Cryptic plasmid and rifampin resistance in Rhizobium meliloti influencing nodulation competitiveness.

An assessment was made of the relative contributions of a spontaneous mutation to rifampin resistance and a cryptic plasmid, pTA2, to competitive nodulation of Medicago sativa by a strain of Rhizobium meliloti. This was facilitated by use of rifampin-resistant derivatives of this strain in which pTA2 was originally present, cured, or reintroduced. Both curing of pTA2 and spontaneous mutation to rifampin resistance significantly influenced nodulating competitiveness, but the effect of rifampin resistance was greater and such that the contribution of pTA2 was evident only in cases in which paired competitors had the common rifampin resistance background. The data suggest that rifampin-resistant derivatives contain an altered RNA polymerase insensitive to the action of rifampin. All R. meliloti derivatives had symbiotic characteristics and phage susceptibility patterns similar to those of the wild type. Plasmid pTA2 transfer or other genetic interchange was not detected in nodules of M. sativa inoculated with paired competitors.     

Structural investigation of inhibitor designs targeting 3-dehydroquinate dehydratase from the shikimate pathway of Mycobacterium tuberculosis

The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form π-stacking interactions with the catalytic Tyr24 have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19-24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.     

Uptake kinetics of 99Tc in common duckweed

The uptake of the nuclear waste product technetium-99 was studied in common duckweed (Lemna minor). In addition to measurements, a model involving two compartments in duckweed with different chemical forms of technetium was derived. The model was tested by chemical speciation, i.e. differentiating between reduced Tc-compounds and Tc(VII)O(4)(-). The TcO(4)(-) concentrations measured were in good agreement with those predicted by the model. Two processes determine technetium uptake: (1) transport of Tc(VII)O(4)(-) across the cell membrane, and (2) reduction of Tc(VII). The TcO(4)(-) concentration in duckweed reaches a steady state within 2 h while reduced Tc-compounds are stored, as a result of absence of release or re-oxidation processes. Bioaccumulation kinetic properties were derived by varying 99Tc concentration, temperature, nutrient concentrations, and light intensity. The reduction of technetium in duckweed was highly correlated with light intensity and temperature. At 25 degrees C the maximum reduction rate was observed at light intensities above 200 μmol m(-2) s(-1) while half of the maximum transformation rate was reached at 41 μmol m(-2) s(-1). Transport of TcO(4)(-) over the cell membrane requires about 9.4 kJ mol(-1), indicating an active transport mechanism. However, this mechanism behaved as first-order kinetics instead of Michaelis-Menten kinetics between 1x10(-14) and 2.5x10(-5) mol l(-1) TcO(4)(-). Tc uptake could not be inhibited by 10(-3) mol l(-1) nitrate, phosphate, sulphate or chloride.     

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Background  

The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes.     

Heterogeneous binding of high mobility group chromosomal proteins to nuclei

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.     

Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells

Introduction

The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.

Methods

Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.

Results

Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.

Conclusions

Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.     

Atmospheric change causes declines in woodland arthropods and impacts specific trophic groups

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Molecular evolution of rodent insulins

Several trees of amino acid sequences of rodent insulins were derived with the maximum-parsimony procedure. Possible orthologous and paralogous relationships were investigated. Except for a recent gene duplication in the ancestor of rat and mouse, there are no strong arguments for other paralogous relationships. Therefore, a tree in agreement with other biological data is the most reasonable one. According to this tree, the capacity to form zinc-binding hexamers was lost once in the ancestor of the hystricomorph rodents, followed by moderately increased evolutionary rates in the lineages to African porcupine and chinchilla but highly increased rates in at least three independent lines to other taxa of this suborder: guinea pig, cuis, and Octodontoidea (coypu and casiragua).