast with freshly isolated C1q, was at least 3 times more active in enhancement of the production of Ig by B cells in culture in the presence of suboptimal concentrations of T cell growth factor. The capability of C1q to stimulate B cells could be ascribed to aggregates of C1q. Monomeric C1q was only marginally active to stimulate B cell Ig production, whereas dimeric and tetrameric C1q were able to enhance Ig production by B cells in relation to their size. Furthermore, aggregation of C1q on soluble aggregates of rabbit IgM also increased its potential to enhance B cell Ig production. The interaction of C1q with the B cells occurs via the collagenous tail of C1q, as suggested by inhibition experiments with purified collagenous tails and globular heads of C1q. These results indicate that triggering of C1qR on B cells positively regulates Ig production in vitro.     

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD^−/− mice. LCAD^−/− mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD^−/− mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD^−/− surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD^−/− lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.     

A biomechanical mathematical model for the collagen bundle distribution-dependent contraction and subsequent retraction of healing dermal wounds

A continuum hypothesis-based, biomechanical model is presented for the simulation of the collagen bundle distribution-dependent contraction and subsequent retraction of healing dermal wounds that cover a large surface area. Since wound contraction mainly takes place in the dermal layer of the skin, solely a portion of this layer is included explicitly into the model. This portion of dermal layer is modeled as a heterogeneous, orthotropic continuous solid with bulk mechanical properties that are locally dependent on both the local concentration and the local geometrical arrangement of the collagen bundles. With respect to the dynamic regulation of the geometrical arrangement of the collagen bundles, it is assumed that a portion of the collagen molecules are deposited and reoriented in the direction of movement of (myo)fibroblasts. The remainder of the newly secreted collagen molecules are deposited by ratio in the direction of the present collagen bundles. Simulation results show that the distribution of the collagen bundles influences the evolution over time of both the shape of the wounded area and the degree of overall contraction of the wounded area. Interestingly, these effects are solely a consequence of alterations in the initial overall distribution of the collagen bundles, and not a consequence of alterations in the evolution over time of the different cell densities and concentrations of the modeled constituents. In accordance with experimental observations, simulation results show furthermore that ultimately the majority of the collagen molecules ends up permanently oriented toward the center of the wound and in the plane that runs parallel to the surface of the skin.

     

Functional role of phenylacetic acid from metapleural gland secretions in controlling fungal pathogens in evolutionarily derived leaf-cutting ants

Fungus-farming ant colonies vary four to five orders of magnitude in size. They employ compounds from actinomycete bacteria and exocrine glands as antimicrobial agents. Atta colonies have millions of ants and are particularly relevant for understanding hygienic strategies as they have abandoned their ancestors'' prime dependence on antibiotic-based biological control in favour of using metapleural gland (MG) chemical secretions. Atta MGs are unique in synthesizing large quantities of phenylacetic acid (PAA), a known but little investigated antimicrobial agent. We show that particularly the smallest workers greatly reduce germination rates of Escovopsis and Metarhizium spores after actively applying PAA to experimental infection targets in garden fragments and transferring the spores to the ants'' infrabuccal cavities. In vitro assays further indicated that Escovopsis strains isolated from evolutionarily derived leaf-cutting ants are less sensitive to PAA than strains from phylogenetically more basal fungus-farming ants, consistent with the dynamics of an evolutionary arms race between virulence and control for Escovopsis, but not Metarhizium. Atta ants form larger colonies with more extreme caste differentiation relative to other attines, in societies characterized by an almost complete absence of reproductive conflicts. We hypothesize that these changes are associated with unique evolutionary innovations in chemical pest management that appear robust against selection pressure for resistance by specialized mycopathogens.     

Plasma Membrane Profiling Defines an Expanded Class of Cell Surface Proteins Selectively Targeted for Degradation by HCMV US2 in Cooperation with UL141

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2’s impact on HCMV pathogenesis.     

The epidemiology of mycobacterium leprae: Recent insight

Abstract Leprosy is still a health problem in many countries. Because the causative organism, Mycobacterium leprae cannot be cultured in vitro, it is virtually impossible to assess exposure, and the onset of infection and disease. As a consequence, the chain of infection, considered as the relationships between M. leprae , transmission and human host, is poorly understood. Here, we discuss a number of organism-, host- and environmental-related factors which may be incriminated in the dynamic process of the development of leprosy disease. The use of modern molecular and immunological tools has become a valuable addition to epidemiological research. Understanding of the epidemiology of leprosy is a prerequisite for effective control of the disease.     

Modification of Brassica napus seed oil by expression of the Escherichia coli fabH gene,encoding 3-ketoacyl-acyl carrier protein synthase III

The Escherichia coli fabH gene encoding 3-ketoacyl-acyl carrier protein synthase III (KAS III) was isolated and the effect of overproduction of bacterial KAS III was compared in both E. coli and Brassica napus. The change in fatty acid profile of E. coli was essentially the same as that reported by Tsay et al. (J Biol Chem 267 (1992) 6807–6814), namely higher C14:0 and lower C18:1 levels. In our study, however, an arrest of cell growth was also observed. This and other evidence suggests that in E. coli the accumulation of C14:0 may not be a direct effect of the KAS III overexpression, but a general metabolic consequence of the arrest of cell division. Bacterial KAS III was expressed in a seed- and developmentally specific manner in B. napus in either cytoplasm or plastid. Significant increases in KAS III activities were observed in both these transformation groups, up to 3.7 times the endogenous KAS III activity in mature seeds. Only the expression of the plastid-targeted KAS III gene, however, affected the fatty acid profile of the storage lipids, such that decreased amounts of C18:1 and increased amounts of C18:2 and C18:3 were observed as compared to control plants. Such changes in fatty acid composition reflect changes in the regulation and control of fatty acid biosynthesis. We propose that fatty acid biosynthesis is not controlled by one rate-limiting enzyme, such as acetyl-CoA carboxylase, but rather is shared by a number of component enzymes of the fatty acid biosynthetic machinery.