Cyanide Production by Rhizobacteria and Potential for Suppression of Weed Seedling Growth

Rhizobacteria strains were characterized for ability to synthesize hydrogen cyanide and for effects on seedling root growth of various plants. Approximately 32% of bacteria from a collection of over 2000 isolates were cyanogenic, evolving HCN from trace concentrations to >30 nmoles/mg cellular protein. Cyanogenesis was predominantly associated with pseudomonads and was enhanced when glycine was provided in the culture medium. Concentrations of HCN produced by rhizobacteria were similar to exogenous concentrations inhibiting seedling growth in bioassays, suggesting that cyanogenesis by rhizobacteria in the rhizosphere can adversely affect plant growth. Growth inhibition of lettuce and barnyardgrass by volatile metabolites of the cyanogenic rhizobacteria confirmed that HCN was the major inhibitory compound produced. Our results suggest that HCN produced in the rhizospheres of seedlings by selected rhizobacteria is a potential and environmentally compatible mechanism for biological control of weeds. Received: 13 December 2000/Accepted: 6 February 2001     

RFAC,a program for automated NMR R-factor estimation

A computer program (RFAC) has been developed, which allows the automated estimation of residual indices (R-factors) for protein NMR structures and gives a reliable measure for the quality of the structures. The R-factor calculation is based on the comparison of experimental and simulated ¹H NOESY NMR spectra. The approach comprises an automatic peak picking and a Bayesian analysis of the data, followed by an automated structure based assignment of the NOESY spectra and the calculation of the R-factor. The major difference to previously published R-factor definitions is that we take the non-assigned experimental peaks into account as well. The number and the intensities of the non-assigned signals are an important measure for the quality of an NMR structure. It turns out that for different problems optimally adapted R-factors should be used which are defined in the paper. The program allows to compute a global R-factor, different R-factors for the intra residual NOEs, the inter residual NOEs, sequential NOEs, medium range NOEs and long range NOEs. Furthermore, R-factors can be calculated for various user defined parts of the molecule or it is possible to obtain a residue-by-residue R-factor. Another possibility is to sort the R-factors according to their corresponding distances. The summary of all these different R-factors should allow the user to judge the structure in detail. The new program has been successfully tested on two medium sized proteins, the cold shock protein (TmCsp) from Termotoga maritima and the histidine containing protein (HPr) from Staphylococcus carnosus. A comparison with a previously published R-factor definition shows that our approach is more sensitive to errors in the calculated structure.     

In activated mast cells, IL-1 up-regulates the production of several Th2-related cytokines including IL-9

Mast cells can play detrimental roles in the pathophysiology and mortality observed in anaphylaxis and other Th2-dominated allergic diseases. In contrast, these cells contribute to protective host defense mechanisms against parasitic worm infections. After IgE/Ag activation, mast cells can produce multiple cytokines that may enhance allergic inflammations, while a similar panel of Th2-related cytokines may support immunological strategies against parasites. Here we report that in primary mouse bone marrow-derived mast cells activated by ionomycin or IgE/Ag, the proinflammatory mediator IL-1 (alpha or beta) up-regulated production of IL-3, IL-5, IL-6, and IL-9 as well as TNF, i.e., cytokines implicated in many inflammatory processes including those associated with allergies and helminthic infections. IL-1 did not induce significant cytokine release in the absence of ionomycin or IgE/Ag, suggesting that Ca-dependent signaling was required. IL-1-mediated enhancement of cytokine expression was confirmed at the mRNA level by Northern blot and/or RT-PCR analysis. Our study reveals a role for IL-1 in the up-regulation of multiple mast cell-derived cytokines. Moreover, we identify mast cells as a novel source of IL-9. These results are of particular importance in the light of recent reports that strongly support a central role of IL-9 in allergic lung inflammation and in host defense against worm infections.     

Cloning and characterization of human histone deacetylase 8

To date, seven different human histone deacetylases (HDACs) have been identified, which fall into two distinct classes. We have isolated and characterized a cDNA encoding a novel human HDAC, which we name HDAC8. HDAC8 shows a high degree of sequence similarity to HDAC1 and HDAC2 and thus belongs to the class I of HDACs. HDAC8 is expressed in a variety of tissues. Human cells overexpressing HDAC8 localize the protein in sub-nuclear compartments whereas HDAC1 shows an even nuclear distribution. In addition, the HDAC8 gene is localized on the X chromosome at position q13, which is close to the XIST gene and chromosomal breakpoints associated with preleukemia.     

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.     

Eelgrass recovery after nutrient enrichment reversal

Mumford Cove, a 48 ha Connecticut embayment on Long Island Sound, has a history of excessive nutrient inputs and corresponding eutrophic conditions with concomitant eelgrass (Zostera marina L.) loss. From 1945 to 1987, a municipal wastewater treatment facility discharged into the cove. In 1987, when the wastewater outfall was diverted to another location, the cove supported a near monoculture of the green algae Ulva lactuca L., covering 74% of the bottom. By 1988, macroalgal areal cover in Mumford Cove had declined to 9%. When we first sampled the cove in 1992, Z. marina and Ruppia maritima L. were present. By 1999, areal distribution of Z. marina and R. maritima had expanded to cover a third of the bottom. Periodic summertime surveys from 2002 through 2004 indicated that Z. marina was present in approximately half of the cove; R. maritima was sparse. Fifteen years after termination of nutrient enrichment, this cove had recovered from 40 years of point source anthropogenic nutrient input, returning from an Ulva-dominated to a Zostera-dominated state.     

Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis

The remarkable survival ability of Mycobacterium tuberculosis in infected hosts is related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate expression of these lipids in response to environmental changes are unknown. Here we demonstrate that the enoyl-ACP reductase activity of InhA, an essential enzyme of the mycolic acid biosynthetic pathway and the primary target of the anti-tubercular drug isoniazid, is controlled via phosphorylation. Thr-266 is the unique kinase phosphoacceptor, both in vitro and in vivo. The physiological relevance of Thr-266 phosphorylation was demonstrated using inhA phosphoablative (T266A) or phosphomimetic (T266D/E) mutants. Enoyl reductase activity was severely impaired in the mimetic mutants in vitro, as a consequence of a reduced binding affinity to NADH. Importantly, introduction of inhA_T266D/E failed to complement growth and mycolic acid defects of an inhA-thermosensitive Mycobacterium smegmatis strain, in a similar manner to what is observed following isoniazid treatment. This study suggests that phosphorylation of InhA may represent an unusual mechanism that allows M. tuberculosis to regulate its mycolic acid content, thus offering a new approach to future anti-tuberculosis drug development.