Production of cellulase using a mutant strain of Trichoderma reesei growing on lactose in batch culture

The production of cellulases in batch culture was studied using a mutant strain of Trichoderma reesei C-5 growing on lactose. Growth kinetic parameters on 2% lactose were studied and the comparative results for growth and enzyme productivities at two different lactose levels are discussed. The cellulase synthesis rate depended on the lactose concentration in the medium. Although growth was favoured at a higher lactose level, the volumetric enzyme productivity did not increase in proportion and the specific enzyme productivity decreased to a certain extent, indicating that partial catabolic inhibition at higher lactose concentrations may be possible. However, it was noted that the mutant strain was highly depressed and capable of synthesising active cellulases on lactose.     

Detection and identification of ferricrocin produced by ectendomycorrhizal fungi in the genusWilcoxina

The ectendomycorrhizal fungiWilcoxina mikolae isolates CSY-14 and RMD-947 andW. rehmii isolate CSY-85 were grown in pure culture under iron-limiting conditions. All three isolates tested positive for siderophore formation using both the ferric perchlorate assay and a sensitive HPLC iron-binding assay. A peptide siderophore was isolated from the culture medium by HPLC and shown to contain the amino acids serine, glycine and ornithine in a 1:2:3 ratio. This siderophore was identified as ferricrocin on the basis of electrospray mass spectroscopy and its co-chromatography in two different HPLC systems with ferricrocin isolated fromAspergillus fumigatus. Ferricrocin was the only siderophore isolated from theseWilcoxina cultures. This is the first report of siderophore formation by ectendomycorrhizal fungi.     

Differential expression of H-Y antigen on lymphocyte subsets: analysis by flow cytometry

Expression of H-Y antigen in human white blood cells was measured using flow cytometry with monoclonal antibodies. In this system, lymphocytes were stained preferentially in the male, and to a lesser extent in the female. Analysis of the lymphocyte subsets with biotinylated H-Y antibody conjugated with streptavidin-fluorescein isothiocyanate (FITC) and subset-specific antibody conjugated with phycoerythrin derivative (RD1) revealed differential expression of H-Y among the subsets of the male. In samples from eight men, 41.1% +/- 21.7% of B cells (B1) were stained, compared with 20.7% +/- 12.8% of cytotoxic-suppressor T cells (T8) and 5.4% +/- 3.0% of helper-inducer T cells (T4). In samples from seven women, 12.4% +/- 10.9% of B cells were stained, but staining of T cells was negligible.     

Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications

Although plant expression systems used for production of therapeutic proteins have the advantage of being scalable at a low price, the downstream processing necessary to obtain pure therapeutic molecules is as expensive as for the traditional Chinese hamster ovary (CHO) platforms. However, when edible plant tissues (EPTs) are used, there is no need for exhaustive purification, because they can be delivered orally as partially purified formulations that are safe for consumption. This economic benefit is especially interesting when high doses of recombinant proteins are required throughout the treatment/prophylaxis period, as is the case for antibodies used for oral passive immunization (OPI). The secretory IgA (SIgA) antibodies, which are highly abundant in the digestive tract and mucosal secretions, and thus the first choice for OPI, have only been successfully produced in plant expression systems. Here, we cover most of the up‐to‐date examples of EPT‐produced pharmaceuticals, including two examples of SIgA aimed at oral delivery. We describe the benefits and drawbacks of delivering partially purified formulations and discuss a number of practical considerations and criteria to take into account when using plant expression systems, such as subcellular targeting, protein degradation, glycosylation patterns and downstream strategies, all crucial for improved yield, high quality and low cost of the final product.     

Neural network‐derived Potts models for structure‐based protein design using backbone atomic coordinates and tertiary motifs

Designing novel proteins to perform desired functions, such as binding or catalysis, is a major goal in synthetic biology. A variety of computational approaches can aid in this task. An energy‐based framework rooted in the sequence‐structure statistics of tertiary motifs (TERMs) can be used for sequence design on predefined backbones. Neural network models that use backbone coordinate‐derived features provide another way to design new proteins. In this work, we combine the two methods to make neural structure‐based models more suitable for protein design. Specifically, we supplement backbone‐coordinate features with TERM‐derived data, as inputs, and we generate energy functions as outputs. We present two architectures that generate Potts models over the sequence space: TERMinator, which uses both TERM‐based and coordinate‐based information, and COORDinator, which uses only coordinate‐based information. Using these two models, we demonstrate that TERMs can be utilized to improve native sequence recovery performance of neural models. Furthermore, we demonstrate that sequences designed by TERMinator are predicted to fold to their target structures by AlphaFold. Finally, we show that both TERMinator and COORDinator learn notions of energetics, and these methods can be fine‐tuned on experimental data to improve predictions. Our results suggest that using TERM‐based and coordinate‐based features together may be beneficial for protein design and that structure‐based neural models that produce Potts energy tables have utility for flexible applications in protein science.     

Malabarase,a serine protease with anticoagulant activity from Trimeresurus malabaricus venom

In the present study we describe the purification and characterization of Malabarase, a serine protease from Trimeresurus malabaricus venom. Purification was achieved by gel-permeation chromatography on Sephadex G-75 followed by ion-exchange chromatography on CM Sephadex C-25. Homogeneity of Malabarase was confirmed by RP-HPLC. Malabarase is a monomer that migrated as a single protein band on SDS-PAGE under both reducing and non-reducing conditions. The molecular mass of Malabarase was determined to be 23.4 kDa using MALDI-TOF mass spectrometry. Malabarase is the first serine protease purified from T. malabaricus venom and is selective for fibrinogen. Malabarase hydrolyzes Aα and Bβ but not γ-chains of fibrinogen similar to the metalloproteases, Malabarin and Trimarin, isolated from the same venom. However, the action of Malabarase on plasma coagulation is opposite than those of Malabarin, Trimarin and the whole venom. Malabarase significantly prolonged plasma coagulation time from 152–341 s; whereas Malabarin, Trimarin, and whole venom, greatly reduce plasma clotting time from 152 to 12, 48, and 14 s, respectively. Malabarase did not show hemorrhagic or myotoxic activity. In contrast, Malabarin, Trimarin and whole venom are highly hemorrhagic and myotoxic. These observations support the specificity of Malabarase towards fibrinogen and its non-toxic nature. In conclusion, Malabarase is a fibrinogen-specific, anti-coagulant, and non-toxic serine protease. Its selective action and non-toxic nature might make it useful for treating thrombotic disorders.     

Piriformospora indica?rescues growth diminution of rice seedlings during high salt stress

Piriformospora indica association has been reported to increase biotic as well as abiotic stress tolerance of its host plants. We analyzed the beneficial effect of P. indica association on rice seedlings during high salt stress conditions (200 and 300 mM NaCl). The growth parameters of rice seedlings such as root and shoot lengths or fresh and dry weights were found to be enhanced in P. indica-inoculated rice seedlings as compared with non-inoculated control seedlings, irrespective of whether they are exposed to salt stress or not. However, salt-stressed seedlings performed much better in the presence of the fungus compared with non-inoculated control seedlings. The photosynthetic pigment content [chlorophyll (Chl) a, Chl b, and carotenoids] was significantly higher in P. indica-inoculated rice seedlings under high salt stress conditions as compared with salt-treated non-inoculated rice seedlings, in which these pigments were found to be decreased. Proline accumulation was also observed during P. indica colonization, which may help the inoculated plants to become salt tolerant. Taken together, P. indica rescues growth diminution of rice seedlings under salt stress.     

Tmem64 Modulates Calcium Signaling during RANKL-Mediated Osteoclast Differentiation

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Highlights? Tmem64-deficient mice show increased bone volume ? Tmem64 deficiency reduces [Ca²⁺]_i oscillation in response to RANKL stimulation ? Tmem64 interacts with SERCA2 ? Tmem64 positively regulates osteoclast formation via SERCA2/Ca²⁺ signaling