Protoplast formation and regeneration in <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii.     

The Small GTPase RhoA Is Required for Proper Locomotor Circuit Assembly

The assembly of neuronal circuits during development requires the precise navigation of axons, which is controlled by attractive and repulsive guidance cues. In the developing spinal cord, ephrinB3 functions as a short-range repulsive cue that prevents EphA4 receptor-expressing corticospinal tract and spinal interneuron axons from crossing the midline, ensuring proper formation of locomotor circuits. Here we report that the small GTPase RhoA, a key regulator of cytoskeletal dynamics, is also required for ephrinB3/EphA4-dependent locomotor circuit formation. Deletion of RhoA from neural progenitor cells results in mice that exhibit a rabbit-like hopping gait, which phenocopies mice lacking ephrinB3 or EphA4. Consistent with this locomotor defect, we found that corticospinal tract axons and spinal interneuron projections from RhoA-deficient mice aberrantly cross the spinal cord midline. Furthermore, we determined that loss of RhoA blocks ephrinB3-induced growth cone collapse of cortical axons and disrupts ephrinB3 expression at the spinal cord midline. Collectively, our results demonstrate that RhoA is essential for the ephrinB3/EphA4-dependent assembly of cortical and spinal motor circuits that control normal locomotor behavior.     

Security framework for smart cyber infrastructure

Cluster Computing - The rapid deployment of the Internet of Things (IoT) devices have led to the development of innovative information services, unavailable a few years ago. To provide these...     

The plant nuclear envelope

This review summarizes our present knowledge about the composition and function of the plant nuclear envelope. Compared with animals or yeast, our molecular understanding of the nuclear envelope in higher plants is in its infancy. However, fundamental differences in the structure and function of the plant and animal nuclear envelope have already been found. Here, we compare and contrast these differences with respect to nuclear pore complexes, targeting of Ran signaling to the nuclear envelope, inner nuclear envelope proteins, and the role and fate of the nuclear envelope during mitosis. Further investigation of the emerging fundamental differences as well as the similarities between kingdoms might illuminate why there appears to be more than one blueprint for building a nucleus.Abbreviations GFP Green fluorescent protein - INE Inner nuclear envelope - LAP Lamina-associated polypeptide - LBR Lamin B receptor - MTOC Microtubule-organizing center - NE Nuclear envelope - NPC Nuclear pore complex - ONE Outer nuclear envelope - RanBP Ran-binding protein - RanGAP Ran GTPase-activating protein - WPP domain Tryptophan–proline–proline domain     

The plant nuclear envelope protein MAF1 has an additional location at the Golgi and binds to a novel Golgi-associated coiled-coil protein

Tomato MAF1 (LeMAF1) is a plant-specific, nuclear envelope (NE)-associated protein. It is the founding member of a group of WPP domain-containing, NE-associated proteins. This group includes the Arabidopsis WPP family, which is involved in cell division, as well as plant RanGAPs. In addition to its NE localization, LeMAF1 accumulates in speckles in the cytoplasm. Here, we show that the LeMAF1-containing speckles are components of the Golgi apparatus. A novel tomato coiled-coil protein was identified that specifically binds to LeMAF1. Tomato WPP domain-associated protein (LeWAP) interacts in yeast and in vitro through its coiled-coil domain with several WPP-domain containing proteins, including AtRanGAP1 and the WPP family (LeMAF, WPP1 and WPP2). Like LeMAF1, LeWAP is localized at the Golgi. Moreover, we present data showing that Arabidopsis WAP is necessary for the existence of a multi-protein complex containing WPP2. Electronic Supplementary Material Supplementary material is available for this article at .     

The SANT2 domain of the murine tumor cell DnaJ-like protein 1 human homologue interacts with alpha1-antichymotrypsin and kinetically interferes with its serpin inhibitory activity

The murine tumor cell DnaJ-like protein 1 or MTJ1/ERdj1 is a membrane J-domain protein enriched in microsomal and nuclear fractions. We previously showed that its lumenal J-domain stimulates the ATPase activity of the molecular chaperone BiP/GRP78 (Chevalier, M., Rhee, H., Elguindi, E. C., and Blond, S. Y. (2000) J. Biol. Chem. 275, 19620-19627). MTJ1/ERdj1 also contains a large carboxyl-terminal cytosolic extension composed of two tryptophan-mediated repeats or SANT domains for which the function(s) is unknown. Here we describe the cloning of the human homologue HTJ1 and its interaction with alpha(1)-antichymotrypsin (ACT), a member of the serine proteinase inhibitor (serpin) family. The interaction was initially identified in a two-hybrid screening and further confirmed in vitro by dot blots, native electrophoresis, and fluorescence studies. The second SANT domain of HTJ1 (SANT2) was found to be sufficient for binding to ACT, both in yeast and in vitro. Single tryptophan-alanine substitutions at two strictly conserved residues significantly (Trp-497) or totally (Trp-520) abolished the interaction with ACT. SANT2 binds to human ACT with an intrinsic affinity equal to 0.5 nm. Preincubation of ACT with nearly stoichiometric concentrations of SANT2 wild-type but not SANT2: W520A results in an apparent loss of ACT inhibitory activity toward chymotrypsin. Kinetic analysis indicates that the formation of the covalent inhibitory complex ACT-chymotrypsin is significantly delayed in the presence of SANT2 with no change on the catalytic efficiency of the enzyme. This work demonstrates for the first time that the SANT2 domain of MTJ1/HTJ1/ERdj1 mediates stable and high affinity protein-protein interactions.     

Autophagy and plant innate immunity

Plant innate immunity is often associated with specialized programmed cell death at or near the site of pathogen infection. Despite the isolation of several lesion mimic mutants, the molecular mechanisms that regulate cell death during an immune response remain obscure. Recently, autophagy, an evolutionarily conserved process of bulk protein and organelle turnover, was shown to play an important role in limiting cell death initiated during plant innate immune responses. Consistent with its role in plants, several studies in animals also demonstrate that the autophagic machinery is involved in innate as well as adaptive immunities. Here, we review the role of autophagy in plant innate immunity. Because autophagy is observed in healthy and dying plant cells, we will also examine whether autophagy plays a protective or a destructive role during an immune response.     

Adaptation of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> to Thermal Stress Yields a Thermotolerant Variant Which Also Exhibits Improved Survival at pH 2

Loss in probiotic viability upon exposure to stressful storage and transport conditions has plagued the probiotic market worldwide. Lactobacillus acidophilus is an important probiotic that is added to various functional foods. It is known to be fairly labile and susceptible to temperature variations that it encounters during processing and storage which increases production cost. It has been repeatedly demonstrated that pre-exposure to sub-lethal doses of stress, particularly, temperature and pH, leads to improved survival of various probiotics when they subsequently encounter the same stress of a much greater magnitude. Attempts to adapt L. acidophilus to temperatures as high as 65 °C to arrive at a thermotolerant variant have not been reported previously. To improve viability at elevated temperatures, we gradually adapted the L. acidophilus NCFM strain to survival at 65 °C for 40 min. Following adaptation, the variant showed a 2-log greater survival compared to wild-type at 65 °C. Interestingly, this thermotolerant variant also demonstrated a 2-log greater stability compared to wild-type at pH 2.0. The improved pH and temperature stress tolerance exhibited by this variant remained unaltered even when the strain was lyophilized. Moreover, the thermotolerant variant demonstrated improved stability compared to wild-type when stored for up to a week at 37 and 42 °C. Probiotic properties of the variant such as adherence to epithelial cells and antibacterial activity remained unaltered. This strain can potentially help address the issue of significant loss in viable cell counts of L. acidophilus which is typically encountered during probiotic manufacture and storage.     

Arabidopsis ATG6 is required to limit the pathogen-associated cell death response

To begin to understand the interplay between autophagy and the hypersensitive response (HR), a type of programmed cell death (PCD) induced during plant innate immunity, we generated ATG6 antisense plants in the genetically tractable Arabidopsis thaliana system. AtATG6 antisense (AtATG6-AS) plants senesce early and are sensitive to nutrient starvation, suggestive of impairment of autophagic function in these plants. Additionally, these plants exhibited multiple developmental abnormalities, a phenomenon not observed in other AtATG mutants. AtATG6-AS plants produced fewer Monodansylcadaverine (MDC) and LysoTracker (LT) stained-autolysosomes in response to carbon and nitrogen starvation indicating that AtATG6 plays a role in the autophagic pathway in Arabidopsis. Interestingly, the level of AtATG6 mRNA in wild type Col-0 Arabidopsis plants is increased during the early phase of virulent and avirulent Pseudomonas syringae pv tomato (Pst) DC3000 infection suggesting that AtATG6 plays an important role during pathogen infection. In AtATG6-AS plants, HR-PCD induced upon infection with avirulent Pst DC3000 carrying the AvrRpm1 effector protein is not able to be contained at the infection site and spreads into uninfected tissue. Additionally, the disease-associated cell death induced by the infection of virulent Pst DC3000 bacteria is also partially misregulated in AtATG6-AS plants. Therefore, the AtATG6 antisense plants characterized here provide an excellent genetic model system to elucidate the molecular mechanisms by which autophagy regulates pathogen-induced cell death.