Identification of 4-hydroxynonenal-modified retinal proteins induced by photooxidative stress prior to retinal degeneration

4-Hydroxynonenal (4-HNE) is a reactive aldehyde species generated endogenously from the nonenzymatic oxidation of n-6 polyunsaturated fatty acids under physiological conditions. We have reported that intense white light exposure increases 4-HNE-protein modification in the retina prior to the onset of photoreceptor cell apoptosis. To understand the molecular mechanism(s) underlying the retinal degeneration induced by photooxidative stress, we identified 4-HNE-modified retinal proteins using a proteomic approach. Albino rats were exposed to 5 k lx white fluorescent light for 3 h and retinas were removed 24 h later and pooled. By Western dot blot analysis, the total intensity of 4-HNE-modified proteins was increased 1.5-fold following the exposure compared to dim light controls. In two independent sets of two-dimensional gel electrophoresis/Western blots followed by peptide mass fingerprinting (PMF), nine proteins including voltage-dependent anion channel, enolase 1, aldolase C, crystallins A and βB3, heterogeneous nuclear ribonucleoprotein A2/B1, albumin, and glutamine synthetase were identified. We observed that 4-HNE modifications of retinal proteins are specific to a particular set of proteins rather than random events on abundant proteins. By immunohistochemistry, localization of 3 identified proteins overlapped with immunoreactivity of 4-HNE-modified proteins in light-exposed retinas. Intense light exposure increases 4-HNE-protein modifications on specific retinal proteins in several functional categories including energy metabolism, glycolysis, chaperone, phototransduction, and RNA processing. Together with previous reports that 4-HNE modification changes protein activities, these results suggest a close association of 4-HNE-protein modifications with the initiation of light-induced retinal degeneration.     

A Hospital-Based Serological Survey of Cryptosporidiosis in the Republic of Korea

The seroprevalence of cryptosporidiosis was examined using patients'' sera collected from hospitals located in 4 different areas of the Republic of Korea. ELISA was used to measure antibody titers against Cryptosporidium parvum antigens from a total of 2,394 serum samples, which were collected randomly from patients in local hospitals; 1) Chungbuk National University Hospital, 2) Konkuk University Hospital, 3) local hospitals in Chuncheon, Gangwon-do (province), 4) Jeonnam National University Hospital, from 2002 through 2003. Of the 2,394 samples assayed, 34%, 26%, and 56% were positive for C. parvum-specific IgG, IgM, and IgA antibodies, respectively. Positive IgG titers were most common in sera from Jeonnam National University Hospital, Gwangju, Jeollanam-do, and positive IgM titers were most common in sera from Chungbuk National University Hospital, Cheongju, Chuncheongbuk-do. The seropositivity was positively correlated with age for both the IgG and IgA antibodies but was negatively correlated with age for the IgM antibodies. Western blotting revealed that 92%, 83%, and 77% of sera positive for IgG, IgM, and IgA ELISA reacted with 27-kDa antigens, respectively. These results suggested that infection with Cryptosporidium in hospital patients occurs more commonly than previously reported in the Republic of Korea.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.     

Protein Extraction of Formalin-fixed, Paraffin-embedded Tissue Enables Robust Proteomic Profiles by Mass Spectrometry

Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples has been confounded by formalin-induced protein cross-linking. The performance of extracted proteins in a liquid chromatography tandem MS format from FFPE samples and extracts from whole and laser capture microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)–embedded matched control rat liver samples were compared. Extracts from FFPE and frozen/OCT–embedded livers from atorvastatin-treated rats were further compared to assess the performance of FFPE samples in identifying atorvastatin-regulated proteins. Comparable molecular mass representation was found in extracts from FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less for the FFPE sample. The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples. Subcellular representation in FFPE samples was similar to OCT-frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin-treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis. (J Histochem Cytochem 57:849–860, 2009)     

A collaboration of aquaporins handles water transport in relation to the estrous cycle in the bitch uterus

Fluid movement through uterine cell membranes is crucial, as it can modulate the tissue imbibition pattern in the different phases of the estrous cycle. To gain insight into the mechanisms underlying steroid-controlled water handling, the presence and distribution of aquaporins (AQPs), integral membrane channel proteins permitting rapid passive water movement, was explored in bitch uterine tissues. Immunohistochemistry and Western immunoblot analysis were used to study the presence of AQP1, AQP2, and AQP5 in the layers of the bitch uterine wall during the different estrous phases. Presence of endothelial nitric oxide-generating enzyme NO synthase (NOS3) was also investigated, as it is known that the vasodilator NOS3 might be involved in the development of uterine edema. The results demonstrated the following: (1) AQP1, AQP2, and AQP5 were present in the uterus of cycling bitches. (2) AQP1 was localized within uterine mesometrial, myometrial, and endometrial blood vessels and in the circular and longitudinal layers of myometrium. AQP1 localization and expression were unaffected by the estrous cycle. (3) The estrogenic milieu was probably at the basis of AQP2 expression in the glandular and luminal epithelium of the endometrium. (4) AQP5 water channels were present in the apical plasma membrane of uterine epithelial cells in coincidence with plasma progesterone increase. (5) NOS3 was localized in the myometrial and epithelial tissues as well as in blood vessels indicating a contribution of this vasoactive peptide to the uterine imbibition processes. Thus, we can hypothesize that a functional and distinctive collaboration exists among diverse AQPs in water handling during the different functional uterine phases.     

Mass spectrometry‐based absolute quantification of microsomal cytochrome P450 2D6 in human liver

We describe the assay of the human cytochrome P450 2D6 in a set of 30 genotyped liver samples using the ‘absolute quantification’ (AQUA) technique. We found approximately 30 fmol CYP2D6 per μg of microsomal protein, with the values spanning from 0 to nearly 80 fmol/μg. This is greater by a factor of 5–10 from the compared to the currently accepted value, which was around 5 fmol/μg. Our results thus suggest that the amount of cytochrome P450 (CYP) enzymes in liver have to be reassessed. We used quantitative Western blotting, calibration standards and activity assays, to validate the results. Our results show, that using the AQUA technique a true assay of CYP2D6 in human liver was possible.     

琼脂糖凝胶中DNA片段的挤压回收法

为了简化琼脂糖凝胶中DNA片段的回收,该文报道一种新的挤压回收法。将含有DNA片段的凝胶块放在折叠的封口膜之间,然后用一小塑料平板将凝胶块中的DNA溶液用力挤出,用移液枪把所有挤压出的DNA溶液放入effendorf管中,然后用常规的苯酚抽提法进行纯化。DNA回收率达到40-60%(w/w)。该方法简单而有效,且回收的DNA能够直接应用于酶切、连接及PCR等各种分子生物学操作。     

Vector fragmentation: Characterizing vector integrity in transfected clones by Southern blotting

The Chinese Hamster Ovary production cell line development process using methotrexate (MTX) amplification is well studied and commonly used for biopharmaceutical processes. However, successful MTX amplification varies from clone to clone and suggested reasons include vector fragmentation during the transfection process and genomic rearrangement of the Chinese Hamster Ovary chromosomes. Here, we elucidated the vector integration patterns of 40 transfected single‐cell clones by Southern blotting and showed that vector fragmentation occurs at a significant level in our experiment. This concurs with MTX amplification studies implying that single‐cell cloning is necessary to ensure a successful amplification process. Truncations at the ends of the integrated vectors were also observed, whereas gross DNA insertions were not detected in our data. This suggests that end deletions are common, whereas insertion events are rare in animal cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A combined RT‐PCR and dot‐blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius)

Aims: To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red‐spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. Methods and Results: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin‐labelled probes resulted in the identification of both genotypes separately. The application of the RT‐PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46·87% of viral nervous necrosis virus carriers. The combination of RT‐PCR and blot hybridization increases the detection rate up to 90·62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56·25%). Conclusions: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. Significance and Impact of the Study: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.     

高效薄层层析-病毒覆盖结合法比较细胞表面神经节苷脂与不同动物源性新城疫病毒结合特性

【目的】神经节苷脂是新城疫病毒入侵宿主细胞的受体,但不同动物源性的新城疫病毒利用受体的特性是否存在差异尚不明确。以鹅源新城疫病毒NA-1株和鸡源新城疫病毒F48E9株为研究对象,比较两株病毒受体结合特性的差异。【方法】提取鸡胚和鹅胚成纤维细胞新城疫病毒受体成分——神经节苷脂,用高效薄层层析法比较两种细胞所含神经节苷脂的类型和含量;通过高效薄层层析-病毒覆盖结合法比较两种病毒与不同细胞神经节苷脂的结合特性,最后通过病毒红细胞吸附抑制试验进一步验证神经节苷脂与病毒之间的相互作用关系。【结果】鸡胚和鹅胚成纤维细胞所含的神经节苷脂成分存在明显差异;高效薄层层析-病毒覆盖结合实验结果显示NA-1和F48E9与神经节苷脂的结合模式不同,NA-1主要与神经节苷脂GD1a结合,即包含双SAα2,3Gal末端的神经节苷脂,而F48E9可与更多类型的神经节苷脂结合,从单唾液酸到三唾液酸形式的神经节苷脂如GM1、GD1a、GD1b、GT1b等。【结论】鹅源新城疫病毒NA-1株和鸡源新城疫病毒F48E9株在入侵靶细胞时优先选择利用的受体不同。