Isolation,Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson’s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.     

Cervical cancer development,chemoresistance, and therapy: a snapshot of involvement of microRNA

Cervical cancer (CC) is one of the leading causes of death in women due to cancer and a major concern in the developing world. Persistent human papilloma virus (HPV) infection is the major causative agent for CC. Besides HPV infection, genetic and epigenetic factors including microRNA (miRNA) also contribute to the malignant transformation. Earlier studies have revealed that miRNAs participate in cell proliferation, invasion and metastasis, angiogenesis, and chemoresistance processes by binding and inversely regulating the target oncogenes or tumor suppressor genes. Based on functions and mechanistic insights, miRNAs have been identified as cellular modulators that have an enormous role in diagnosis, prognosis, and cancer therapy. Signatures of miRNA could be used as diagnostic markers which are necessary for early diagnosis and management of CC. The therapeutic potential of miRNAs has been shown in CC; however, more comprehensive clinical trials are required for the clinical translation of miRNA-based diagnostics and therapeutics. Understanding the molecular mechanism of miRNAs and their target genes has been useful to develop miRNA-based therapeutic strategies for CC and overcome chemoresistance. In this review, we summarize the role of miRNAs in the development, progression, and metastasis of CC as well as chemoresistance. Further, we discuss the diagnostic and therapeutic potential of miRNAs to overcome chemoresistance and treatment of CC.

     

In vitro propagation of the medicinal plant Plumbago zeylanica L. Through nodal explants

Summary A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 μM adenine sulfate +2.46 μM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 μM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60–65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.     

Arbuscular Mycorrhizal Fungi Community Structure,Abundance and Species Richness Changes in Soil by Different Levels of Heavy Metal and Metalloid Concentration

Arbuscular Mycorrhizal Fungi (AMF) play major roles in ecosystem functioning such as carbon sequestration, nutrient cycling, and plant growth promotion. It is important to know how this ecologically important soil microbial player is affected by soil abiotic factors particularly heavy metal and metalloid (HMM). The objective of this study was to understand the impact of soil HMM concentration on AMF abundance and community structure in the contaminated sites of South Korea. Soil samples were collected from the vicinity of an abandoned smelter and the samples were subjected to three complementary methods such as spore morphology, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) for diversity analysis. Spore density was found to be significantly higher in highly contaminated soil compared to less contaminated soil. Spore morphological study revealed that Glomeraceae family was more abundant followed by Acaulosporaceae and Gigasporaceae in the vicinity of the smelter. T-RFLP and DGGE analysis confirmed the dominance of Funneliformis mosseae and Rhizophagus intraradices in all the study sites. Claroideoglomus claroideum, Funneliformis caledonium, Rhizophagus clarus and Funneliformis constrictum were found to be sensitive to high concentration of soil HMM. Richness and diversity of Glomeraceae family increased with significant increase in soil arsenic, cadmium and zinc concentrations. Our results revealed that the soil HMM has a vital impact on AMF community structure, especially with Glomeraceae family abundance, richness and diversity.     

Effect of Melatonin on Glutamate: BDNF Signaling in the Cerebral Cortex of Polychlorinated Biphenyls (PCBs)—Exposed Adult Male Rats

Neurochemical Research - Various epidemiological survey suggests that the central nervous system is the target for many environmental contaminants. One among them is Aroclor 1254,...     

Brevibacterium frigoritolerans a novel entomopathogen of Anomala dimidiata and Holotrichia longipennis (Scarabaeidae: Coleoptera)

We describe the isolation, biochemical characterization, phylogenetic analysis, and pathogenecity of a novel entomopathogenic bacterium Brevibacterium frigoritolerans to first instar larvae of Anomala dimidiata and Holotrichia longipennis. The almost full length 16S rRNA sequence of the bacterium has 99% identity with the type strain of B. frigoritolerans, while phylogenetic analysis revealed that the isolate formed a tightly linked branch with the type strain of B. frigoritolerans. Under in vitro bioassay conditions, the isolate infected and caused 89±5.4 and 74±7.7% mortality, in first instar larvae of A. dimidiata and H. longipennis, respectively. The infected larvae exhibited bacteremia like symptoms and mortality occurred between the second and fifth weeks after inoculation. This is an early report on the entomopathogenic potential of the hitherto lesser-known bacterium Brevibacterium frigoritolerans.     

Mountain Aspect Influences the Genetic Clustering of Psychrotolerant Phosphate Solubilizing <Emphasis Type="Italic">Pseudomonads</Emphasis> in the Uttarakhand Himalayas

Fourteen cold tolerant phosphate solubilizing bacteria isolated from high altitude representative locations of the two major mountain aspects of the Uttarakhand Himalayas (cooler north and warmer south facing slopes) were selected for this study. The tricalcium phosphate (TCP) solubilizing abilities of the isolates were estimated at three different incubation temperatures viz., 4, 15, and 30°C under in vitro conditions. Irrespective of their geographical origin, all the isolates recorded maximum P release values at 30°C. At 4°C, the isolates from the north facing slope were found to release significantly higher levels of P, as compared to the isolates from the south facing slopes. Alternatively at 15°C, the isolates from the south facing slope were found to release significantly higher levels of P. Initial confirmation of their genus level identity as Pseudomonads was arrived by amplification of a 990 bp fragment of the 16S rRNA gene using genus specific primers. Further putative species level identification was arrived by sequencing of the 16S rRNA gene. The diversity among the isolates was determined by rep-PCR using the primers BOX, ERIC, and ERIC2. A composite dendrogram constructed using the rep-PCR profiles revealed that the isolates from the north and south mountain aspects formed separate major clusters. The extent of diversity was greater among the isolates from the south mountain aspect. This study reveals the potential of rep-PCR in determining the genetic diversity among Pseudomonads selected for a single functional trait, but varying in their geographical origin.     

Structural insights into the aggregation behavior of Murraya koenigii miraculin‐like protein below pH 7.5

Murraya koenigii miraculin‐like protein (MKMLP) gradually precipitates below pH 7.5. Here, we explore the basis for this aggregation by identifying the aggregation‐prone regions via comparative analysis of crystal structures acquired at several pH values. The prediction of aggregation‐prone regions showed the presence of four short peptides either in beta sheets or loops on surface of the protein. These peptides were distributed in two patches far apart on the surface. Comparison of crystal structures of MKMLP, determined at 2.2 Å resolution in pH 7.0 and 4.6 in the present study and determined at 2.9 Å in pH 8.0 in an earlier reported study, reveal subtle conformational differences resulting in gradual exposure of aggregation‐prone regions. As the pH is lowered, there are alterations in ionic interactions within the protein interactions of the chain with water molecules and exposure of hydrophobic residues. The analysis of symmetry‐related molecular interfaces involving one patch revealed shortening of nonpolar intermolecular contacts as the pH decreased. In particular, a decrease in the intermolecular distance between Trp103 of the aggregation‐prone peptide WFITTG (103–108) unique to MLPs was observed. These results demonstrated that aggregation occurs due to the cumulative effect of the changes in interactions in two aggregation‐prone defined regions. Proteins 2014; 82:830–840. © 2013 Wiley Periodicals, Inc.     

3-hydroxyisobutyrate dehydrogenase-I from Pseudomonas denitrificans ATCC 13867 degrades 3-hydroxypropionic acid

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant (k _cat/K _m) of 604.1 ± 71.1 mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD⁺ over NADP⁺ as a cofactor for its catalytic activity. The k _cat/K _m determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD⁺ at 37°C and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl-d,l-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k _cat/K _m values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn²⁺ increased the activity of 3HIBDHI significantly, whereas the presence of Fe²⁺, Hg²⁺ and Ag⁺ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.     

Dietary Administration of Probiotic Aeromonas veronii V03 on the Modulation of Innate Immunity,Expression of Immune-Related Genes and Disease Resistance Against Aeromonas hydrophila Infection in Common Carp (Cyprinus carpio)

This study investigates the effects of dietary Aeromonas veronii V03 supplementation on growth performances, innate immunity, and expression of immune-related genes in lymphoid organs of Cyprinus carpio and resistance to Aeromonas hydrophila infection. Fish were fed for 4 weeks with basal diet (BD; without probiotic), and experiment diet containing different doses of A. veronii V03 at 3.2 × 10⁷ (DI) and 3.5 × 10⁹ (DII) CFU g⁻¹ of diet. At the end of the probiotic feeding trial, fish were challenged with A. hydrophila, and the percentage of survival rates was recorded over 7 days. Results revealed that fish fed with A. veronii V03 demonstrated a significant improvement in growth and enhancement of innate immunity, including respiratory burst, myeloperoxidase, and lysozyme activities, and total immunoglobulin level compared with BD fed to fish. Relatively, expression of cytokines (MyD88, IL-1β1, IL-8, and IL-10) and c- and g-type lysozymes were significantly up- and downregulated in lymphoid organs of fish. Moreover, dietary supplementation of A. veronii V03 exhibited significantly (p < 0.001) higher survival rates of DI (90%) and DII (96.66%) compared with BD (53.33%) fed fish against A. hydrophila infection. These findings help to understand the effects of probiotic A. veronii V03 administrated feed influences on growth and ailment resistance to A. hydrophila infection by regulating innate and systemic immunity in common carp fish.