Cell biology in China: Focusing on the lysosome

The view that lysosomes are merely the recycling bins of the cell has changed greatly during recent years. Lysosomes are now known to play a central role in signal transduction, cellular adaptation, plasma membrane repair, immune responses and many other fundamental cellular processes. In conjunction with the seminal discoveries made by international colleagues, many important questions regarding lysosomes are being addressed by Chinese scientists. In this review, we briefly summarize recent exciting findings in China on lysosomal signaling, biogenesis, integrity and physiological functions.     

Pathogenic Streptococcus strains employ novel escape strategy to inhibit bacteriostatic effect mediated by mammalian peptidoglycan recognition protein

Pathogenic streptococcal species are responsible for some of the most lethal and prevalent animal and human infections. Previous reports have identified a candidate pathogenicity island (PAI) in two highly virulent clinical isolates of Streptococcus suis type 2, a causative agent of high‐mortality streptococcal toxic shock syndrome. This PAI contains a type‐IVC secretion system C subgroup (type‐IVC secretion system) that is involved in the secretion of unknown pathogenic effectors that are responsible for streptococcal toxic shock syndrome caused by highly virulent strains of S. suis. Both virulence protein B4 and virulence protein D4 were demonstrated to be key components of this type‐IVC secretion system. In this study, we identify a new PAI family across 3 streptococcal species; Streptococcus genomic island contains type‐IV secretion system, which contains a genomic island type‐IVC secretion system and a novel PPIase molecule, SP1. SP1 is shown to interact with a component of innate immunity, peptidoglycan recognition protein (PGLYRP‐1) and to perturb the PGLYRP‐1‐mediated bacteriostatic effect by interacting with protein PGLYRP‐1. Our study elucidates a novel mechanism by which bacteria escape by components of the innate immune system by secretion of the SP1 protein in pathogenic Streptococci, which then interacts with PGLYRP‐1 from the host. Our results provide potential targets for the development of new antimicrobial drugs against bacteria with resistance to innate host immunity.     

A P4‐ATPase subunit of the Cdc50 family plays a role in iron acquisition and virulence in Cryptococcus neoformans

The pathogenic fungus Cryptococcus neoformans delivers virulence factors such as capsule polysaccharide to the cell surface to cause disease in vertebrate hosts. In this study, we screened for mutants sensitive to the secretion inhibitor brefeldin A to identify secretory pathway components that contribute to virulence. We identified an ortholog of the cell division control protein 50 (Cdc50) family of the noncatalytic subunit of type IV P‐type ATPases (flippases) that establish phospholipid asymmetry in membranes and function in vesicle‐mediated trafficking. We found that a cdc50 mutant in C. neoformans was defective for survival in macrophages, attenuated for virulence in mice and impaired in iron acquisition. The mutant also showed increased sensitivity to drugs associated with phospholipid metabolism (cinnamycin and miltefosine), the antifungal drug fluconazole and curcumin, an iron chelator that accumulates in the endoplasmic reticulum. Cdc50 is expected to function with catalytic subunits of flippases, and we previously documented the involvement of the flippase aminophospholipid translocases (Apt1) in virulence factor delivery. A comparison of phenotypes with mutants defective in genes encoding candidate flippases (designated APT1, APT2, APT3, and APT4) revealed similarities primarily between cdc50 and apt1 suggesting a potential functional interaction. Overall, these results highlight the importance of membrane composition and homeostasis for the ability of C. neoformans to cause disease.     

里氏木霉VPS13基因缺失对菌丝分支、生孢和纤维素酶产量的影响

【目的】丝状真菌里氏木霉是纤维素酶生产的主要工业真菌。纤维素酶分泌过程中的蛋白运输途径是控制大量纤维素酶成功输出的重要环节,因此,研究蛋白分泌途径的特定靶标基因功能将有助于鉴定纤维素酶运输分泌过程的关键调控因子。本研究借助基因敲除方法将里氏木霉液泡蛋白分选相关基因VPS13缺失,分析了该基因缺失对菌株生长、生孢尤其是纤维素酶分泌的影响。【方法】利用Double-joint PCR技术和同源重组策略构建里氏木霉VPS13基因缺失突变株,通过菌丝培养、显微观察、生孢检测、蛋白与酶活测定,系统比较VPS13基因敲除前后菌株的生长特征、菌丝形态、孢子形成、蛋白分泌以及纤维素酶活等。【结果】成功获得两株VPS13基因缺失株。与出发菌株相比,该基因突变后菌丝蔓延速率明显减慢,但菌体生物量在对数生长期后显著增多。通过显微观察,发现该基因缺失株菌丝更加密集,分支明显增多。此外,该基因缺失也导致菌株生孢延迟。纤维素底物平板分析发现VPS13基因缺失株菌落周围透明圈更加清晰,且透明圈圈径比是出发菌株的4倍,说明降解纤维素的能力有明显提高。进一步的液体发酵实验结果显示,该基因缺失导致蛋白产量及纤维素酶活力分别提高16.4%和21.9%。【结论】里氏木霉VPS13基因在菌丝生长、生孢、蛋白分泌等不同生物学过程中具有功能多样性,且该基因在菌种改良上可以作为提高纤维素酶产量的重要靶点。     

Autophagy is required for gamete differentiation in the moss Physcomitrella patens

Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abiotic stresses (through the induction or repression of programmed cell death, PCD) as well as to promotion of developmentally regulated PCD, starch degradation or caloric restriction important for life span. Much less is known regarding its role in plant cell differentiation. Here we show that macroautophagy, the autophagy pathway driven by engulfment of cytoplasmic components by autophagosomes and its subsequent degradation in vacuoles, is highly active during germ cell differentiation in the early diverging land plant Physcomitrella patens. Our data provide evidence that suppression of ATG5-mediated autophagy results in reduced density of the egg cell-mediated mucilage that surrounds the mature egg, pointing toward a potential role of autophagy in extracellular mucilage formation. In addition, we found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility. The similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking, strongly pointing toward an ancestral function of autophagy not only as a protector against nutrient stress, but also in gamete differentiation.     

广州地区红火蚁工蚁的巢外活动及有翅蚁的婚飞规律

为了阐明红火蚁Solenopsis invicta Buren的发生危害规律,为制定红火蚁的监测与防治措施提供科学依据,本研究调查了广州地区（113°45′E,22°43′N）红火蚁工蚁巢外活动日节律和季节性变化,以及有翅蚁的婚飞活动。结果表明,工蚁巢外活动日节律随季节或月份的不同而存在着明显差异：其中12-2月份的日活动节律为单峰型,即在中午温度较高时数量较大,而在5-10月份的日活动节律为双峰型,即在上午或下午工蚁数量较大,其余月份的活动为不明显的双峰型。工蚁巢外活动数量和时间随季节的不同而有显著差异。其中以6月和10月份的日活动数量最多,而在1月和2月份的活动数量最少,时间最短,其余月份活动数量居中。工蚁在阵雨前后的活动数量明显多于晴天,但处于降雨时刻的巢外工蚁数量极少。蚁巢受侵扰后出巢工蚁数量在30 s内最大,之后便随时间的延长而逐渐减少,该种现象可以用房室函数进行描述。另外,工蚁的巢外数量会随侵扰强度的加大而增加。在试验区内全年都可见到红火蚁婚飞,但婚飞活动主要集中在3-5月份,每日婚飞活动主要发生在下午,并主要发生在雨前或雨后。上述结果对于了解我国红火蚁的发生危害规律和提升其监控技术水平有较大参考价值。     

Relationships between stream macroinvertebrate communities and new flood‐based indices of glacial influence

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Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time

Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 10⁶ spermatozoa/mL in a TES–Tris–Fructose–based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at −196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin–fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.     

Human deciduous mandibular molar incremental enamel development

Quantitative studies of incremental markings retained within human enamel have reconstructed the duration and rate (crown and cusp formation times, initiation and completion, daily enamel secretion rates) of permanent tooth development. This approach has provided one way of estimating human age‐at‐death, and facilitated comparative dental studies of primate evolution. Similar applications from deciduous enamel are inhibited because developmental reconstructions from incremental markings for these teeth are less frequently reported in the literature. This study quantified the duration and rate of enamel development for mesial (protoconid, metaconid) and distal cusps (hypoconid, entoconid) for first (dm1) and second (dm2) deciduous mandibular molars from an archaeological sample of modern human juveniles. Crown formation time can be calculated from the dm1 protoconid because growth initiates and completes in this cusp, and from the dm2 protoconid combined with the final period of hypoconid growth. The dm1 postnatal crown formation time included the time taken for the tubercle of Zuckerkandl to develop, and differed slightly compared to radiographic methods. The majority of dm1 protoconid cuspal (occlusal region) enamel formed before birth. The dm2 entoconid enamel formed mainly after birth. Birth reduced daily enamel secretion rates, changed the visibility of incremental markings, and disrupted enamel growth for 3 to 8 days. Findings presented here can contribute to age‐at‐death estimates for human infants aged 13‐postnatal months or less, and should facilitate comparisons of primate deciduous incremental enamel development in an evolutionary context. Regression equations are included so that cuspal formation time can be estimated from enamel thickness. Am J Phys Anthropol, 2011. © 2010 Wiley‐Liss, Inc.