Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway

Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.     

Involvement of pigment epithelium-derived factor,docosahexaenoic acid and neuroprotectin D1 in corneal inflammation and nerve integrity after refractive surgery

Alterations in corneal innervations result in impaired corneal sensation, severe dry eye and damage to the epithelium that may in turn lead to corneal ulcers, melting and perforation. These alterations can occur after refractive surgery. We have discovered that pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA or the docosanoid bioactive neuroprotectin D1 (NPD1)) induces nerve regeneration after corneal surgery that damages the stromal nerves. We found that PEDF is released from corneal epithelial cells after injury, and when DHA is provided to the cells it stimulates the biosynthesis of NPD1 by an autocrine mechanism. The combination of PEDF plus DHA also decreased the production of leukotriene B4 (LTB4), a neutrophil chemotactic factor, thereby decreasing the inflammation induced after corneal damage. These studies suggest that PEDF plus DHA and its derivative NPD1 hold promise as a future treatment to restore a healthy cornea after nerve damage.     

单气囊小肠镜在诊断胶囊内镜检查阴性的可疑小肠疾病患者中的应用价值

目的：探讨单气囊小肠镜（single-balloonenteroscop,SBE）在胶囊内镜检查阴性的可疑小肠疾病患者中的应用价值。方法：选取在我院行胶囊内镜检查无异常发现,后行单气囊小肠镜检查的可疑小肠疾病患者24例,分析后者对胶囊内镜检查阴性可疑小肠疾病患者的阳性发现率和病因的分布特点。结果：24例行SBE检查者有22例（91．7％）获得成功,2例失败,10例被检出阳性病变,其中间质瘤5例,小肠息肉2例,过敏性紫癜1例,血管畸形2例,SBE对胶囊内镜检查阴性的患者小肠疾病的再检出率为41．7％。结论：胶囊内镜和SBE在小肠疾病的诊断上有着各自的优缺点,对于胶囊内镜检查阴性的可疑小肠疾病患者进一步行SBE检查有助于确诊。     

Spectrophotometric measurements of human tissues for the detection of subjacent blood vessels in an endonasal endoscopic surgical approach

Thin slices of human tissues are characterized concerning reflection and transmission in a wavelength range from 400 to 1700 nm. The results are primarily useful to find a wavelength for the detection of subjacent blood vessels during surgical procedures, especially neurological surgery. The measurements have been conducted using a customized measuring station, utilizing two halogen bulb lamps and two spectrometers. This paper focuses on creating a data base with the optical properties of artery, brain, bone, nasal mucosa, and nerve. The spectral distributions are compared among each other, similarities and differences are pointed out. Each tissue has got unique spectral characteristics, whereas typical absorption bands can be found in the overall tissues, especially hemoglobin and water absorption bands. The reflectivity maxima are typically located in the red or near‐infrared. All the transmission maxima are located between 1075 nm and 1100 nm. The measurements have been conducted at the Institute of Anatomy at the University of Leipzig. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

蓬莱玉参早期发育的形态学观察

本研究对仿刺参(Apostichopus japonicus)的一个特殊品系蓬莱玉参产卵、受精及胚胎和幼体发育过程进行显微观察,并与普通仿刺参进行比较。在19~21℃水温下,蓬莱玉参受精卵分别在受精12 min和24 min后释放第一、二极体,1 h后卵裂成2细胞期,之后每30 min左右完成一次卵裂,6 h后进入囊胚期,19 h后发育成原肠胚;40 h后进入耳状幼体阶段,在其后侧臂的一端出现一个不规则形的石灰质骨片,并发现其位置与水体腔处于同一侧这一规律;5 d和8 d后发育为中耳状幼体和大耳状幼体,10 d后变态发育为樽形幼体,骨片由不规则状发育为齿轮状,并出现第二个石灰质骨片;12 d后发育为五触手,14 d后发育成稚参,40 d后发育为幼参。蓬莱玉参胚胎和幼体发育时序与当前已报道的仿刺参无显著差异,但从幼参开始蓬莱玉参通体始终为白色,而普通仿刺参在45日龄时体表局部出现色素,疣足处较为明显,60日龄幼体一半以上全身布满色素。蓬莱玉参因通体纯白色而受到了众多养殖企业和研究领域的关注,本文的结果为其今后的研究奠定了可靠的理论基础。     

A readily usable two‐photon fluorescence lifetime microendoscope

A two‐photon fluorescence lifetime (2P‐FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence, at sub‐cellular resolution, is demonstrated. It exhibits readily usable characteristics such as convenient endoscope probe diameter (≈2 mm), fiber length (>5 m) and data accumulation rate (16 frames per second (fps)), leading to a FLIM refreshing rate of ≈0.1 to 1 fps depending on the sample. The spiral scanning image formation does not influence the instrument response function (IRF) characteristics of the system. Near table‐top microscope performances are achieved through a comprehensive system including a home‐designed spectro‐temporal pulse shaper and a custom air‐silica double‐clad photonic crystal fiber, which enables to reach up to 40 mW of ≈100 fs pulses @ 760 nm with a 80 MHz repetition rate. A GRadient INdex (GRIN) lens provides a lateral resolution of 0.67 μm at the focus of the fiber probe. Intracellular NADH fluorescence lifetime data are finally acquired on cultured cells at 16 fps.      

Oxygen‐dependent delayed fluorescence of protoporphyrin IX measured in the stomach and duodenum during upper gastrointestinal endoscopy

Protoporphyrin IX‐triplet state lifetime technique (PpIX‐TSLT) is a method used to measure oxygen (PO₂) in human cells. The aim of this study was to assess the technical feasibility and safety of measuring oxygen‐dependent delayed fluorescence of 5‐aminolevulinic acid (ALA)‐induced PpIX during upper gastrointestinal (GI) endoscopy. Endoscopic delayed fluorescence measurements were performed 4 hours after oral administration of ALA in healthy volunteers. The ALA dose administered was 0, 1, 5 or 20 mg/kg. Measurements were performed at three mucosal spots in the gastric antrum, duodenal bulb and descending duodenum with the catheter above the mucosa and while applying pressure to induce local ischemia and monitor mitochondrial respiration. During two endoscopies, measurements were performed both before and after intravenous administration of butylscopolamine. Delayed fluorescence measurements were successfully performed during all 10 upper GI endoscopies. ALA dose of 5 mg/kg showed adequate signal‐to‐noise ratio (SNR) values >20 without side effects. All pressure measurements showed significant prolongation of delayed fluorescence lifetime compared to measurements performed without pressure (P < .001). Measurements before and after administration of butylscopolamine did not differ significantly in the duodenal bulb and descending duodenum. Measurements of oxygen‐dependent delayed fluorescence of ALA‐induced PpIX in the GI tract during upper GI endoscopy are technically feasible and safe.     

Purification and sequence determination of a yellow protein from sexually mature males of the desert locust, Schistocerca gregaria

A yellow protein from abdominal cuticle of the desert locust, Schistocerca gregaria, has been purified and its amino acid sequence determined. The yellow color comes from bound carotene, the protein is only deposited in the epidermis and cuticle of male locusts during their sexual maturation, and the deposition is dependent upon a sufficiently high titer of juvenile hormone. The sequence of the protein is atypical for a cuticular protein, but it has some similarity to a putative juvenile hormone binding protein from Manduca sexta. It is suggested that the protein is involved in the transport of carotenes from internal tissues to epidermis and cuticle of the locust.     

Extraction and quantitative analysis of the blue-green pigment “marennine” synthesized by the diatom Haslea ostrearia

This study reports further information on mareninne, awater-soluble blue-green pigment synthesized by the diatom Hasleaostrearia, which is essential to the greening of maturing oysters inFrench production areas. The extraction process is reported, as well aspreliminary characterization of a partially purified marennine extract,including quantitative spectrophotometric analysis of intracellular pigment. Amean specific extinction coefficient, E ^1% _1cm = 17.2 at 669 nm, is proposed. Results forquantitative determination of marennine accumulated in cells during batchcultures are presented, and their importance for pigment production isconsidered in relation to potential industrial applications.     

Expression of pigmentation genes following electroporation of albino<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

A UV-induced albino strain of Monascus purpureus was subjected to electroporation in the presence of genomic DNA from a wild-type red strain of the fungus. Eight colonies expressed color after several weeks of growth. The growth rates of all eight color variants were significantly greater than the recipient and donor strains under some culture conditions. Spectrophotometric analysis of the pigments extracted from the color variants revealed the pigments had absorbance spectra different from the DNA donor strain. These color variants may have resulted from transformation with wild-type DNA, mutation reversion, or activation of alternative pathway(s)—i.e., new mutations—that resulted in pigment production.