Effect of porcine placenta steroid extract on myogenic satellite cell proliferation, transdifferentiation, and lipid accumulation

Intramuscular long-chain fatty acids (LCFAs) play an important role in energy production and initiation of mitochondrial oxidation of lipids. Herein, we report a natural porcine placenta steroid extract (PPSE) that stimulates transdifferentiation and lipid accumulation in bovine myogenic satellite cells (MSCs). The steroids hormones in PPSE were analyzed using enzyme-linked immunosorbant assay and presence of LCFA was established using gas chromatography. At 70% confluent growth, cells were treated with PPSE, LCFAs, transdifferentiation cocktail and commercially available steroid hormones. The working concentrations of all chemicals were manipulated similar to PPSE. The cells were observed for morphological changes and subjected to quantitative analysis of lipid deposition on Days 2, 4, and 6 of treatment. PPSE-treated MSCs exclusively transformed into lipid-accumulated adipose-like cells (ALCs). However, myotubes or adipocytes were formed in cells treated with other chemicals. Expression of different genes was studied to ascertain the molecular mechanism involved in ALC formation. CD36, fatty acid binding protein 4, and peroxisome proliferator-activated receptor-gamma were up-regulated. The expression of CD36 was established through immunocyto-chemical analysis. A viability assay was used to confirm the effect of PPSE on proliferation of MSCs. Hence, a natural steroid extract from porcine was found as a nontoxic mixture, which induces lipid accumulation and transdifferentiation of MSCs to ALCs. From the gene expression studies, it was established that the extract works almost in homogenous manner with other lipid inducers.     

Porcine testicular extract inhibits T cell proliferation by blocking cell cycle transition from G1 phase to S phase

Since T cells express diverse sex steroid hormone receptors, they might be a good model to evaluate the effects of sex steroid hormones on immune modulation. Porcine testicular extract contains several sex steroid hormones and may be useful to study the effects of sex steroid hormones during T cell activation. We have examined the effects of the porcine testicular extract on T cell activation: proliferation and secretion of cytokines (IL-2 and IFN-γ) by activated T cells were severely decreased after treatment with porcine testicular extract. The extract produced an immunosuppressive effect and inhibited the proliferation of activated T cells by blocking the cell cycle transition from the G(1) phase to S phase. These effects were mediated by a decrease in the expression of cyclin D1 and cyclin E and constitutive expression of p27(KIP1) after T cell activation.     

Generation and evaluation of the efficacy of rhesus monkey soluble cytotoxic T lymphocyte-associated antigen-4 in the allogeneic mixed lymphocyte reaction

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is a transmembrane protein that is structurally similar to CD28. As CTLA-4 has a much higher binding affinity to B7 than CD28, several approaches using soluble CTLA-4 have been tried to down-regulate T cell activity by blocking the interaction between CD28 and B7. We constructed soluble rhesus monkey CTLA-4 immunoglobulin (CTLA-4Ig) containing a critical binding site to B7 combined with a constant Ig heavy chain region in a mammalian system. Flow cytometry analyses indicated that soluble rhesus monkey CTLA-4Ig bound to rhesus monkey CD86 (B7.2). Moreover, soluble rhesus monkey CTLA-4Ig more effectively blocked the rhesus monkey–rhesus monkey allogeneic mixed lymphocyte reaction compared with that of humans. These results indicate that soluble rhesus monkey CTLA-4Ig may be useful in preclinical trials in a rhesus monkey model.     

Differential effects of two period genes on the physiology and proteomic profiles of mouse anterior tibialis muscles

The molecular components that generate and maintain circadian rhythms of physiology and behavior in mammals are present both in the brain (suprachiasmatic nucleus; SCN) and in peripheral tissues. Examination of mice with targeted disruptions of either mPer1 or mPer2 has shown that these two genes have key roles in the SCN circadian clock. Here we show that loss of the clock gene mPer2 affects forced locomotor performance in mice without altering muscle contractility. A proteomic analysis revealed that the anterior tibialis muscles of the mPer2 knockout mice had higher levels of glycolytic enzymes such as triose phosphate isomerase and enolase than those of either the wild type or mPer1 knockout mice. In addition, the level of expression of HSP90 in the mPer2 mutant mice was also significantly higher than in wildtype mice. These results suggest that the reduced locomotor endurance of the mPer2 knockout mice reflects a greater dependence on anaerobic metabolism under stress conditions, and that the two canonical clock genes, mPer1 and mPer2, play distinct roles in the physiology of skeletal muscle.     

Preclinical mouse model of optical coherence tomography for subcortical brain imaging without dissection

The purpose of this study was to investigate the feasibility of using optical coherence tomography (OCT) to identify internal brain lesions, specifically intracerebral hemorrhage, without dissection. Mice with artificially injected brain hematomas were used to test the OCT system, and the recorded images were compared with microscopic images of the same mouse brains after hematoxylin and eosin staining. The intracranial structures surrounding the hematomas were clearly visualized by the OCT system without dissection. These images reflect the ability of OCT to determine the extent of a lesion in several planes. OCT is a useful technology, and these findings could be used as a starting point for future research in intraoperative imaging.     

Molecular mechanism underlying muscle mass retention in hibernating bats: Role of periodic arousal

Hibernators like bats show only marginal muscle atrophy during prolonged hibernation. The current study was designed to test the hypothesis that hibernators use periodic arousal to increase protein anabolism that compensates for the continuous muscle proteolysis during disuse. To test this hypothesis, we investigated the effects of 3‐month hibernation (HB) and 7‐day post‐arousal torpor (TP) followed by re‐arousal (RA) on signaling activities in the pectoral muscles of summer‐active (SA) and dormant Murina leucogaster bats. The bats did not lose muscle mass relative to body mass during the HB or TP‐to‐RA period. For the first 30‐min following arousal, the peak amplitude and frequency of electromyographic spikes increased 3.1‐ and 1.4‐fold, respectively, indicating massive myofiber recruitment and elevated motor signaling during shivering. Immunoblot analyses of whole‐tissue lysates revealed several principal outcomes: (1) for the 3‐month HB, the phosphorylation levels of Akt1 (p‐Akt1) and p‐mTOR decreased significantly compared to SA bats, but p‐FoxO1 levels remained unaltered; (2) for the TP‐to‐RA period, p‐Akt1 and p‐FoxO1 varied little, while p‐mTOR showed biphasic oscillation; (3) proteolytic signals (i.e., atrogin‐1, MuRF1, Skp2 and calpain‐1) remained constant during the HB and TP‐to‐RA period. These results suggest that the resistive properties of torpid bat muscle against atrophy might be attained primarily by relatively constant proteolysis in combination with oscillatory anabolic activity (e.g., p‐mTOR) corresponding to the frequency of arousals occurring throughout hibernation. J. Cell. Physiol. 222: 313–319, 2010. © 2009 Wiley‐Liss, Inc.     

The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity

The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity.     

Gene expression profiles analyzed by DNA sequencing of cDNA clones constructed from porcine preadipocytes and adipocytes

As a step towards understanding the molecular mechanism of adipogenesis in pigs, preadipocytes purified from the back fat of 1 day-old female piglets were used for in vitro culture. Normalized cDNA libraries were constructed with 1.6×10⁷ and 1.1×10⁷ independent clones from preadipocyte and mature adipocyte mRNAs, respectively. Polymerase chain reaction (PCR) result using primers T3 and T7 (universal primer) confirmed the presence of the insert in the vector. Sequencing of 2,112 randomly selected clones from each cDNA library identified 217 clusters, 1,169 singletons, and 216 contigs in preadipocytes and 231 clusters, 1,100 singletons, and 233 contigs in mature adipocytes. Expressed sequence tag (EST) identified 24 genes with known annotation highly expressed in adipocytes and 21 in preadipocytes by at least four EST number. Among those 45 genes, when analyzed by real time RT-PCR, 76% of the gene showed significant difference between preadipocytes and mature adipocytes. Highly expressed genes in mature adipocytes were related to adipogenesis, extracellular matrix control and oncogenes, whereas cytoskeleton-related genes were down-regulated. An interesting similarity found during gene profile studies indicated a correlation between cancer and adipogenesis.     

Penta-peptide ATN-161 based neutralization mechanism of SARS-CoV-2 spike protein

SARS-CoV-2 has become a big challenge for the scientific community worldwide. SARS-CoV-2 enters into the host cell by the spike protein binding with an ACE2 receptor present on the host cell. Developing safe and effective inhibitor appears an urgent need to interrupt the binding of SARS-CoV-2 spike protein with ACE2 receptor in order to reduce the SARS-CoV-2 infection. We have examined the penta-peptide ATN-161 as potential inhibitor of ACE2 and SARS-CoV-2 spike protein binding, where ATN-161 has been commercially approved for the safety and possess high affinity and specificity towards the receptor binding domain (RBD) of S1 subunit in SARS-CoV-2 spike protein. We carried out experiments and confirmed these phenomena that the virus bindings were indeed minimized. ATN-161 peptide can be used as an inhibitor of protein-protein interaction (PPI) stands as a crucial interaction in biological systems. The molecular docking finding suggests that the binding energy of the ACE2-spike protein complex is reduced in the presence of ATN-161. Protein-protein docking binding energy (-40.50 kcal/mol) of the spike glycoprotein toward the human ACE2 and binding of ATN-161 at their binding interface reduced the biding energy (-26.25 kcal/mol). The finding of this study suggests that ATN-161 peptide can mask the RBD of the spike protein and be considered as a neutralizing candidate by binding with the ACE2 receptor. Peptide-based masking of spike S1 protein (RBD) and its neutralization is a highly promising strategy to prevent virus penetration into the host cell. Thus masking of the RBD leads to the loss of receptor recognition property which can reduce the chance of infection host cells.