A Novel Domain of Amino-Nogo-A Protects HT22 Cells Exposed to Oxygen Glucose Deprivation by Inhibiting NADPH Oxidase Activity

This study aimed to investigate the protective effect of the M9 region (residues 290–562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia–reperfusion induced by oxygen–glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4 μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.     

Characterization of the N-methyltransferase CalM involved in calcimycin biosynthesis by Streptomyces chartreusis NRRL 3882

Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the calcimycin biosynthesis gene cluster is required for calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl calcimycin to calcimycin in vitro. The enzyme was determined to have a kinetic parameter of K_m 276 μM, k_cat 1.26 min⁻¹ and k_cat/K_m 76.2 M⁻¹ s⁻¹. These results proved that CalM is a N-methyltransferase that is required for calcimycin biosynthesis, and they set the stage for generating much desired novel calcimycin derivatives by rational genetic and chemical engineering.     

hABCF3, a TPD52L2 interacting partner, enhances the proliferation of human liver cancer cell lines in vitro

In the present study, we characterized an evolutionarily conserved non-transmembrane ATP-binding cassette protein: hABCF3. Subcellular immunofluorescence staining demonstrated that hABCF3 localizes preferentially in cytoplasm, unlike its paralog protein hABCF1, which localizes in both cytoplasm and nucleus. Quantitative realtime PCR analysis revealed that hABCF3 is expressed in all tissues examined, with high expression level in heart, liver, and pancreas. Interestingly, ectopic hABCF3 promoted proliferation of human liver cancer cell lines. Moreover, knock down of hABCF3 protein expression by siRNA inhibited cell proliferation. In addition, we identified TPD52L2 (Tumor Protein D52-like 2) as a hABCF3 interacting protein via yeast two-hybrid. This interaction was further confirmed by in vivo co-immunoprecipitation and co-localization assays. Furthermore, we identified the interactional region of hABCF3 to be the first 200 amino acids uncharacterized region. Notably, the truncated version of hABCF3, which lacks the TPD52L2 binding region, remarkably impaired hABCF3-mediated cell proliferation. Taken together, these findings suggest that hABCF3 positively regulates cell proliferation, at least partially through the interaction with a tumor protein D52 protein family member: TPD52L2.     

Neuroprotective effect of 2-(4-methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-d-pyranoside against sodium nitroprusside-induced neurotoxicity in HT22 cells

In the present investigation, we used directed evolution approach to engineer a lipase from metagenomic origin. A variant S311C, was generated, characterized in detail and compared with wild type. Wild type and variant lipases were overexpressed and purified to homogeneity. The temperature optima of the purified lipases (Variant and wild type) were almost same, and found to be 45 and 50 °C, respectively. The variant protein was highly thermostable (54 times) as compared with the wild type at 60 °C. The variant displayed very high kinetic efficiency over the wild type protein. Analysis of the homology models of wild type and variant lipase showed that the substitution is on the surface of the protein. This substitution, along with hydrophobic residues in near vicinity may be involved in formation of strong hydrophobic channel leading to active site. This study identifies the role of hydrophobic interactions in protein stability along with enhancement of enzyme activity.     

Structural development of aleurone and its function in common wheat

The wheat aleurone is formed from surface endosperm cells, and its developmental status reflects its biogenesis, structural characteristics, and physiological functions. In this report, wheat caryopses at different development stages were embedded in Spurr’s low-viscosity embedding medium for observation of the development of aleurone cells (ACs) by light microscopy, scanning electron microscopy, and fluorescence microscopy, respectively. According to their structures and physiological characterization, the ACs development process was divided into five stages: endosperm cellulization, spherosome formation, aleurone grain formation, filling material proliferation, and maturation. Furthermore, ACs in different parts of the caryopsis formed differently. ACs near the vascular bundle developed earlier and formed transfer cells, but other ACs formed slowly and did not form transfer cells. ACs on the caryopsis backside were a regular square shape; however, ACs in the caryopsis abdomen were mainly irregular. There were also differences in development between wheat varieties. ACs were rectangular in hard wheat but square in soft wheat. ACs were larger and showed a greater degree of filling in hard compared to soft wheat. The storage materials in ACs were different compared to inner endosperm cells (IECs). The concentrations of minerals such as sodium, magnesium, silicon, phosphorus and potassium were higher in ACs than in IECs. ACs contained many aleurone grains and spherosomes, which store lipids and mineral nutrients, respectively. The cell nucleus did not disappear and the cells were still alive during aleurone maturation. However, IECs were dead and mainly contained amyloplast and protein bodies, which store starch and protein, respectively. Overall, the above results characterized major structural features of aleurone and revealed that the wheat aleurone has mainly four functions.     

Isolation and characterization of Histone1 gene and its promoter from tea plant (Camellia sinensis)

One 1.2 kbp long sequence was cloned by using PCR with primers that were designed from cDNA sequence of CsH1 gene (Genbank: EU716314) from tea plant (Camellia sinensis). According to the 1.2 kbp sequence, a 0.6 kbp sequence was isolated from tea plant genomic DNA using DNA Walking Method. Sequence analysis revealed that the 1.2 kbp sequence is a CsH1 gene consisting of 1 exon and 2 introns, the border of exton and intron sequences conforming to the GT–AG rule, and the 0.6 kbp sequence was found to be the promoter of CsH1 gene which contains basic promoter elements, TATA-box and CAAT-box. Abscisic acid responsiveness cis-acting element, elictor-responsive element, GA response element, light response cis-acting element and TC-rich repeats were also represented. To further study the activity of this promoter, the sequence was used to drive a GUS fusion gene in Agrobacterium-mediated transformation of tea plant somatic embryos, leaf discs and calli of tobacco (Nicotiana tabacum L.) where a high level of GUS expression was both observed in the tobacco calli and tea plant somatic embryos. These results suggest that the CsH1 gene promoter isolated is capable of conferring nuclear gene expression.     

Association of angiotensinogen M235T gene polymorphism with end-stage renal disease risk: a meta-analysis

Association between angiotensinogen (AGT) M235T gene polymorphism and end-stage renal disease (ESRD) risk is still controversial. This meta-analysis was performed to evaluate the association of AGT M235T gene polymorphism with ESRD susceptibility. A predefined literature search and selection of eligible relevant studies were performed to collect data from electronic databases of PubMed, Embase and Cochrane Library. Sixteen literatures were identified for the analysis of association of AGT M235T gene polymorphism with ESRD risk. T allele and TT genotype were associated with ESRD susceptibility in Caucasians (T: OR = 1.13, 95 % CI: 1.02–1.25, P = 0.02; TT: OR = 1.22, 95 % CI: 1.03–1.45, P = 0.02). However, MM genotype might not play a protective role against ESRD risk in Caucasians. Furthermore, there was no a markedly positive association between AGT M235T gene polymorphism and ESRD susceptibility in overall populations, Asians and Africans. In conclusion, T allele or TT homozygote is associated with the onset of ESRD in Caucasians. However, more studies should be performed in the future.     

Gangrenous Cutaneous Mucormycosis Caused by Rhizopus oryzae: A Case Report and Review of Primary Cutaneous Mucormycosis in China Over Past 20 Years

Cutaneous mucormycosis is a rare opportunistic infection caused by zygomycetes that can be rapidly fatal if unrecognized. We describe the clinical, histopathological, fungal and molecular features of a case of gangrenous cutaneous mucormycosis. The patient presented with great necrosis on his right forearm at the site of detained intravenous cannula needle. He had type II diabetes and chronic renal insufficiency. KOH mount of black eschar showed many broad, aseptate fungal hyphae with right-angle branching. PAS staining of the tissue sample revealed similar broad hyphae in the dermis and cutis. Fungal culture and ITS sequence analysis identified this fungus as Rhizopus oryzae. As no organ involvement was detected, the patient was diagnosed with primary cutaneous mucormycosis. Considering the poor state of the patient, complete excision of the infectious tissue was performed without skin graft instead of amputation. At the same time, intravenous liposomal amphotericin B was given, starting from a small dosage and increased to a total dosage amount of 5.45 g. The wound recovered well with granulation. We emphasize that early recognition and prompt therapy including the control of the primary diseases were important. In this article, we also reviewed the features of primary cutaneous mucormycosis reported in China over the last 20 years.