Transduced Tat-glyoxalase protein attenuates streptozotocin-induced diabetes in a mouse model

Diabetes mellitus (DM) is characterized by hyperglycemia. Glyoxalase 1 (GLO) has considerable potential as a possible therapeutic agent for DM. However, the precise action of GLO remains unclear in DM. In this study, we examined the protective effects of GLO protein in a streptozotocin (STZ)-induced diabetes animal model using cell-permeable Tat-GLO protein. Purified Tat-GLO protein was efficiently transduced into RINm5F cells in a time- and dose-dependent manner and protected cells against sodium nitroprusside (SNP)-induced cell death and DNA fragmentation. Furthermore, Tat-GLO protein significantly inhibited blood glucose levels and altered the serum biochemical parameters in STZ-induced diabetic mice. These results demonstrate that transduced Tat-GLO protein protects pancreatic cells by the inhibition of STZ-mediated toxicity. Therefore, Tat-GLO protein could be useful as a therapeutic agent against DM.     

The Rho1 GTPase acts together with a vacuolar glutathione S-conjugate transporter to protect yeast cells from oxidative stress

Maintenance of redox homeostasis is critical for the survival of all aerobic organisms. In the budding yeast Saccharomyces cerevisiae, as in other eukaryotes, reactive oxygen species (ROS) are generated during metabolism and upon exposure to environmental stresses. The abnormal production of ROS triggers defense mechanisms to avoid the deleterious consequence of ROS accumulation. Here, we show that the Rho1 GTPase is necessary to confer resistance to oxidants in budding yeast. Temperature-sensitive rho1 mutants (rho1(ts)) are hypersensitive to oxidants and exhibit high accumulation of ROS even at a semipermissive temperature. Rho1 associates with Ycf1, a vacuolar glutathione S-conjugate transporter, which is important for heavy metal detoxification in yeast. Rho1 and Ycf1 exhibit a two-hybrid interaction with each other and form a bimolecular fluorescent complex on the vacuolar membrane. A fluorescent-based complementation assay suggests that the GTP-bound Rho1 associates with Ycf1 and that their interaction is enhanced upon exposure to hydrogen peroxide. The rho1(ts) mutants also exhibit hypersensitivity to cadmium, while cells carrying a deletion of YCF1 or mutations in a component of the Pkc1-MAP kinase pathway exhibit little or minor sensitivity to oxidants. We thus propose that Rho1 protects yeast cells from oxidative stress by regulating multiple downstream targets including Ycf1. Since both Rho1 and Ycf1 belong to highly conserved families of proteins, similar mechanisms may exist in other eukaryotes.     

An assessment of orchids’ diversity in Penang Hill,Penang, Malaysia after 115 years

A comprehensive study on the orchid diversity in Penang Hill, Penang, Malaysia was conducted from 2004 to 2008 with the objective to evaluate the presence of orchid species listed by Curtis (J Strait Br R Asiat Soc 25:67–173, 1894) after more than 100 years. A total of 85 species were identified during this study, of which 52 are epiphytic or lithophytic and 33 are terrestrial orchids. This study identified 57 species or 64.8% were the same as those recorded by Curtis (1894), and 78 species or 66.1% of Turner’s (Gardens’ Bull Singap 47(2):599–620, 1995) checklist of 118 species for the state of Penang including 18 species which were not recorded by Curtis (1894) and the current study but are actually collected from Penang Hill. A comparison table of the current findings against Curtis (1894) and Turner (1995) is provided which shows only 56 species were the same in all three studies. The preferred account for comparison was Curtis’ (1894) list as his report was specifically for the areas around Penang Island especially Penang Hill, Georgetown and Ayer Itam areas. Our study reveals that about 50% of Curtis’ collection localities have been converted to residential areas and agricultural land, and this probably explains the decreasing numbers of species found in the current study especially for the terrestrial species as epiphytic species have better adaptation capabilities towards environmental changes. Seven species were identified as new records to Penang Hill as they were not recorded by Curtis (1894). None of the three species recorded as endemic to Penang by Turner (1995) was recollected during the current study, of which only Zeuxine rupestris was in Curtis’ (1894) list. Overall, we concluded that Penang hill harbours at least 136 species of orchids of which 78 species or 57.4% were recollected in this study. This also indicates that this area is still suitable for orchid growth even though it is surrounded by rapid development and mass conversion of forested land into fruit orchards and residential area. The designation of Penang Hill as a Permanent Forest Reserve would better guarantee the survival of some orchid species unless human interventions and climatic changes occur.     

PEP-1-PON1 Protein Regulates Inflammatory Response in Raw 264.7 Macrophages and Ameliorates Inflammation in a TPA-Induced Animal Model

Paraoxonase 1 (PON1) is an antioxidant enzyme which plays a central role in various diseases. However, the mechanism and function of PON1 protein in inflammation are poorly understood. Since PON1 protein alone cannot be delivered into cells, we generated a cell permeable PEP-1-PON1 protein using protein transduction domains, and examined whether it can protect against cell death in lipopolysaccharide (LPS) or hydrogen peroxide (H₂O₂)-treated Raw 264.7 cells as well as mice with 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced skin inflammation. We demonstrated that PEP-1-PON1 protein transduced into Raw 264.7 cells and markedly protected against LPS or H₂O₂-induced cell death by inhibiting cellular reactive oxygen species (ROS) levels, the inflammatory mediator’s expression, activation of mitogen-activated protein kinases (MAPKs) and cellular apoptosis. Furthermore, topically applied PEP-1-PON1 protein ameliorates TPA-treated mice skin inflammation via a reduction of inflammatory response. Our results indicate that PEP-1-PON1 protein plays a key role in inflammation and oxidative stress in vitro and in vivo. Therefore, we suggest that PEP-1-PON1 protein may provide a potential protein therapy against oxidative stress and inflammation.     

PEP-1–metallothionein-III protein ameliorates the oxidative stress-induced neuronal cell death and brain ischemic insults

Background

Oxidative stress is considered to be involved in a number of human diseases including ischemia. Metallothioneins (MT)-III can protect neuronal cells from the cytotoxicity of reactive oxygen species (ROS). However, MT-III proteins biological function is unclear in ischemia. Thus, we examined the protective effects of MT-III proteins on oxidative stress-induced neuronal cell death and brain ischemic insult.

Methods

A human MT-III gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1–MT-III protein. PEP-1–MT-III protein was purified using affinity chromatograph. Transduced PEP-1–MT-III proteins were detected by Western blotting and immunoflourescence. Cell viability and DNA fragmentation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay and terminal dexoynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Brain ischemic injury was detected with immunohistochemistry.

Results

Purified PEP-1–MT-III proteins transduced into astrocytes in a time- and dose-dependent manner and protected against oxidative stress-induced cell death. Also, transduced PEP-1–MT-III proteins efficiently protected cells against DNA fragmentation. Furthermore, immunohistochemical analysis revealed that PEP-1–MT-III prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. We demonstrated that transduced PEP-1–MT-III protein protects against oxidative stress induced cell death in vitro and in vivo.

General significance

Transduced PEP-1–MT-III protein has neuroprotective roles as an antioxidant in vitro and in vivo. PEP-1–MT-III protein is a potential therapeutic agent for various human brain diseases such as stroke, Alzheimer's disease, and Parkinson's disease.     

Capparis dongvanensis sp. nov. (Capparaceae) from Vietnam

Capparis dongvanensis (Capparaceae) is described as a new species and illustrated from Ha Giang Province of Vietnam. It is morphologically similar to C. lanceolaris and C. fengii, but differs in the number of flowers on peduncles, length of spine and petiole, apex of the leaf, type of inflorescence, petal color, cylindrical ovary without any beak, fruit shape and number of seeds per fruit. Diagnostic morphological characters along with pollen and seed SEM images of C. dongvanensis and allied species are provided. Its ecology, habitat and conservation status are also described.     

Human brain pyridoxal-5'-phosphate phosphatase (PLPP):protein transduction of PEP-1-PLPP into PC12 cells

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin B(6) precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin B(6).     

Organization of calbindin D28K-immunoreactive neurons in the dog superior colliculus

We localized calbindin D28K-immunoreactive (IR) neurons in the superior colliculus (SC) of the dog and studied the distribution and effect of enucleation on the distribution of this protein. We also compared this labeling to that of GABA. Calbindin D28K was localized with antibody immunocytochemistry. Calbindin D28K-IR neurons formed three laminar tiers in the SC, one within the lower superficial gray layer (SGL), the second within the upper intermediate gray layers (IGL), and the third within the deep gray layer (DGL). The third tier was not very distinctive when compared with the other two tiers. Calbindin D28K-IR neurons in the SC varied dramatically in morphology and size, and included round/oval, vertical fusiform, stellate, pyriform, and horizontal neurons. Neurons with varicose dendrite were also labeled in the IGL. Enucleation appeared to have no effect on the distribution of calbindin D28K-IR neurons in the contralateral SC. Two-color immunofluorescence revealed that a small percentage (11.20%) of calbindin D28K-IR neurons co-localized with GABA. The current results demonstrate that the patterned distribution of calbindin D28K-IR neurons in the intermediate and deep SC is comparable with other animals, but that the distribution of this protein in the superficial SC is strikingly different from that in previously studied animals. The results also suggest that retinal projection may not control the activity of the expression of calbindin D28K in the dog SC. These results will not only provide valuable knowledge of the basic neurochemical architecture of the dog visual system, but also provide clues for the understanding of the similarities and differences among species.