Hemopexin Modulates Expression of Complement Regulatory Proteins in Rat Glomeruli

In systemic hemolysis and in hematuric forms of kidney injury, the major heme scavenging protein, hemopexin (HPX), becomes depleted, and the glomerular microvasculature (glomeruli) is exposed to high concentrations of unbound heme, which, in addition to causing oxidative injury, can activate complement cascades; thus, compounding extent of injury. It is unknown whether unbound heme can also activate specific complement regulatory proteins that could defend against complement-dependent injury. Isolated rat glomeruli were incubated in media supplemented with HPX-deficient (HPX⁻) or HPX-containing (HPX⁺) sera as a means of achieving different degrees of heme partitioning between incubation media and glomerular cells. Expression of heme oxygenase (HO)-1 and of the complement activation inhibitors, decay-accelerating factor (DAF), CD59, and complement receptor-related gene Y (Crry), was assessed by western blot analysis. Expression of HO-1 and of the GPI-anchored DAF and CD59 proteins increased in isolated glomeruli incubated with HPX⁻ sera with no effect on Crry expression. Exogenous heme (hemin) did not further induce DAF but increased Crry expression. HPX modulates heme-mediated induction of complement activation controllers in glomeruli. This effect could be of translational relevance in glomerular injury associated with hematuria.     

Unconventional autophagy mediated by the WD40 domain of ATG16L1 is derailed by the T300A Crohn disease risk polymorphism

A coding polymorphism of the critical autophagic effector ATG16L1 (T300A) increases the risk of Crohn disease, but how this mutation influences the function of ATG16L1 has remained unclear. In a recent report, we showed that the A300 allele alters the ability of the C-terminal WD40 domain of ATG16L1 to interact with proteins containing a specific amino acid motif able to recognize this region. This defect impairs the capacity of the motif-containing transmembrane molecule TMEM59 to induce the unconventional autophagic labeling of the same single-membrane vesicles where this protein is located. Such alteration derails the intracellular trafficking of TMEM59 and the xenophagic response against bacterial infection. In contrast, canonical autophagy remains unaffected in the presence of ATG16L1^T300A. These data argue that the T300A polymorphism impairs the unconventional autophagic activities carried out by the WD40 domain, a region of ATG16L1 whose function has remained poorly understood.     

Analysis of population cytokinetics of chick myocardial cells in tissue culture

The growth of embryonic chick cardiac myocytes and fibroblasts in tissue culture was evaluated by the kinetics of nuclear labeling during continuous exposure to [3H]thymidine. The fraction of mitotically active cells, the mean intermitotic period and the population doubling times were determined in each cell type during 3 weeks in culture. After 24 hr in culture, 90% of the muscle cells were mitotically active with minimal population doubling times of 65 hr. By 17 days in culture only 5% of the myocytes continued to divide with population doubling times greater than 3000 hr. Primarily, the lengthening of doubling times was due to a withdrawal of cells from the mitotic cycle and much less to a lengthening of the intermitotic period. Growth of cardiac muscle cells from embryonic hearts from 4 to 10 days of development was also compared. Muscle cells from younger hearts displayed greater mitotic activity than those from older hearts at equivalent times in culture.     

Endogenous iron excretion

The effects of supplemental oral (0, 40, and 400 ppm) and parenteral iron (0 and 2.72 mg Fe iv given initially as a single dose) on iron absorption, excretion, and retention were determined in 30 rats. Endogenous fecal iron excretion was determined by the radioisotope dilution technique after im injection of 80 kBq Fe-59, using blood and certain body tissues as reference sources for the estimation of the specific activity (Bq Fe-59/micrograms Fe) of endogenous iron. The basal diet contained 3.6 ppm Fe. Fe(III)-hydroxide-polymaltose was used as the sole iron source in oral, iv, and im iron treatments. Iron balance as determined from day 14 to 20 of the experiment was not significantly affected by iv iron administration. Nevertheless, a temporarily reduced retention should have occurred, since differences in final body iron contents were lower than 2.72 mg, as compared to the respective untreated groups. Apparent iron absorption and iron retention increased with surplus oral iron, and the efficiency rates were highest with adequate iron supply (40 ppm). True absorption rates of iron were similar without any, and with 40 ppm Fe amounting 40 to 50% of the intake. In the iron deficient rats, half of the actually absorbed iron (about 16 micrograms/d) was lost by endogenous fecal re-excretion, and another 3 micrograms/d by the urinary route. Endogenous loss with feces and with urine increased with further oral iron supply, but at a considerably lower rate as total fecal excretion. Parenterally administered iron did not affect endogenous loss at all. The results indicate that endogenous excretion cannot be regarded as a means to eliminate excessive iron, and might actually be an inevitable loss.     

The mechanism of inhibition and "reversal" of mitogen-induced lymphocyte activation in a model of adenosine deaminase deficiency

The biochemical mechanism of lymphocyte dysfunction with adenosine deaminase deficiency has been investigated using cultured phytohemagglutinin stimulated normal peripheral blood lymphocytes and the adenosine deaminase (ADA) inhibitor 2'-deoxycoformycin. The addition of deoxyadenosine to ADA-inhibited (but not to uninhibited) cells generated increased dATP pools (up to 50-fold greater than controls) and depressed the mitogen response. dATP Accumulation was accompanied by depletion of the other three deoxynucleoside triphosphate (dNTP) pools (dTTP, dCTP, and dGTP). Suppression of the mitogen response could be prevented ("reversed") to 90% of control levels by the addition of deoxynucleoside precursors for the depleted dNTPs at the initiation of mitogen stimulation. "Reversal" restored the dTTP and possibly the dGTP pools. Thus the mechanism of toxicity in this model appears to be inhibition of ribonucleotide reductase by massive accumulation of dATP, resulting in starvation for the other three deoxyribonucleoside triphosphates. "Reversibility" of this toxicity by providing sources for the missing three deoxynucleoside triphosphates argues for ribonucleotide reductase inhibition rather than other mechanisms of deoxyadenosine toxicity in this model.     

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged¹. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact². We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use³. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm^-1) is lower than that of water (72 dynes cm^-1) and also below the limit (25 - 30 dyne cm^-1) for stomatal penetration⁴, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO₂ and O₂, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation^5,6, surgery⁷, artificial blood⁸, oxygenation of growth media⁹, and studies of ice crystal formation in plants¹⁰. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.     

H7N9禽流感病毒裂解佐剂(MF59)疫苗稳定性研究

目的评价H7N9禽流感病毒裂解佐剂(MF59)疫苗的稳定性。方法采用国内自主构建的重组H7N9禽流感病毒疫苗株毒种制备单价疫苗原液,制备含MF59佐剂的成品疫苗。按照企业注册质量标准进行检定,分别放置于(37±2)℃、(25±2)℃和(5±3)℃环境下,观察不同时间疫苗的稳定性。结果经检定,成品疫苗的外观、装量、无菌检查、异常毒性检测、细菌内毒素含量、渗透压质量摩尔浓度及其他检定项目均符合企业注册质量标准。H7N9禽流感病毒裂解佐剂(MF59)疫苗在(37±2)℃保存3 d、在(25±2)℃保存3个月、在(5±3)℃保存30个月,疫苗各项指标均符合企业注册质量标准。结论 H7N9禽流感病毒裂解佐剂(MF59)疫苗具有良好的稳定性。     

The pathogen reduction treatment of platelets with S-59 HCl (Amotosalen) plus ultraviolet A light: Genotoxicity profile and hazard assessment

Despite restrictive donor criteria and screening procedures, infections resulting from the transfusion of bacterially contaminated platelet products continue to occur. Pathogen reduction technologies targeting nucleic acids have been developed. However, concerns about the safety of these procedures exist; the main concern being the possible mutagenic and carcinogenic effects of the pathogen-inactivated preparation in the recipient. This report reviews the genotoxicity profile of the S-59 (Amotosalen) plus long wavelength ultraviolet light (UVA) pathogen reduction technology, and assesses the mutagenic and carcinogenic hazards in recipients of treated platelets. S-59, a synthetic heterocyclic psoralen, non-covalently intercalates into the nucleic acids of pathogens and forms crosslinks when UVA photoactivated. Before clinical use, the levels of residual S-59 and free photoproducts are greatly reduced using a ‘compound adsorption device’ (CAD). In vitro, S-59 is mutagenic in Salmonella typhimurium and mouse lymphoma L5178Y TK^+/− cells, and is clastogenic in CHO cells. There is reduced activity (Salmonella, CHO cells) or no activity (mouse lymphoma cells) with metabolic activation (S9 mix). When tested up to toxic dose levels, S-59 was negative in the mouse bone marrow micronucleus assay and the rat hepatocyte unscheduled DNA synthesis (UDS) test. Based on comparative studies conducted with S-59 plus UVA-treated platelets (up to 25 times without CAD), any genotoxic effects can be attributed to residual S-59. Considering (1) the known genotoxic mechanism of action for S-59, (2) the negative in vivo studies for S-59 at multiples >40,000× over clinical peak plasma levels, and (3) the fact that the positive in vitro genotoxicity effects for the end product seem due to residual S-59, any mutagenic hazard to a recipient of S-59 plus UVA-treated platelets is negligible and there is no concern about a carcinogenic potential as a consequence of a mutagenic activity. This conclusion is supported by a negative p53^+/− mouse carcinogenicity study.     

Mutational analysis of the T4 gp59 helicase loader reveals its sites for interaction with helicase, single-stranded binding protein, and DNA

Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.