A new approach of successful denaturation of human sperm chromatin without undergoing decondensation treatment

Due to the highly folded chromatin in human sperm, a proper nuclear swelling was highly required to localize certain DNA inside the sperm nuclei. Therefore, previous method for denaturation of sperm chromatin had to adopt chemical agents of decondensation treatment using Heparin/DTT or LIS, directly applied into the sperm cell before further examinations by FISH. Nevertheless, authors still had questions arising on the efficiency of decondensation process which is directly related to the quality of fluorescence signals, which, in turn, underlies the reliability of the results in frequencies and compositions as that still not a proper solution to overcome the major limitation in sperm studies. In this study, we approached a newly improved denaturation process of sperm chromatin without undergoing decondensation treatments that intact human sperms were used as the first time to localize examined DNA, and also two rounds of sequential FISH was carried out in the same sperm cell for the first time to investigate an idea of nullisomy of given chromosomes. From the results, all the variable centromeric compositions of sperm chromosomes 7, 8, and sex chromosomes revealed with significantly given frequencies of monosomy, disomy and nullisomy. Moreover, nullisomy was identified as a true absence of given chromosome rather than technical error of hybridization failure under decondensation. From the findings by our modified denaturation of human sperm chromatin without undergoing decondensation treatment, we strongly believe that more advanced and deep studies in human sperm of nuclear architecture and frequencies can be progressed with significantly reliable results.     

Sequence polymorphisms in ribosomal RNA genes and variations in chromosomal loci of Oenothera odorata and O. laciniata

Numerous studies on Oenothera species have been investigated for the physiological and ecological characteristics. However, no such an information based on molecular cytogenetic has yet been introduced, in turn, is very essential for identifying sequence polymorphisms of rRNA genes with their loci on mitotic phases for further biological researches. In this study, sequence variations of rRNA genes in Oenothera odorata and O. laciniata were examined to identify informative factors as unique or repeat sequences in intra- and inter-specific variations. Intra-specific variation revealed that the sequences of the rRNA genes including the spacer regions were highly conserved revealing only a few variations. From the inter-specific variation, spacer regions of species were completely different as (1) non-homologous sequences in NTS and (2) different type repeat sequences in ITS 1, 2 and 5.8S rRNA, whereas the remaining coding regions were highly conserved. FISH was carried out on mitotic phases using the 5S rDNA of the analyzed sequences. From the interphase and metaphase chromosomes of the examined species, two loci of 5S rDNA in O. odorata and four loci in O. laciniata were confirmed on the telomeric region of the short arm. Due to the small size and unclear centromere of the chromosomes, karyotype could not be completed. However, we confirmed that the chromosomes are organized by meta- and acrocentric chromosomes and the chromosomes with identified loci were assumed to be paired by the location of loci at the telomeric region on the short arm with relative lengths.     

Gender Difference in the Epidemiological Association between Metabolic Syndrome and Olfactory Dysfunction: The Korea National Health and Nutrition Examination Survey

Metabolic syndrome (MetS) is associated with a higher risk of morbidity and/or mortality for various chronic diseases. The aim of this study was to investigate the relationships of MetS and its components with olfactory dysfunction in a representative Korean population. We analyzed the data from the Korean National Health and Nutrition Examination Survey (2008–2010). A total of 11,609 adults who underwent otolaryngological examination were evaluated. The olfactory function was classified as normosmia or hyposmia by a self-report questionnaire according to the sense problems of smell during the past 3 months. MetS was diagnosed if a participant had at least three of the following: (1) WC ≥90 cm in men and ≥80 cm in women; (2) fasting blood sugar ≥ 100 mg/dL or medication use for elevated glucose; (3) fasting triglyceride ≥ 150 mg/dL or cholesterol-lowering medication use; (4) HDL-cholesterol <40 mg/dL in men and <50 mg/dL in women or cholesterol-lowering medication use; and (5) SBP ≥ 130 mmHg and/or DBP ≥ 85 mmHg or antihypertensive drug use for patients with a history of hypertension. The prevalence of olfactory dysfunction in the study population was 6.3%. The prevalence of olfactory dysfunction was significantly higher in older people with MetS than in those without MetS in both sexes (male, 42.0 ± 3.4% vs. 34.7 ± 0.9%, p = 0.0354; female, 46.2 ± 2.8% vs. 37.8 ± 0.8%, p = 0.0026). However, elevated waist circumference, elevated fasting glucose, elevated triglycerides, reduced HDL cholesterol, elevated blood pressure, severe stress, depressed mood, and suicidal ideation were significantly associated with olfactory dysfunction only in women. After controlling for confounders, olfactory dysfunction was significantly associated with MetS (odds ratio, 1.352; 95% confidence interval, 1.005–1.820) only in women. MetS are associated with olfactory dysfunction only in Korean women.     

Oxidative Stress Triggers Body-Wide Skipping of Multiple Exons of the Spinal Muscular Atrophy Gene

Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), the most frequent genetic cause of infant mortality. While SMN2 cannot compensate for the loss of SMN1 due to predominant skipping of exon 7, correction of SMN2 exon 7 splicing holds the promise of a cure for SMA. Previously, we used cell-based models coupled with a multi-exon-skipping detection assay (MESDA) to demonstrate the vulnerability of SMN2 exons to aberrant splicing under the conditions of oxidative stress (OS). Here we employ a transgenic mouse model and MESDA to examine the OS-induced splicing regulation of SMN2 exons. We induced OS using paraquat that is known to trigger production of reactive oxygen species and cause mitochondrial dysfunction. We show an overwhelming co-skipping of SMN2 exon 5 and exon 7 under OS in all tissues except testis. We also show that OS increases skipping of SMN2 exon 3 in all tissues except testis. We uncover several new SMN2 splice isoforms expressed at elevated levels under the conditions of OS. We analyze cis-elements and transacting factors to demonstrate the diversity of mechanisms for splicing misregulation under OS. Our results of proteome analysis reveal downregulation of hnRNP H as one of the potential consequences of OS in brain. Our findings suggest SMN2 as a sensor of OS with implications to SMA and other diseases impacted by low levels of SMN protein.     

Constitutive expression of a fungus-inducible carboxylesterase improves disease resistance in transgenic pepper plants

Main conclusion

Resistance against anthracnose fungi was enhanced in transgenic pepper plants that accumulated high levels of a carboxylesterase, PepEST in anthracnose-susceptible fruits, with a concurrent induction of antioxidant enzymes and SA-dependent PR proteins. A pepper esterase gene (PepEST) is highly expressed during the incompatible interaction between ripe fruits of pepper (Capsicum annuum L.) and a hemibiotrophic anthracnose fungus (Colletotrichum gloeosporioides). In this study, we found that exogenous application of recombinant PepEST protein on the surface of the unripe pepper fruits led to a potentiated state for disease resistance in the fruits, including generation of hydrogen peroxide and expression of pathogenesis-related (PR) genes that encode mostly small proteins with antimicrobial activity. To elucidate the role of PepEST in plant defense, we further developed transgenic pepper plants overexpressing PepEST under the control of CaMV 35S promoter. Molecular analysis confirmed the establishment of three independent transgenic lines carrying single copy of transgenes. The level of PepEST protein was estimated to be approximately 0.002 % of total soluble protein in transgenic fruits. In response to the anthracnose fungus, the transgenic fruits displayed higher expression of PR genes, PR3, PR5, PR10, and PepThi, than non-transgenic control fruits did. Moreover, immunolocalization results showed concurrent localization of ascorbate peroxidase (APX) and PR3 proteins, along with the PepEST protein, in the infected region of transgenic fruits. Disease rate analysis revealed significantly low occurrence of anthracnose disease in the transgenic fruits, approximately 30 % of that in non-transgenic fruits. Furthermore, the transgenic plants also exhibited resistance against C. acutatum and C. coccodes. Collectively, our results suggest that overexpression of PepEST in pepper confers enhanced resistance against the anthracnose fungi by activating the defense signaling pathways.

     

Retraction Note to: Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis

Applied Microbiology and Biotechnology -