Single step recombinant human growth hormone (rhGH) purification from milk by peptide affinity chromatography

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:999–1005, 2018     

Engineering and monitoring cellular barrier models

Epithelia and endothelia delineate tissue compartments and control their environments by regulating the passage of ions and solutes. This barrier function is essential for the development and maintenance of multicellular organisms, and its dysfunction is associated with numerous human diseases. Recent advances in biomaterials and microfabrication technologies have evolved in vitro approaches for modelling biological barriers. Current microphysiological systems have become more efficient and reliable in mimicking the cell microenvironment. Additionally, methods for the quantification of barrier permeability have long provided significant insight into their underlying mechanisms. In this review, we outline the current techniques to quantify the barrier function of engineered tissues, and we also give an overview of recent microphysiological systems of biological barriers that emulate the microenvironment and microarchitecture of native tissues.     

The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications

Plant calcium-dependent protein kinases (CDPKs) are key proteins implicated in calcium-mediated signaling pathways of a wide range of biological events in the organism. The action of each particular CDPK is strictly regulated by many mechanisms in order to ensure an accurate signal translation and the activation of the adequate response processes. In this work, we investigated the regulation of a CDPK involved in rice cold stress response, OsCPK17, to better understand its mode of action. We identified two new alternative splicing (AS) mRNA forms of OsCPK17 encoding truncated versions of the protein, missing the CDPK activation domain. We analyzed the expression patterns of all AS variants in rice tissues and examined their subcellular localization in onion epidermal cells. The results indicate that the AS of OsCPK17 putatively originates truncated forms of the protein with distinct functions, and different subcellular and tissue distributions. Additionally, we addressed the regulation of OsCPK17 by post-translational modifications in several in vitro experiments. Our analysis indicated that OsCPK17 activity depends on its structural rearrangement induced by calcium binding, and that the protein can be autophosphorylated. The identified phosphorylation sites mostly populate the OsCPK17 N-terminal domain. Exceptions are phosphosites T107 and S136 in the kinase domain and S558 in the C-terminal domain. These phosphosites seem conserved in CDPKs and may reflect a common regulatory mechanism for this protein family.     

Behavioral and Biochemical Effects of N-Acetylcysteine in Zebrafish Acutely Exposed to Ethanol

Neurochemical Research - Alcohol hangover refers to unpleasant symptoms experienced as a direct consequence of a binge drinking episode. The effects observed in this condition are related to the...     

Carotenoids in roots indicated the level of stress induced by mannitol and sodium azide treatment during the early stages of maize germination

Chemical mutagens, such as sodium azide, have attracted the interest of plant breeders. Azide creates DNA point mutations and affects plant growth and development, disturbs metabolic activity and inhibits protein and DNA replication, whereas mannitol is used to simulate drought stresses in tissue culture. To identify biochemical markers for stress tolerance, maize seeds were germinated under mannitol and sodium azide induced stress in controlled conditions for 7 days. Then levels of chlorophyll, carotenoids, phenolics and aldehydes produced were subsequently determined. Germination percentage was not affected by either mannitol or sodium azide and was always above 85%. However, total fresh weight decreased by 50% with the application of 153.4 mM mannitol and 0.26 mM azide in combination. This treatment significantly reduced plantlet growth from 0.94 g in the control to 0.53 g in the treated materials. Root weight reduced by 68.1%, cotyledons by 14.3%, stems by 65.0% and leaves by 70.0% in treated samples. The level of carotenoids in roots was the clearest biochemical indicator of stress produced by the mannitol and sodium azide treatment. Carotenoids increased from 0.01 µg g^??1 fresh weight in the control to 9.03 µg g^??1 fresh weight in the treated materials. A large-scale seed treatment with mannitol and sodium azide was carried out. 2296 seeds were placed in magenta containers with 153.4 mM mannitol and 0.26 mM NaN₃. At 7 days of germination, the heaviest seedlings (450) (450/2296?=?20%) were transferred to soil environment. Forty-two plants (42/450?=?9.3%) were off-type phenotypes at 45 days. Genetic variants may have been obtained following the novel procedure described here which combines chronic treatment with sodium azide and selection pressure with mannitol to simulate drought conditions.     

Zebrafish “personality” influences sensitivity to magnetic fields

How animals integrate different sensory information for orientation is a complex process involving interactions between a variety of internal and external factors. Due to this complexity, each component of a suite of factors is typically studied in isolation. Here, we examine how an internal factor (personality of fish) influences the response of zebrafish (Danio rerio) to the magnetic field, while swimming in a flow chamber. Our previous work demonstrated that the orientation to the water current (rheotaxis) of zebrafish individuals is influenced by variations of the magnetic field only when fish are part of a shoal. In this study, we evaluated the rheotactic behavior of 20 fish, grouped in shoals of “proactive” or “reactive” individuals, under magnetic fields of different directions. We found that the magnetic field influenced at which water speed rheotaxis was elicited in zebrafish with “reactive” personality, but not in those with “proactive” personality. These results suggest that fish personality influences response to or weighing of sensory inputs and provides some insight on the variation in behavioral responses to environmental stimuli in both laboratory and natural settings.     

Polyclonal origin of medullary carcinoma of the thyroid in multiple endocrine neoplasia type 2

Multiple endocrine neoplasia type 2 (MEN 2) is a dominantly inherited cancer syndrome characterized by medullary thyroid carcinoma (MTC) and other tumors. Since MTC can also occur in a sporadic form and as familial medullary thyroid carcinoma, this neoplasm offers a unique opportunity to investigate the difference of origin, if any, between the sporadic and the hereditary forms of a tumor. While sporadic malignancies have usually been found to result from a mutational event occurring at the single-cell level and are therefore monoclonal, studies on hereditary neoplasms have been scarce and often produced conflicting results. In order to determine the clonal origin of sporadic MTCs and of those occurring in MEN 2 syndromes we used a clonality assay based on a polymorphic trinucleotide repeat of the X-linked human androgen-receptor gene. We found that 10 out of 11 MTCs expressed a polyclonal pattern of X inactivation, including a significant percentage of the cases clinically defined as sporadic. Received: 21 May 1996 / Revised: 14 August 1996     

Expression of a Truncated, Kinase-Defective TGF-β Type II Receptor in Mouse Skeletal Tissue Promotes Terminal Chondrocyte Differentiation and Osteoarthritis

Members of the TGF-β superfamily are important regulators of skeletal development. TGF-βs signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-βs in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space.

We then tested the hypothesis that TGF-β is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-β may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-β promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.

     

β-Amino acid residues: Conformational characterization of an N- and C-protected homo-β-(S)-leucine

Summary An N- and C-protected derivative ofhomo-β-leucine, Fmoc-homo-β-(S)-leucine methyl ester, synthesized from the corresponding proteinogenic parent α-amino acid in enantiopure form has been fully characterized in the solid state by X-ray diffraction analysis. The crystal conformation of this new residue indicates and extended conformation for thishomo-β-residue, with the ϕ torsion angle being more constrained than the μ and ψ angles.