<Emphasis Type="Italic">Glycine max</Emphasis> non-nodulation locus <Emphasis Type="Italic">rj1</Emphasis>: a recombinogenic region encompassing a SNP in a lysine motif receptor-like kinase (<Emphasis Type="Italic">GmNFR1α</Emphasis>)

The rj1 mutation of soybean is a simple recessive allele in a single line that arose as a spontaneous mutation in a population; it exhibits non-nodulation with virtually all Bradyrhizobium and Sinorhizobium strains. Here, we described fine genetic and physical mapping of the rj1 locus on soybean chromosome 2. The initial mapping of the rj1 locus using public markers indicated that A343.p2, a sequence-based marker that contains sequence similar to a part of the LjNFR1 gene regulating nodule formation as a member of lysin motif-type receptor-like kinase (LYK) family, maps very close to or cosegregates with the rj1 locus. The sequence of A343.p2 is 100% identical to parts of two BAC clone sequences (GM_WBb0002O19 and GM_WBb098N11) that contain three members of the LYK family. We analyzed the sequence contig (262 kbp) of the two BAC clones by resequencing and subsequent fine genetic and physical mapping. The results indicated that rj1 is located in a gene-rich region with a recombination rate of 120 kbp/cM: several fold higher than the genome average. Among the LYK genes, NFR1α is most likely the gene encoded at the Rj1 locus. The non-nodulating rj1 allele was created by a single base-pair deletion that results in a premature stop codon. Taken together, the fine genetic and physical mapping of the Rj1-residing chromosomal region, combined with the unexpected observation of a putative recombination hotspot, allowed us to demonstrate that the Rj1 locus most likely encodes the NFR1α gene.     

New basal media for half-anther culture of <Emphasis Type="Italic">Anthurium andreanum</Emphasis> Linden ex André cv. Tropical

A successful protocol for high frequency callus induction and plant regeneration from Anthurium andreanum Linden ex André cv. Tropical half-anthers is described. Different variables using Winarto and Teixeira and Murashige and Skoog basal media supplemented with several plant growth regulators [2,4-dichlorophenoxy acetic acid (0.1–1.0 mg/l), α-naphthalene acetic acid (0.01–0.2 mg/l), thidiazuron (0.5–2.0 mg/l), 6-benzylaminopurine (0.5–1.0 mg/l), and kinetin (0.5–1.0 mg/l)] were tested for their ability to induce high frequency callusing in half-anthers, indirect regeneration and rooting of shoots. Basal medium, as well as the combination and concentration of hormones applied, had a significant effect on callus formation, shoot regeneration and adventitious root formation. Winarto and Teixeira-1, an original basal medium containing 0.01 mg/l α-naphthalene acetic acid, 0.5 mg/l thidiazuron and 1.0 mg/l 6-benzylaminopurine was suitable for callus formation while an improved basal medium i.e., New Winarto–Teixeira-3 supplemented with 0.25 mg/l 2,4-dichlorophenoxy acetic acid, 0.02 mg/l α-naphthalene acetic acid, 1.5 mg/l thidiazuron and 0.75 mg/l 6-benzylaminopurine enhanced callus formation. High shoot regeneration and multiplication was also possible on New Winarto–Teixeira-3. Shoots formed a strong adventitious root system on New Winarto–Teixeira-3 containing 0.2 mg/l α-naphthalene acetic acid and 1.0 mg/l kinetin. Plantlets that varied in size and performance were successfully acclimatized and adapted to ex vitro conditions. Cytological analysis of 180 acclimatized-plantlets ex vitro revealed that 34 were haploid (n = 14–18), 15 aneuploid (n = 20–26), 126 diploid (n = 28–34) and 5 triploid (n = 45–57). The potential use of this protocol for developing half-anther culture of other Anthurium species or cultivars is discussed.     

Microspore culture protocol for Indonesian <Emphasis Type="Italic">Brassica oleracea</Emphasis>

A microspore culture protocol for Brassica oleracea of Indonesian origin (cv. ‘Kemeh’) has been successfully established. A high number of embryos formed with high microspore density i.e. 15 × 10⁴ cells/ml. Embryo formation was improved by using flower buds (4.5–4.6 mm in length) as explants, a temperature treatment at 30.5°C for 48 h and then transfer to 25°C continuously until embryos formed. A total of 295 embryos were obtained from 189 buds, 30% of which were abnormal (i.e. with an abnormal cotyledon or lacking hypocotyls). All normal embryos that grew and survived, 165 in total, were successfully transferred to soil and grew well in plastic bags (15 cm in diameter) containing a mixture of burned-rice husk and organic manure (1:1, v/v).     

Screening of commercial cyclic peptide as inhibitor NS5 methyltransferase of Dengue virus through Molecular Docking and Molecular Dynamics Simulation

Dengue has become a major global health threat, especially in tropical and subtropical regions. The development of antiviral agent targeting viral replication is really needed at this time. NS5 methyltransferase presents as a novel antiviral target. This enzyme plays an important role in the methylation of 5''-cap mRNA. Inhibition of the NS5 methyltransferase could inhibit dengue virus replication. In this research, two sites of NS5 methyltransferase (S-Adenosyl methionine/SAM binding site and RNA-cap site) were used as targets for inhibition. As much as 300 commercial cyclic peptides were screened to these target sites by means of molecular docking. Analysis of ligand-enzyme binding free energy and pharmacological prediction revealed two best ligands, namely [Tyr123] Prepro Endothelin (110-130), amide, human and Urotensin II, human. According to molecular dynamic simulation, both ligands maintain a stable complex conformation between enzyme and ligand at temperature 310 K and 312 K. Hence, Urotensin II, human is more reactive at 312 K than at 310 K. However, both ligands can be used as potential inhibitor candidates against NS5 methyltransferase of dengue virus with Urotensin II, human exposes more promising activity at 312 K.     

Decreased expression of Apaf‐1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease‐activating factor‐1 (Apaf‐1) is a cell death effector that acts with cytochrome c and caspase‐9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf‐1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf‐1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf‐1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf‐1 expression was observed when comparing nevi and melanomas (chi‐square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf‐1 (chi‐square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf‐1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf‐1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf‐1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Effect of external stresses on protein conformation: a computer modelling study

The increasing use of digital technologies such as mobile phones has led to major health concerns about the effects of non-ionizing pulsed radiation exposure. We believe that the health implications of exposure to radiation cannot be fully understood without establishing the molecular mechanisms of biological effects of pulsed microwaves. We aim to establish methods for studying the molecular mechanisms of protein structural and energetic changes occurring due to external stresses related to non-ionizing radiation by using a combination of experimental and theoretical approaches. In this paper, we present the results from our fully atomistic simulation study of chemical and thermal stress response of a prototype protein, insulin. We performed a series of molecular dynamics simulations of insulin in solution under equilibrium conditions, under chemical stress (imitated by reducing the disulfide bonds in the protein molecule), and under short-lived thermal stress (imitated by increasing simulation temperature for up to 2 ns). The resultant protein conformational behaviour was analysed for various properties with the aim of establishing analysis routines for classification of protein unfolding pathways and associated molecular mechanisms.Submitted as a record of the 2002 Australian Biophysical Society meeting     

<Emphasis Type="Italic">Ex vitro</Emphasis> rooting using a mini growth chamber increases root induction and accelerates acclimatization of Kopyor coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) embryo culture-derived seedlings

Ex vitro rooting using a mini growth chamber to maintain relative humidity has been used to mass produce true-to-type Kopyor coconut (Cocos nucifera L.) seedlings through embryo culture. This new process was found to (1) improve the proportion of seedlings successfully transferred to soil (from 40 to 90% for seedlings with roots); (2) achieve this step in the shortest time possible (a reduction from 10 to 4 mo in in vitro culture); (3) improve root formation using indolebutyric acid (IBA; an improvement in the number of primary roots from 2 to 5); and (4) improve the vigor of seedlings ex vitro (improvements of fresh weight, shoot length, number of opened leaves, leaf thickness, amount of epicuticular wax, and stomatal density). The best ex vitro rooting and rapid acclimatization protocol was obtained when 4-mo-old seedlings with two opened leaves were kept in the mini growth chamber for 3 mo before being transferred into soil, and when the mini growth chamber was flooded with a quarter strength hybrid embryo culture (HEC) medium with 1 μM IBA but depleted of vitamins and sugar. This protocol was efficient in delivering high-value Kopyor seedlings to the field (90% success rate), with a decreased risk of contamination and lower labor cost. The improved process was found applicable to both tall and dwarf Kopyor and other coconut types.     

Responses of Mature and Aged Sympathetic Neurons to Laminin and NGF: An in Vitro Study

Whilst the potent effects of NGF and laminin on developing neurons are well documented, relatively little is known about the effects of, or altered availability of or altered responsiveness to, these substances on the growth of adult neurons. We have therefore examined this question using explant cultures of sympathetic neurons from the superior cervical ganglion (SCG) of mature and aged rats. Explants were grown on substrata containing different doses of laminin, either with or without added NGF in culture medium containing FCS. Individually, laminin and NGF had relatively small effects on neurite outgrowth and length, which tended to be reduced in old neurons. In contrast, laminin in the presence of exogenous NGF exerted a powerful effect on nerve growth which was substantially greater than the sum of the effects of the individual factors. This synergy was evident in all experimental groups and was greatest in old explants at high doses of laminin, where growth was comparable to that of mature neurons. The dose-response curve of old neurons to laminin in the presence of added NGF indicated reduced responsiveness. These results suggest that variations in the availability of laminin and/or exogenous NGF, together with altered patterns of neuronal responsiveness, may contribute to impaired neuronal plasticity in old age.     

Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning

Different protocols based on Illumina high-throughput DNA sequencing and denaturing gradient gel electrophoresis (DGGE)-cloning were developed and applied for investigating hot spring related samples. The study was focused on three target genes: archaeal and bacterial 16S rRNA and mcrA of methanogenic microflora. Shorter read lengths of the currently most popular technology of sequencing by Illumina do not allow analysis of the complete 16S rRNA region, or of longer gene fragments, as was the case of Sanger sequencing. Here, we demonstrate that there is no need for special indexed or tailed primer sets dedicated to short variable regions of 16S rRNA since the presented approach allows the analysis of complete bacterial 16S rRNA amplicons (V1–V9) and longer archaeal 16S rRNA and mcrA sequences. Sample augmented with transposon is represented by a set of approximately 300 bp long fragments that can be easily sequenced by Illumina MiSeq. Furthermore, a low proportion of chimeric sequences was observed. DGGE-cloning based strategies were performed combining semi-nested PCR, DGGE and clone library construction. Comparing both investigation methods, a certain degree of complementarity was observed confirming that the DGGE-cloning approach is not obsolete. Novel protocols were created for several types of laboratories, utilizing the traditional DGGE technique or using the most modern Illumina sequencing.     

Identification of pericytes in the central nervous system by silver staining of the basal lamina

Vibratome sections obtained from perfusion-fixed rat brains were stained by means of silver impregnation and physical development according to Gallyas (1970). Small pieces of the cerebral cortex were postfixed with buffered osmium tetroxide solution and processed for electron microscopy to examine the localization of the silver deposit at the cellular level. The cell surfaces of pericytes and smooth muscle cells were completely outlined by silver grains. Endothelial cells and perivascular astrocytes, however, showed an asymmetric distribution of the silver deposit, i.e., the deposit was restricted to the abluminal endothelial surface and to the astrocytic membrane adjacent to the vessel wall, respectively. The method allowed a clear-cut distinction between perikarya of endothelial cells and pericytes as well as glial cells in perivascular position, even at the light-microscopic level.