Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus

The envelope protein of human immunodeficiency virus (HIV) is synthesized as a polyprotein (gp160) and cleaved intracellularly to a gp120-gp41 heterodimer. In this study, the tryptic-like endoproteolytic cleavage site was removed by site-directed mutagenesis and replaced with a chymotryptic-like site. The resultant mutant, RIP7/mut10, was found to be indistinguishable from wild-type HIV when analyzed at the level of proviral replication, RNA processing, protein expression, and viral assembly. However, the gp160 polyprotein was not cleaved and the mutated virions were biologically inactive, until and unless they were exposed to limiting concentrations of chymotrypsin. As is the case for other enveloped mammalian viruses, endoproteolytic cleavage of the HIV envelope protein and release of a unique hydrophobic domain appear to be necessary for the full expression of viral infectivity.     

ATP-induced Secretion in PC12 Cells and Photoaffinity Labeling of Receptors

Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P₂-type pur-inergic receptor. ATP-induced norepinephrine release was inhibited 80% when extracellular Ca²⁺ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2-methylthio-ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP-induced secretion was inhibited by Mg²⁺, and this inhibition was overcome by the addition of excess ATP suggesting that ATP^4-was the active ligand. ATP-induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12- O -tetradecanoylphorbol 13-acetate. The stimulatory effects of 12- O -tetradecanoyl-phorbol 13-acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α-³²P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP-γS and BzATP but not GTP blocked labeling of the proteins by [³²P]BzATP. Labeling of the 50-kDa protein was more sensitive to competition by 2-methylthio-ATP than the other labeled proteins, suggesting that the 50-kDa protein represents the P₂ receptor responsible for ATP-stimulated secretion in these cells.     

黑皮质素受体-4调节通路的研究进展

黑皮质素系统来自阿片-促黑素细胞皮质素原,在中枢摄食行为和能量平衡代谢中起到重要作用,此系统生理功能的发挥主要通过与下丘脑神经元细胞上特定膜受体（黑皮质素受体）结合完成。黑皮质素受体（MCR）有五种亚型（MC1R-MC5R）,其中参与体重调节的受体主要是黑皮质素受体3（MC3R）和黑皮质素受体4（MC4R）。MC4R属于G蛋白耦联受体,具有七次跨膜结构。作为一种膜受体,MC4R发挥体重调节作用,一方面受外界激动剂或拮抗剂的调节;另一方面,此受体活化后会影响到细胞内的信号调节通路。研究MC4R的功能首先要了解受体的结构,本文对G蛋白耦联受体的结构进行了较详细的叙述,MC4R经信号调节通路,激活腺苷酸环化酶,增加cAMP的浓度,最终通过影响细胞内基因的转录和翻译,来调节体重和能量的消耗。     

Expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand- and cell-dependent

Recent genome-wide association studies suggest distinct roles for 12 human interferon-alpha (IFN-α) and 3 IFN-λ subtypes that may be elucidated by defining the expression patterns of these sets of genes. To overcome the impediment of high homology among each of the sets, we designed a quantitative real-time PCR assay that incorporates the use of molecular beacon and locked nucleic acid (LNA) probes, and in some instances, LNA oligonucleotide inhibitors. We then measured IFN subtype expression by human peripheral blood mononuclear cells and by purified monocytes, myeloid dendritic cells (mDC), plasmacytoid dendritic cells (pDC), and monocyte-derived macrophages (MDM), and -dendritic cells (MDDC) in response to poly I:C, lipopolysaccharide (LPS), imiquimod and CpG oligonucleotides. We found that in response to poly I:C and LPS, monocytes, MDM and MDDC express a subtype pattern restricted primarily to IFN-β and IFN-λ1. In addition, while CpG elicited expression of all type I IFN subtypes by pDC, imiquimod did not. Furthermore, MDM and mDC highly express IFN-λ, and the subtypes of IFN-λ are expressed hierarchically in the order IFN-λ1 followed by IFN-λ2, and then IFN-λ3. These data support a model of coordinated cell- and ligand-specific expression of types I and III IFN. Defining IFN subtype expression profiles in a variety of contexts may elucidate specific roles for IFN subtypes as protective, therapeutic or pathogenic mediators.     

Analysis of RNA secondary structure by photochemical reversal of psoralen crosslinks.

Aminomethyltrioxsalen (AMT), a psoralen, is known to cause interstrand crosslinks in double stranded nucleic acids. We have demonstrated the photochemical reversal of this reaction, and have used this result to develop a method for identification of specific sequences which are adjacent because of RNA secondary structure formation. E. coli 5S rRNA is used as a model system. We isolated and characterized a product that is derived from the stem region of 5S RNA.     

Modulation of interleukin 2 release from a primate lymphoid cell line in serum-free and serum-containing media

The ability to grow a clone of the cell line, MLA144, which is a constitutive producer of interleukin 2 (IL-2), in serum-free medium permitted the study of the direct effect of various agents on cell growth and IL-2 production in a homogeneous population. Bovine serum albumin (BSA) at 4 mg/ml was optimal for cell growth and IL-2 production. Selenium at 10 ng/ml enhanced IL-2 production nearly twofold and lithium at 42 ng/ml also enhanced IL-2 production by nearly twofold. Neither compound at these levels altered cellular proliferation. Two other compounds, iron and zinc, known to be associated with cellular proliferation and/or immunoregulation did not alter IL-2 production. Catalase or horseradish peroxidase was able to substitute for BSA and maintain the long-term growth of the MLA144 clone with only a 30% decrease in the rate of cellular proliferation and a 50% decrease in IL-2 production compared to cells maintained in the serum-free formulation with BSA. Addition of 0.5 mg of BSA to the catalase serum-free formulation increased the production of IL-2 to 70% of that of cells cultured in the BSA-containing serum-free formulation. The catalase-containing serum-free formulation has the advantage of consisting of only three proteins, catalase, insulin, and transferrin, at a very low protein content. The catalase-containing serum-free medium also supported the long-term growth of a human T-cell line, HSB-2.     

Vitrification of Carotid Artery Segments: An Integrated Study of Thermophysical Events and Functional Recovery Toward Scale-Up for Clinical Applications

In recent years, ice-free cryopreservation by vitrification has been demonstrated to provide superior preservation of tissues compared with conventional freezing methods. To date, this has been accomplished almost exclusively for small model systems, whereas cryopreservation of large tissue samples-of a clinically useful size-continues to be hampered by thermomechanical effects that compromise the structure and function of the tissue. Reduction of mechanical stress is an integral condition of successful cryopreservation of large specimens. The current study focuses on the impact of sample size on both the physical events, observed by cryomacroscopy, and on the outcome on tissue function. To this end, the current study sought to address the question of functional recovery of vitrified carotid artery segments, processed as either artery rings (3-4 mm long) or segments (25 mm long) as selected models; the latter model represents a significant increase in sample size for evaluating the effects of vitrification. Tissue vitrification using an 8.4 M cryoprotectant cocktail solution (VS55) was achieved in 1-ml samples by imposing either a high (50-70 °C/min) or a low (2-3 °C/min) cooling rate, between -40°C and -100°C, and a high rewarming rate between -100°C and -40°C. Following cryoprotectant removal, the artery segments were cut into 3 to 4-mm rings for function testing on a contractility apparatus by measuring isometric responses to four agonist and antagonists (norepinephrine, phenylepinephrine, calcium ionophore, and sodium nitroprusside). In addition, nonspecific metabolic function of the vessel rings was determined using the REDOX indicator alamarBlue. Contractile function in response to the agonists norepinephrine and phenylepinephrine was maintained at the same level (350%) for the segments as for the rings, when compared with noncryopreserved control samples. Relaxation in response to the antagonists calcium ionophore and sodium nitroprusside was maintained at between 75% and 100% of control levels, irrespective of cooling rate or sample size. No evidence of macroscopic crystallization or fractures was observed by cryomacroscopy at the above rates in any of the samples. In conclusion, this study verifies that the rate of cooling and warming can be reduced from our baseline vitrification technique such that the function of larger tissue samples is not significantly different from that of smaller blood vessel rings. This represents a step toward the goal of achieving vitreous cryopreservation of large tissue samples without the destructive effect of thermal stresses.