Resistance to the cytolytic action of lymphotoxin and tumor necrosis factor coincides with the presence of gap junctions uniting target cells

The resistance of target cells to the cytolytic action of lymphotoxin (LT) and recombinant tumor necrosis factor (rTNF) has been investigated by using clonally derived cell lines with defined gap junction-mediated, intercellular communication properties. Gap junction-competent Chinese hamster ovary cells are normally insensitive to the action of LT/TNF. However, treatment with 12-o-tetradecanoylphorbol-13-acetate, which promotes the loss of gap junctions, or culturing at low cell density to reduce intercellular contacts, significantly increased their sensitivity to LT/TNF. The LT/TNF-sensitive murine CL-1D and L929 cell lines, which in normal culture conditions are unable to form gap junctions, were not changed in their susceptibility to LT/TNF after treatment with phorbol ester or low culture density. However, the formation of gap junctions by CL-1D can be promoted by treatment with 8-bromo-cyclic adenosine monophosphate (1 mM), and this treatment completely suppressed the ability of LT and rTNF to kill CL-1D. Additionally, the LA25-normal rat kidney cell line, which is infected with a temperature-sensitive mutant of Rous sarcoma virus (LA25), is gap junction-competent and resistant to the effects of LT at the restrictive temperature (39 degrees C). However, when shifted to the permissive temperature (33 degrees C), LA25-normal rat kidney cells express the pp60v-src viral gene product, lose their ability to form gap junctions, and become sensitive to the lytic activity of LT. The results demonstrate that the expression of the retroviral pp60v-src, a tyrosine protein kinase, is sufficient to render cells susceptible to the lytic effects of LT and rTNF. Collectively, these experiments demonstrate a strong correlation between the resistance of target cells to the action of LT/TNF and their ability to cooperate metabolically through gap junctions. The results do not completely exclude the possibility that other mechanisms, such as LT receptor modulation, are also occurring under these experimental conditions. These data also suggest that a possible physiologic function of the stable cytotoxic lymphokines is to induce cytolysis/cytostasis of cells that have lost gap junctional contact, such as those in the process of mitosis or metastasis that have separated from the main tissue mass.     

Studies of myofibrillar ATPase in ragweed-sensitized canine pulmonary smooth muscle

The maximal shortening velocities of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized dogs were significantly higher than those of muscles from their littermate controls. Myofibrils of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized and control dogs were obtained with use of Triton X-100 homogenizing solution. The myofibrillar adenosinetriphosphatase (ATPase) activities of the sensitized tissues were significantly higher (P less than 0.05) than those of their respective controls.     

Properties of a prolactin receptor from the rabbit mammary gland

Receptors for human, simian, ovine, bovine and murine prolactin, human growth hormone and human placental lactogen have been identified in plasma-membrane-containing subcellular particles isolated from rabbit mammary glands. The association and dissociation of (125)I-labelled prolactin are time- and temperature-dependent processes, both being maximal at 37 degrees C. (125)I-labelled prolactin prepared by the enzymic iodination procedure with lactoperoxidase binds better to receptors than does the preparation obtained by using chloramine-t as the oxidizing agent. The binding of (125)I-labelled prolactin to receptors is strongly influenced by pH and ionic composition but not by many low-molecular-weight compounds tested, e.g. steroids, nucleotides and several drugs. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipid moieties are essential for the binding of (125)I-labelled prolactin. The binding of (125)I-labelled prolactin to receptors is a saturable and reversible process. Scatchard and Lineweaver-Burk analyses suggest that (125)I-labelled prolactin has a high affinity for its receptor. Binding of (125)I-labelled prolactin to receptors does not result in the destruction of the hormone. Considerable prolactin-binding activity is also observed in subcellular fractions isolated from the adrenal gland, liver, ovary and kidney of the pregnant rabbit, a finding that is consistent with other reported actions of prolactin in these organs.     

Induction of two transformation-sensitive membrane polypeptides in normal fibroblasts by a block in glycoprotein synthesis or glucose deprivation.

Following transformation of chick embryo fibroblasts (CEF) by avian RNA tumor viruses, two membrane polypeptides with apparent molecular weights of 90,000 and 75,000 daltons have been found to be increased (Stone, Smith and Joklik, 1974). We find that this alteration in membrane proteins is not directly related to transformation.The 90,000 and 75,000 dalton proteins are present in increased amounts in a 3T3 fibroblast mutant (AD6) defective in glycoprotein synthesis. Feeding the mutant N-acetylglucosamine, a metabolite that bypasses the metabolic block, restores the amount of these two proteins to the levels found in normal cells. The 75,000 dalton protein is markedly reduced, and the 90,000 dalton protein disappears and is replaced by a fully glycosylated derivative with a molecular weight of 92,000 daltons.Two glucose derivatives, glucosamine and 2-deoxyglucose, are known to interfere with the glycosylation process. The addition of these substances to normal CEF and 3T3 cells specifically induces the accumulation of the 90,000 and 75,000 dalton membrane polypeptides.Finally, the deprivation of glucose for 24–48 hr also induces the synthesis of the 90,000 and 75,000 dalton polypeptides in normal fibroblasts. The induction of these two proteins by glucose starvation suggests that they have a role in glucose utilization.     

Identification of a transformation-specific protein induced by a Rous sarcoma virus.

Using antisera obtained from rats bearing Schmidt-Ruppin strain Rous sarcoma virus-induced tumors, we have idnetified a protein with an apparent molecular weight of 56,000 daltons and an isoelectric point of 6.3 in extracts of chick embryo fibroblasts transformed by a wild-type nondefective Rous sarcoma virus (Schmidt-Ruppin strain). This protein was not found in cells infected by trnasformation-defective mutants with either a partial or complete deletion of the src gene, nor in cells infected by a nontransforming avian leukosis virus. The 56,000 dalton molecular weight protein was found to be synthesized at both the permissive and nonpermissive temperatures in cells infected by either of two conditionallethal mutants that are temperature-sensitive in cell transformation. The amount of this protein, however, accumulated in cells infected by these temperature-sensitive mutants, relative to the structural polypeptides, differed significnatly from that seen with the nondefective virus. Pulsechase experiments indicate that the protein is extremely unstable, with a half-life of about 20 min, and does not serve as a precursor to any of the detectable virion polypeptides. Furthermore, incubation of the rat antiserum with purified, disrupted virus did not affect its immunoreactivity to this particular protein. We conclude that this 56,000 dalton molecular weight protein is a nonstructural protein specific to cells transformed by Rous sarcoma virus.     

The characterization of growth factor activity in human brain

The purification of fibroblast growth factor from bovine brain has been reported (Gospodarowicz, D., Bialecki, H., and Greenberg, G. (1978) J. Biol. Chem. 253, 3736-3743). Westall et al. (Westall, F. C., Lennon, V. A., and Gospodarowicz, D. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4675-4678) showed that bovine brain fibroblast growth factor was composed of three fragments derived by limited proteolysis from myelin basic protein. In the present study using similar purification methods, we isolated a fraction enriched in growth factor activity from human brain. The mitogenic activity could not be resolved from myelin basic protein by chromatographic procedures but, upon isoelectric focusing, the mitogen and myelin basic protein were readily dissociated. At least two potent growth factors (pI values 7.2 to 7.4 and 8.1 to 8.6) were identified. Studies of a relatively crude basic extract of human brain suggested that the brain may contain a number of growth factors.     

灵芝线粒体及其对菌丝生长、主要活性成分影响的分析

线粒体是为细胞提供能量（ATP）的细胞器,携带着自己的DNA——mtDNA,已有多种灵芝属菌株的线粒体基因组相继报道,但对于种内的线粒体基因组的分析较少,对核相同、线粒体不同的菌株间差异的研究也鲜有报道。本研究对两株灵芝线粒体基因组进行组装注释,根据差异片段构建分子标记进行种间分类。结果显示：两株灵芝线粒体基因组大小分别为49 233bp、61 563bp的闭环结构,含有15个常见蛋白编码基因,rRNA大小亚基基因及26个携带氨基酸的tRNA基因,其差异区段主要为内含子序列、大亚基及基因间区。根据cob、cox2基因序列能够进行灵芝种间区分,用于灵芝种间鉴定。本研究还根据灵芝119、灵芝无孢的单核菌株构建同核异质体（TY-119、TY-W）,分析线粒体对菌落形态、菌丝生长速度及多糖、三萜成分的影响,结果显示：同核异质体TY-W与TY-119菌落形态上有一定差异,同核异质体TY-W菌丝生长速度为4.77mm/d,是TY-119菌丝生长速度4.50mm/d的1.06倍,同核异质体TY-119菌丝、子实体阶段多糖含量分别为4.45mg/g、12.14mg/g是TY-W菌丝体多糖含量（3.23mg/g）的1.38倍、子实体多糖含量（10.24mg/g）的1.19倍;同核异质体TY-W菌丝、子实体阶段的三萜含量分别为6.82mg/g、11.45mg/g是同核异质体TY-119菌丝体三萜含量（9.26mg/g）的0.74倍,子实体三萜含量（9.10mg/g）的1.26倍。利用高效液相色谱分析同核异质体中灵芝酸含量显示同核异质体TY-W灵芝酸A、灵芝酸E、灵芝酸F含量分别为3.77μg/mL、14.29μg/mL、12.91μg/mL;是TY-119灵芝酸A含量（2.59μg/mL）的1.46倍、灵芝酸E含量（13.65μg/mL）的1.17倍、灵芝酸F含量（12.72μg/mL）的1.06倍。对同核异质体菌丝、子实体阶段多糖、三萜合成通路关键基因（pgm、ugp、gls、hmgs、hmgr、mvd、fps、sqs）表达量进行检测,显示两菌株间多数基因具有显著性差异。结果表明线粒体的不同会影响灵芝菌落形态、菌丝生长速度及多糖、三萜的含量,有助于我们进一步研究线粒体基因组。     

Identifying Primate ACE2 Variants That Confer Resistance to SARS-CoV-2

SARS-CoV-2 infects humans through the binding of viral S-protein (spike protein) to human angiotensin I converting enzyme 2 (ACE2). The structure of the ACE2-S-protein complex has been deciphered and we focused on the 27 ACE2 residues that bind to S-protein. From human sequence databases, we identified nine ACE2 variants at ACE2–S-protein binding sites. We used both experimental assays and protein structure analysis to evaluate the effect of each variant on the binding affinity of ACE2 to S-protein. We found one variant causing complete binding disruption, two and three variants, respectively, strongly and mildly reducing the binding affinity, and two variants strongly enhancing the binding affinity. We then collected the ACE2 gene sequences from 57 nonhuman primates. Among the 6 apes and 20 Old World monkeys (OWMs) studied, we found no new variants. In contrast, all 11 New World monkeys (NWMs) studied share four variants each causing a strong reduction in binding affinity, the Philippine tarsier also possesses three such variants, and 18 of the 19 prosimian species studied share one variant causing a strong reduction in binding affinity. Moreover, one OWM and three prosimian variants increased binding affinity by >50%. Based on these findings, we proposed that the common ancestor of primates was strongly resistant to and that of NWMs was completely resistant to SARS-CoV-2 and so is the Philippine tarsier, whereas apes and OWMs, like most humans, are susceptible. This study increases our understanding of the differences in susceptibility to SARS-CoV-2 infection among primates.     

Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

Background

Nasopharyngeal carcinoma (NPC) is known for its high metastatic potential and locoregional recurrence, although the molecular alterations that are driving NPC metastasis remain unclear at this time. This study aimed to examine the expression of fibulin-5 in NPC, correlate the results with clinicopathological variables and survival, and to investigate the role of fibulin-5 in human NPC cell lines.

Material and Methods

Standard semi-quantitative-RT-PCR, quantitative-RT-PCR, immunoblotting, and immunohistochemistry were used to investigate the mRNA and protein expression profiles of fibulin-5 in normal and NPC tissues. Immunohistochemistry of fibulin-5 was correlated with clinicopathological characteristics by univariate analyses. NPC cells overexpressing fibulin-5 or fibulin-5-siRNA cells were generated by stable transfection to characterize the molecular mechanisms of fibulin-5-elicited cell growth and metastasis.

Results

Our results demonstrated that fibulin-5 overexpression in NPC specimens and significantly correlated with advanced tumor metastasis indicating a poor 5-year overall survival. Fibulin-5 was mainly expressed in the nucleus in human NPC specimens and cell lines. Functionally, fibulin-5 overexpression yielded fast growth in NPC cells. In addition, fibulin-5 promotes cell metastasis in NPC cells through increased FLJ10540 and phosphor-AKT activity. In contrast, siRNA depletion of fibulin-5 suppressed FLJ10540 expression and phosphor-AKT activity. Suppression of either fibulin-5 or FLJ10540 can cause significant inhibition with regards to cell motility in NPC cells. Finally, immunohistochemical analysis of human aggressive NPC specimens showed a significant and positive correlation between fibulin-5 and FLJ10540 expression.

Conclusion

Higher fibulin-5 expression is not only an important indicator of poor survival, but also contributes to the development of new therapeutic strategies in the FLJ10540/AKT pathway for NPC treatment.     

Moderate Alcohol Use and Cardiovascular Disease from Mendelian Randomization

Background

Observational studies show moderate alcohol use negatively associated with ischemic heart disease (IHD) and cardiovascular disease (CVD). However, healthier attributes among moderate users compared to never users may confound the apparent association. A potentially less biased way to examine the association is Mendelian randomization, using alcohol metabolizing genes which influence alcohol use.

Methods

We used instrumental variable analysis with aldehyde dehydrogenase 2 (ALDH2) genotypes (AA/GA/GG) as instrumental variables for alcohol use to examine the association of alcohol use (10 g ethanol/day) with CVD risk factors (blood pressure, lipids and glucose) and morbidity (self-reported IHD and CVD) among men in the Guangzhou Biobank Cohort Study.

Results

ALDH2 genotypes were a credible instrument for alcohol use (F-statistic 74.6). Alcohol was positively associated with HDL-cholesterol (0.05 mmol/L per alcohol unit, 95% confidence interval (CI) 0.02 to 0.08) and diastolic blood pressure (1.15 mmHg, 95% CI 0.23 to 2.07) but not with systolic blood pressure (1.00 mmHg, 95% CI -0.74 to 2.74), LDL-cholesterol (0.03 mmol/L, 95% CI -0.03 to 0.08), log transformed triglycerides (0.03 mmol/L, 95% CI -0.01 to 0.08) or log transformed fasting glucose (0.01 mmol/L, 95% CI -0.006 to 0.03), self-reported CVD (odds ratio (OR) 0.98, 95% CI 0.76 to 1.27) or self-reported IHD (OR 1.10, 95% CI 0.83 to 1.45).

Conclusion

Low to moderate alcohol use among men had the expected effects on most CVD risk factors but not fasting glucose. Larger studies are needed to confirm the null associations with IHD, CVD and fasting glucose.