Initiation Factor-2 (eIF-2) Activity in Barley Embryos

The activity of eukaryotic initiation factor-2 (eIF-2), a polypeptidechain initiation factor, which forms a ternary complex withthe eukaryotic initiator methionyl-tRNA (Met-tRNAi) and GTPand which promotes the binding of Met-tRNAi to the 40Sribosomalsubunit, increases several fold in developing barley embryosduring the late stages of embryogenesis. The maximum increasetook place during the desiccation phase and, as a result, dryembryos contain a very large amount of eIF-2 activity. Thisactivity decreases and reaches a level that sustains proteinsynthesis during germination of the embryos. (Received October 19, 1982; Accepted December 30, 1982)     

A complete map of the Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) signaling pathway

Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is a serine/threonine-protein kinase belonging to the Ca²⁺/calmodulin-dependent protein kinase subfamily. CAMKK2 has an autocatalytic site, which gets exposed when Ca²⁺/calmodulin (CAM) binds to it. This results in autophosphorylation and complete activation of CAMKK2. The three major known downstream targets of CAMKK2 are 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPKα), calcium/calmodulin-dependent protein kinase 1 (CAMK1) and calcium/calmodulin-dependent protein kinase 4 (CAMK4). Activation of these targets by CAMKK2 is important for the maintenance of different cellular and physiological processes within the cell. CAMKK2 is found to be important in neuronal development, bone remodeling, adipogenesis, and systemic glucose homeostasis, osteoclastgensis and postnatal myogensis. CAMKK2 is reported to be involved in pathologies like Duchenne muscular dystrophy, inflammation, osteoporosis and bone remodeling and is also reported to be overexpressed in prostate cancer, hepatic cancer, ovarian and gastric cancer. CAMKK2 is involved in increased cell proliferation and migration through CAMKK2/AMPK pathway in prostate cancer and activation of AKT in ovarian cancer. Although CAMKK2 is a molecule of great importance, a public resource of the CAMKK2 signaling pathway is currently lacking. Therefore, we carried out detailed data mining and documentation of the signaling events associated with CAMKK2 from published literature and developed an integrated reaction map of CAMKK2 signaling. This resulted in the cataloging of 285 reactions belonging to the CAMKK2 signaling pathway, which includes 33 protein–protein interactions, 74 post-translational modifications, 7 protein translocation events, and 22 activation/inhibition events. Besides, 124 gene regulation events and 25 activator/inhibitors involved in CAMKK2 activation were also cataloged. The CAMKK2 signaling pathway map data is made freely accessible through WikiPathway database (https://www.wikipathways.org/index.php/Pathway:WP4874). We expect that data on a signaling map of CAMKK2 will provide the scientific community with an improved platform to facilitate further molecular as well as biomedical investigations on CAMKK2 and its utility in the development of biomarkers and therapeutic targets.Electronic supplementary materialThe online version of this article (10.1007/s12079-020-00592-1) contains supplementary material, which is available to authorized users.     

Pregnancies following long luteal phases in southern white rhinoceros (Ceratotherium simum simum)

All extant species in the Rhinocerotidae family are experiencing escalating threats in the wild, making self-sustaining captive populations essential genetic reservoirs for species survival. Assisted reproductive technologies (ARTs) will become increasingly important for achieving and maintaining ex situ population sustainability and genetic diversity. Previous reports have shown that a large proportion of captive southern white rhinoceros (SWR) females are irregularly cyclic or acyclic, and that cycling females display two different estrous cycle lengths of approximately 30 or 70 days. It has been suggested that the longer estrous cycle length is infertile or subfertile, as no term pregnancies have been observed following long cycles. Here we report the achievement of two pregnancies following long luteal phases, using ovulation induction and artificial insemination with either fresh or frozen-thawed semen. One female SWR conceived on the first insemination attempt and gave birth to a live offspring. A second female conceived twice in consecutive long cycles although the first embryo was resorbed by 33 days post-insemination. A pregnancy from this female's second insemination is ongoing with expected parturition in November 2019. Whether prolonged estrous cycles in SWR are subfertile or infertile in natural breeding situations remains unclear. However, our findings demonstrate that the application of ARTs following prolonged cycles can result the successful establishment of pregnancies in SWR. Therefore, with ARTs, female SWR otherwise considered nonreproductive due to long estrous cycles may still have the potential for representation and contribution to the ex situ population.     

Induction of Periostin-like Factor and Periostin in Forearm Muscle, Tendon, and Nerve in an Animal Model of Work-related Musculoskeletal Disorder

Work-related musculoskeletal disorders (WMSDs), also known as repetitive strain injuries of the upper extremity, frequently cause disability and impairment of the upper extremities. Histopathological changes including excess collagen deposition around myofibers, cell necrosis, inflammatory cell infiltration, and increased cytokine expression result from eccentric exercise, forced lengthening, exertion-induced injury, and repetitive strain–induced injury of muscles. Repetitive tasks have also been shown to result in tendon and neural injuries, with subsequent chronic inflammatory responses, followed by residual fibrosis. To identify mechanisms that regulate tissue repair in WMSDs, we investigated the induction of periostin-like factor (PLF) and periostin, proteins induced in other pathologies but not expressed in normal adult tissue. In this study, we examined the level of PLF and periostin in muscle, tendon, and nerve using immunohistochemistry and Western blot analysis. PLF increased with continued task performance, whereas periostin was constitutively expressed. PLF was located in satellite cells and/or myoblasts, which increased in number with continued task performance, supporting our hypothesis that PLF plays a role in muscle repair or regeneration. Periostin, on the other hand, was not present in satellite cells and/or myoblasts. (J Histochem Cytochem 57:1061–1073, 2009)     

Development of a Rapid Streptavidin Capture-Based Assay for the Tyrosine Phosphorylated CSF-1R in Peripheral Blood Mononuclear Cells

A novel assay was developed to measure ratio of p-FMS (phospho FMS) to FMS using the Meso Scale Discovery^® (MSD) technology and compared to the routinely used, IP-Western based approach. The existing IP-Western assay used lysed PBMCs (Peripheral Blood Mononuclear Cells) that were immunoprecipitated (IP) overnight, and assayed qualitatively by Western analysis. This procedure takes three days for completion. The novel IP-MSD method described in this paper employed immunoprecipitation of the samples for one hour, followed by assessment of the samples by a ruthenium labeled secondary antibody on a 96-well Streptavidin-coated MSD plate. This IP-MSD method was semi-quantitative, could be run in less than a day, required one-eighth the volume of sample, and compared well to the IP-Western method. In order to measure p-FMS/FMS, samples from healthy volunteers (HV) were first stimulated with CSF-1(Macrophage colony-stimulating factor) to initiate the changes in the phosphotyrosyl signaling complexes in FMS. The objective of the present work was to develop a high throughput assay that measured p-FMS/FMS semi-quantitatively, with minimal sample requirement, and most importantly compared well to the current IP-Western assay.     

Metabolic utilization of human osteoblast cell line hFOB 1.19 under normoxic and hypoxic conditions: A phenotypic microarray analysis

Osteoblasts play an important role in bone regeneration and repair. The hypoxia condition in bone occurs when bone undergoes fracture, and this will trigger a series of biochemical and mechanical changes to enable bone repair. Hence, it is interesting to observe the metabolites and metabolism changes when osteoblasts are exposed to hypoxic condition. This study has looked into the response of human osteoblast hFOB 1.19 under normoxic and hypoxic conditions by observing the cell growth and utilization of metabolites via Phenotype MicroArrays™ under these two different oxygen concentrations. The cell growth of hFOB 1.19 under hypoxic condition showed better growth compared to hFOB 1.19 under normal condition. In this study, osteoblast used glycolysis as the main pathway to produce energy as hFOB 1.19 in both hypoxic and normoxic conditions showed cell growth in well containing dextrin, glycogen, maltotriose, D-maltose, D-glucose-6-phospate, D-glucose, D-mannose, D-Turanose, D-fructose-6-phosphate, D-galactose, uridine, adenosine, inosine and α-keto-glutaric acid. In hypoxia, the cells have utilized additional metabolites such as α-D-glucose-1-phosphate and D-fructose, indicating possible activation of glycogen synthesis and glycogenolysis to metabolize α-D-glucose-1-phosphate. Meanwhile, during normoxia, D-L-α-glycerol phosphate was used, and this implies that the osteoblast may use glycerol-3-phosphate shuttle and oxidative phosphorylation to metabolize glycerol-3-phosphate.