Cyclase‐associated protein 1 is a key negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells

Adenylyl cyclase‐associated protein (CAP) is a highly conserved protein. Previous reports have suggested that CAP1 may be a negative regulator of cellular proliferation, migration, and adhesion and the development of cell carcinomas. The molecular mechanism of CAP1 regulation of downstream pathways, as well as how CAP1 is regulated by environmental stimuli and upstream signalling, is not well understood. In this present study, we assessed the role of CAP1 in milk synthesis and proliferation of bovine mammary epithelial cells. Using gene overexpression and silencing methods, CAP1 was found to negatively regulate milk synthesis and proliferation of cells via the PI3K‐mTOR/SREBP‐1c/Cyclin D1 signalling pathway. Hormones, such as prolactin and oestrogen, and amino acids, such as methionine and leucine, stimulate MMP9 expression and trigger CAP1 degradation, and thus, abrogate its inhibition of synthesis of milk protein, fat, and lactose by and proliferation of bovine mammary epithelial cells. The results of our study help deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in characterizing the molecular mechanisms of CAP1. Previous reports have suggested that CAP1 is a negative regulator of cellular proliferation and anabolism, but the molecular mechanisms are largely unknown. In this present study, we identified CAP1 as a negative regulator of milk synthesis and proliferation of bovine mammary epithelial cells. Our results will deepen our understanding of the regulatory mechanisms underlying milk synthesis and aid in exploring the molecular mechanisms of CAP1.     

Ginseng stem‐leaf saponins in combination with selenium enhance immune responses to an attenuated pseudorabies virus vaccine

Pseudorabies, a herpesvirus infection, is mainly controlled by using attenuated live vaccines. In this study, the effect of ginseng stem and leaf saponins (GSLS) in combination with selenium (Se; in the form of sodium selenite) on vaccination against attenuated pseudorabies virus (aPrV) was evaluated. It was found that GSLS and Se have an adjuvant effect and that a combination of GSLS and Se stimulates significantly enhanced immune responses than does GSLS or Se alone. Following oral administration of GSLS, mice immunized with an attenuated PrV vaccine diluted in Se‐containing physiological saline solution (PSS) provoked a significantly stronger gB‐specific serum antibodies response (IgG, IgG1 and IgG2a), enhanced lymphocyte proliferation and cytolytic activity of NK cells, along with higher production of cytokines (IFN‐γ, IL‐12, IL‐5 and IL‐10) by splenocytes. Notably, the combination of GSLS and Se conferred a much higher resistance to fPrV challenge after immunization of the mice with aPrV vaccine. This study offers convincing experimental evidence that an injection of Se with oral GSLS is a promising adjuvant combination that improves the efficacy of vaccination against PrV and deserves further study regarding improvement of responses to other animal vaccines.     

Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

M(en)TORship lessons on life and death by the integrated stress response

Cells employ pro-survival and pro-adaptive pathways to cope with different forms of environmental stress. When stress is excessive, and the damage caused by it is unsustainable, cells engage pro-death pathways, which are in place to protect the host from the deleterious effects of harmed cells. Two important pathways that determine the balance between survival and death of stressed cells are the integrated stress response (ISR) and the mammalian target of rapamycin (mTOR), both of which converge at the level of mRNA translation. The two pathways have established avenues of communication to control their activity and determine the fate of stressed cells in a context-dependent manner. The functional interplay between the ISR and mTOR may have significant ramifications in the development and treatment of human diseases such as diabetes, neurodegeneration and cancer.     

Proteomic Profiling of Purified Rabies Virus Particles

While host proteins incorporated into virions during viral budding from infected cell are known to play essential roles in multiple process of the life cycle of progeny virus, these characteristics have been largely neglected in studies on rabies virus(RABV). Here, we purified the RABV virions with good purity and integrity, and analyzed their proteome by nano LC–MS/MS, followed by the confirmation with immunoblot and immuno-electronic microscopy. In addition to the 5 viral proteins, 49 cellular proteins were reproducibly identified to be incorporated into matured RABV virions. Function annotation suggested that 24 of them were likely involved in virus replication. Furthermore, cryo-EM was employed to observe the purified RABV virions, generating high-resolution pictures of the bullet-shaped virion structure of RABV. This study has provided new insights into the host proteins composition in RABV virion and shed the light for further investigation on molecular mechanisms of RABV infection, as well as the discovery of new anti-RABV therapeutics.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

Sugarcane mosaic virus in plantlets regenerated from diseased leaf tissue

Plantlets produced from sugarcane leaf tissue were examined to determine the effect of propagation on the frequency of occurrence of sugarcane mosaic virus (SCMV).Explants from immature leaf tissues of the sugarcane variety CP 72-356 (Saccharum interspecific hybrid), healthy or SCMV-infected, were cultured on Murashige-Skoog medium to which a combination of cytokinin and auxin had been added. Plantlets developed on healthy and infected leaf tissue within 6 weeks. The juice from plantlets was assayed for SCMV on Rio sorghum (Sorghum bicolor (L.) Moench, var. Rio) seedlings and on sugarcane varieties CP 31-294 and CO 31-588 for SCMV-strain identification. Results indicated that SCMV strain H was transmitted from the donor tissue to the regenerated plantlets. Observation on plantlets reared in the greenhouse showed that 23% had symptoms of SCMV. In a second replicated experiment, the leaf tissue from plants of POJ 234 free of mosaic or infected with SCMV strain A, B, D, H, or I was cultured. Each of the five strains was transmitted from donor to plantlet as indicated by assays on sorghum and sugarcane varieties. From 11 to 88% of the plantlets had mosaic symptoms, depending on the strain infecting the donor plant. In this experiment, SCMV-strain M was transmitted from an unidentified donor variety to 23% of the regenerated plantlets.Portions of this paper have been presented to the American Society of Sugar Cane Technologists, at the meeting in Clearwater, Florida in June, 1984.