Role of the interchain interaction domain of chain a in viscumin cytotoxicity

The sequence coding for the viscumin (mistletoe lectin I, MLI) A-chain (MLA) was cloned from Viscum album genomic DNA with the use of synthetic primers. This yielded three recombinant (r) MLA variants differing in number of amino acid substitutions. The rMLA structure and properties were probed using monoclonal antibodies against native MLA. Native MLI B-chain (MLB) was shown to facilitate the rMLA folding. Native MLI and chimeric proteins consisting of rMLA and native MLB did not differ in cytotoxic effect on 3T3 fibroblastoid cells. Residues were identified that are located in the MLB-contacting region and have a considerable effect on the immunochemical and cytotoxic properties of rMLA.     

Improved electrotransformation frequencies of Corynebacterium glutamicum using cell-surface mutants

The electrotransformation efficiency for homologously- and heterologously-derived plasmid DNA was determined for two families of Corynebacterium glutamicum strains derived from ATCC13059 (AS019 and auxotrophic, cell surface mutants MLB133 and MLB194) and ATCC13032 (parent strain and restriction-minus mutants RM3 and RM4), following their growth in LBG supplemented with glycine plus isonicotinic acid hydrazide (INH). Electrotransformation efficiencies of MLB133 were up to 100-fold higher than for strain ASO19 and, when using heterologously-derived plasmid DNA, MLB133 showed efficiencies comparable to or better than strains RM3 and RM4, demonstrating the relative importance of cell surface structures in impeding DNA uptake in C. glutamicum. Transmission electron microscopy analysis of cell surface structures showed that strain MLB133 has a thin cell wall relative to AS019 and growth in either glycine or INH further diminished its thickness. Both RM3 and RM4 were more sensitive to INH than ATCC13032 and growth in glycine plus INH further improved transformation efficiency. The mycolic acid composition of these strains is described and the impact of glycine and INH on this is reported.     

Recruitment limitation in secondary forests dominated by an exotic tree

Question: What factors limit woody plant recruitment in a mosaic landscape where former agricultural lands are dominated by the invasive tree Ligustrum lucidum (Oleaceae)? Location: Subtropical northwestern Argentina. Methods: In secondary forest patches, we measured (1) tree, shrub and liana abundance in different size classes; (2) seed rain of Ligustrum and two native trees and (3) topographic, soil and light variables. We used spatial autoregressive models to test for effects of Ligustrum dominance and environment on native plant abundance in each size class. We used multiple regression on resemblance matrices to quantify the relative importance of spatial (e.g. dispersal) and environmental effects on native species composition. Results: Native tree abundance in the smallest size class was unrelated to Ligustrum canopy dominance, while native tree abundance in larger size classes and native liana abundance were negatively correlated with Ligustrum dominance. Native species composition was both environmentally and spatially structured, suggesting that some species are dispersal limited. Seed rain was spatially correlated with conspecific basal area for one of two native species, but not for Ligustrum. Conclusions: Native tree recruitment appears to be limited primarily by sapling mortality in patches dominated by the invasive Ligustrum. Ligustrum does not appear to be dispersal limited in our study area and is likely to continue spreading. Invaded patches may persist for hundreds of years.     

Cloning and Expression of Catalytic Subunit of MLIII, the Ribosome-Inactivating Protein from Viscum album

We have cloned the gene encoding a precursor of mistletoe (Viscum album) toxin MLIII. Analyses of nucleotide and deduced amino acid sequences of this gene revealed significant differences between MLI and MLIII preprotoxin genes. Immunochemical properties of recombinant A-subunit expressed in Escherichia coli and renatured were investigated using a panel of monoclonal antibodies raised against three mistletoe toxins (MLI, MLII, and MLIII). Ribosome-inactivating activity of recombinant MLIII A-subunit was detected in cell-free lysate of rabbit reticulocytes.     

A comparison of the CHO/HGPRT+ and the L5178Y/TK+/− mutation assays using suspension treatment and soft agar cloning: Results for 10 chemicals

The mouse lymphoma assay (MLA) and Chinese hamster ovary (CHO) cell assay are sensitive indicators of mutagenicity. The CHO assay has been modified technically to permit treatment in suspension and soft agar cloning comparable to the MLA. This methodology eliminates the risk of metabolic cooperation and the trauma of trypsinization. In addition, a larger population of cells can be treated and cloned for mutant selection. In order to compare the effectiveness of the test systems, 10 chemicals were evaluated for the induction of forward mutations in the CHO and MLA. Several of these chemicals have been reported as clastogenic; therefore, abbreviated colony sizing was performed to gauge the extent of genetic damage to the MLA cells. Both test systems detected benzo[a]pyrene, mitomycin C, acridine orange, and proflavin, and, with the exception of proflavin, more large colonies were present than small colonies. The suspect clastogen, phenytoin, was not mutagenic in the MLA and produced inconclusive results in the CHO. Ethidium bromide, a clastogen and a bacterial mutagen, was not mutagenic in either the MLA or CHO. Four compounds (p-aminophenol, benzoin, methoxychlor, and pyrene) were positive in the MLA, generally inducing a large number of small colonies, while demonstrating no mutagenic activity in the CHO assay. They have also been shown to be generally nongenotoxic in other test systems. Overall, the modified CHO assay did not appear to be better than the MLA for the detection of mutagenic agents. However, the MLA does appear to have lower specificity.Abbreviations AO acridine orange - BAP benzo[a]pyrene - BZN benzoin - CHO Chinese hamster ovary cell assay - DPH diphenylhydantoin - EB ethidium bromide - EMS ethylmethanesulfonate - 3MC 3-methylcholanthrene - MLA mouse lymphoma asay - MMC mitomycin C - MXC methoxychlor - PAP p-aminophenol - PRO proflavin - PYR pyrene     

Magnesium Lithospermate B Reduces Inflammatory Response in a Mouse Model of Hepatic Ischemia–Reperfusion Injury

It has been well proved that acute inflammatory response and hepatocellular apoptosis contributed to the pathogenesis of liver ischemia reperfusion (IR) injury. A vast amount of research has demonstrated that magnesium lithospermate B (MLB) has potent anti-apoptosis and potential anti-inflammatory pharmacological properties. However, it has not previously been examined whether MLB can attenuate hepatic IR injury. Firstly, the optimal dose of MLB to protect against hepatic IR injury was determined using hepatic IR model in mice. Then, the effect of MLB on IR-induced inflammatory response was detected in detail. We found that MLB exhibited protective effect from the beginning of 50 mg/kg and culminated at the doses of 100 and 200 mg/kg. The alanine aminotransferase and aspartate aminotransferase levels in MLB group were markedly decreased. Severe hepatocellular swelling/necrosis, sinusoidal/vascular congestion and inflammatory cell infiltration were seen and a large number of apoptotic cells were found in the liver samples from Saline group, while minimal damage and very few apoptotic cells were noted in the samples from MLB group. Kuppfer cells infiltration, myeloperoxidase activity and mRNA level of CD11b in MLB group was significantly decreased. Serum TNF-a and IL-6, and mRNA expression of CXCL-10 and ICAM-1 was markedly decreased in the samples from MLB group. Inflammatory signaling pathway activation was largely prevented in MLB group. MLB can significantly attenuate IR-induced hepatocellular damage and hepatocellular apoptosis by preventing inflammatory signaling pathways activation, inflammatory mediators expression and macrophage and neutrophil infiltration.     

Methyllycaconitine: a selective probe for neuronal α-bungarotoxin binding sites

The ability of methyllycaconitine (MLA) to inhibit the binding of [¹²⁵I]α-bungarotoxin to rat brain membranes, frog and human muscle extracts and the human muscle cell line TE671 has been measured. MLA showed a markedly higher affinity for the rat brain site (K_i 1.4 × 10⁻⁹ M) than for the muscle receptors (K_i; 10⁻⁵-10⁻⁶ M). Structure modelling techniques were used to fit the structure of MLA to a nicotinic pharmacophore model. MLA is the first low molecular weight ligand to be shown to discriminate between muscle nicotinic receptors and their α-bungarotoxinbinding counterpart in the brain, and as such may be a useful structural probe for pursuing the structural and functional properties of the neuronal protein.     

Pyramiding QTL for multiple lateral branching in cucumber using inbred backcross lines

Multiple lateral branching (MLB) is a quantitatively inherited trait associated with yield in cucumber (Cucumis sativus L.; 2n = 2x = 14). Although quantitative trait loci (QTL) have been identified for MLB and QTL-marker associations have been verified by marker-assisted selection, the individual effects of these QTL have not been characterized. To test the effects of pyramiding QTL for MLB, molecular genotyping was utilized to create two sets (standard- and little-leaf types) of inbred backcross (IBC) lines possessing various numbers of QTL that promote branching. These IBC lines were evaluated for lateral branch number in two Wisconsin environments at three plant densities. Highly significant differences in the number of primary lateral branches were detected between spacings, leaf types, and lines, but not between locations. Lateral branch number decreased at higher plant densities in all genotypes, while genotype by environment and QTL by environment interactions were marginally non-significant. As the number of QTL increased among IBC lines, the number of branches did not generally change in the little-leaf lines, but decreased in the standard-leaf lines, demonstrating an epistatic effect related to genetic background during lateral branch development. The genomic location with the greatest effect on MLB was confirmed as the QTL that was previously mapped near the little-leaf locus (ll), while the addition of one specific QTL consistently decreased the number of lateral branches in standard-leaf lines. Although pyramiding QTL for MLB did not uniformly increase the number of lateral branches, pyramiding QTL in IBC lines allowed further characterization of individual QTL involved in MLB. Our results, coupled with those of previous studies indicate that lateral branch development in cucumber is determined by growing environment (i.e., plant spacing), genetic background, and QTL composition.     

Plant growth‐promoting effects of native Pseudomonas strains on Mentha piperita (peppermint): an in vitro study

Plant growth‐promoting rhizobacteria (PGPR) affect growth of host plants through various direct and indirect mechanisms. Three native PGPR (Pseudomonas putida) strains isolated from rhizospheric soil of a Mentha piperita (peppermint) crop field near Córdoba, Argentina, were characterised and screened in vitro for plant growth‐promoting characteristics, such as indole‐3‐acetic acid (IAA) production, phosphate solubilisation and siderophore production, effects of direct inoculation on plant growth parameters (shoot fresh weight, root dry weight, leaf number, node number) and accumulation and composition of essential oils. Each of the three native strains was capable of phosphate solubilisation and IAA production. Only strain SJ04 produced siderophores. Plants directly inoculated with the native PGPR strains showed increased shoot fresh weight, glandular trichome number, ramification number and root dry weight in comparison with controls. The inoculated plants had increased essential oil yield (without alteration of essential oil composition) and biosynthesis of major essential oil components. Native strains of P. putida and other PGPR have clear potential as bio‐inoculants for improving productivity of aromatic crop plants. There have been no comparative studies on the role of inoculation with native strains on plant growth and secondary metabolite production (specially monoterpenes). Native bacterial isolates are generally preferable for inoculation of crop plants because they are already adapted to the environment and have a competitive advantage over non‐native strains.     

Field Studies on Cross-protection against Japanese yam mosaic virus in Chinese yam (Dioscorea opposita) with an Attenuated Strain of the Virus

An attenuated strain of Japanese yam mosaic virus (JYMV), designated T‐3, was evaluated for its cross‐protection efficacy against virulent (native) strains of JYMV in Chinese yam (Dioscorea opposita) grown in farmers’ fields in Japan. Native strains of JYMV were detected by a polymerase chain reaction‐based assay in all the Chinese yam plants grown from virus‐free tubers in the first growing season in the fields. In contrast, the virus was detected in only one of fifty plants grown from tubers preinoculated with T‐3 during the experiments for 6 years, suggesting that T‐3 consistently cross‐protected against native JYMV in Chinese yam in the field. Chinese yam plants preinoculated with T‐3 produced significantly greater yield of tubers per plant compared with non‐inoculated plants.     

Impact of fire and the recovery of molluscs in south‐east Australian salt marsh

Fire has long been recognised as a natural force in structuring Northern Hemisphere salt marshes, yet little is known about the impact of fire on molluscs and native vegetation dynamics of Southern Hemisphere coastal salt marshes. Following a fire at Ash Island, Hunter River New South Wales, Australia in the summer 2012, we assessed patterns of recovery through time of gastropod populations and resident salt marsh vegetation including biomass for three keystone native plant species, Native Rush (Juncus kraussii Hochst.), a chenopod (Sarcocornia quinqueflora Bunge ex Ungen‐Sternberg A.J. Scott), Salt Couch (Sporobolus virginicus, L. Kunth) and the invasive Spiny Rush (Juncus acutus). In temperate east‐coast Australian salt marshes, Spiny Rush is displacing native salt marsh vegetation. After twelve months, the biomass of Native Rush recovered to similar pre‐burn levels. While fire affected the abundance, richness and composition of the gastropod assemblage differences were also largely driven by spatial variability. Gastropod assemblages associated with two of the higher elevation native species (Native Rush and Salt Couch) were impacted the most by fire. Greater abundance (between 1 and 5 orders of magnitude difference in abundance) and richness of gastropods were found in unburnt compared with burnt Native Rush and Salt Couch vegetation, while more gastropods were found in Spiny Rush in one site. Species prevalent in burnt vegetation included larger species of gastropods Ophicardelus ornatus (Ferussac, 1821) and Phallomedusa solida (Martens, 1878) with an unexpected spike in number of the smaller gastropod Tatea huonensis (Tenison‐Woods, 1876) in the spiny rush at one site only. In salt marsh habitats, many gastropods have planktonic larval dispersal stages which are dependent on the tidal height for transport and the structural complexity provided by vegetation at settlement. Since fire appears to negatively affect salt marsh gastropod populations within structurally complex Native Rush and Salt Couch, due consideration of the importance of these refuges for gastropods is recommended when fire or other disturbances occur in ecologically endangered salt marsh in the Southern Hemisphere. Managers need to consider spatial heterogeneity of molluscs and their recovery in the event of fire in Southern Hemisphere salt marshes.     

Carrageenan of <Emphasis Type="Italic">Eucheuma isiforme</Emphasis> (Solieriaceae,Rhodophyta) from Nicaragua

The yield and physicochemical properties of native and alkali treated carrageenan from Eucheuma isiforme harvested from the Nicaraguan coast were investigated. The native carrageenan yield was 57.2% of dry weight and decreased to 43.5% when the alga was alkali treated. Native carrageenan viscosities showed significant differences between native (144.6 ± 3.3 cPs) and treated carrageenan (113.9 ± 2.6 cPs) (p < 0.01). Alkali treatment reduced carrageenan sulphate content by 19.3% and increased 3,6 AG content by 13%. Alkali-treated carrageenan formed very weak gels in 1.5% solutions (<50 g cm⁻²). Chemical analysis and FTIR spectra revealed that Eucheuma isiforme from Nicaragua is a good source of relatively pure iota-carrageenan with sufficient quality to serve as a substitute for traditional iota-carrageenan sources.     

Site-specific mutagenesis of mistletoe lectin: the role of RIP activity in apoptosis.

Recombinant mistletoe lectin (rML) belongs to the class of type II ribosome-inactivating proteins (RIP) composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding properties. To investigate the contribution of the enzymatic activity of the rML A-chain to the observed cytotoxic and apoptotic effects, an rMLA E166Q R169Q molecule was developed by means of site-specific mutagenesis. Following heterologous expression, the activity of mutant rMLA was measured in a cell-free assay for rRNA-N-glycosidase activity. Moreover, after generation of heterodimer, the activities of mutant rML E166Q R169Q and rML wild type were determined in a cytotoxicity and apoptosis assay. Although the reduction of activity as measured in the cell-free RIP assay was more pronounced (factor 237) than in both cellular assays (factors 20-22), the data clearly indicate a close correlation between cytotoxicity, apoptosis, and the enzymatic activity of the rML A-chain. Thus, RIP activity is an essential feature of rML and therefore a prerequisite for its biological function as an anticancer agent.     

Plasma levels and biochemical characterisation of circulating met-enkephalin in canine endotoxin shock

Endogenous opioid peptides have been implicated in the pathophysiology of shock (1-5). In anaesthetised mongrel dogs, administration of E coli endotoxin caused a rise in plasma met-enkephalin-like immunoreactivity (MLI). Biochemical characterisation of MLI by gel filtration chromatography revealed various molecular forms: 31K, 8K, 3-5K and the native pentapeptide in approximately equal amounts. After enzymatic treatment of column fractions the 31K form predominated (90.7%). This is the first demonstration of elevated MLI in endotoxin shock.     

SPECIFIC positive and negative selection of rat lymphocytes reactive to strong histocompatibility antigens: activation with alloantigens in vitro and in vivo.

This study compares the functional properties of rat thoracic duct lymphocytes (TDL) after stimulation with strong alloantigens of the major histocompatibility complex (MHC) either in vitro in preparative mixed lymphocyte interactions (MLI) or in vivo in systemic graft-vs-host (GVH) reactions. Comparisons were made of PHA responses and reactivity to the specific priming haplotypes or to third party haplotypes in analytical MLI and in GVH reactions either before or after the activated populations were "parked" in syngenetic T cell-deprived (B) rats. These comparisons can be summarized as follows: 1) TDL populations primed in bulk MLI cultures (MLI-TDL) slowed some evidence of specific positive selection when tested immediately; MLI responses to specific alloantigens were both relatively large and accelerated in tempo, whereas responses to third party alloantigens were diminished but also accelerated in tempo. Specific GVH responses were more marked than in third party recipients but they were also decreased relative to normal, and displayed an abberant dose/response slope. MLI-TDL populations tested after they had been stored in syngeneic B rats showed clear evidence of stable-specific positive selection; specific MLI and GVH responses were enriched relative to third party responses and also in comparison to normal, unselected TDL populations. This finding indicates that GVH and MLI reactivity are probably both functional capacities of the same lymphocyte subpopulation since positive selection by one function (MLI) also enriched for a second (GVH). 2) Parental strain TDL activated in vivo in the systemic GVH reaction in irradiated F1 animals and recovered from the thoracic duct 3 to 4 days later (late GVH-TDL) consisted mainly of blast cells, however, in contrast to MLI-TDL these populations showed no evidence of positive selection when tested before or after parking in B rats. MLI responses to specific alloantigens were minimal, and greatly reduced in magnitude compared to normal. GVH responses to specific haplotypes could be detected, but these were not enriched compared to normal, despite the content in the late GVH-TDL populations of a significant proportion of blast cells presumably activated by host alloantigens. 3) Early collections (less than 40 hr) of parental strain GVH-TDL collected from F1 recipients contained no blast cells and showed impressive degrees of negative selection; they were markedly depleted of both GVH and MLI activity to specific alloantigens but displayed normal reactivity to third party alloantigens. Moreover, specific negative selection was persistent in these populations parked for several weeks in B rats, and indication that a specific subpopulation of reactive cells had been physically eliminated. 4) PHA responses of both MLI- and GVH-activated TDL populations tested either before or after parking in B rats were approximately normal on a per T cell basis...     

Y chromosome polymorphisms in Native American and Siberian populations: identification of Native American Y chromosome haplotypes

We have initiated a study of ancient male migrations from Siberia to the Americas using Y chromosome polymorphisms. The first polymorphism examined, a C→T transition at nucleotide position 181 of the DYS199 locus, was previously reported only in Native American populations. To investigate the origin of this DYS199 polymorphism, we screened Y chromosomes from a number of Siberian, Asian, and Native American populations for this and other markers. This survey detected the T allele in all five Native American populations studied at an average frequency of 61%, and in two of nine native Siberian populations, the Siberian Eskimo (21%) and the Chukchi (17%). This finding suggested that the DYS199 T allele may have originated in Beringia and was then spread throughout the New World by the founding populations of the major subgroups of modern Native Americans. We further characterized Native American Y chromosome variation by analyzing two additional Y chromosome polymorphisms, the DYS287 Y Alu polymorphic (YAP) element insertion and a YAP-associated A→G transition at DYS271, both commonly found in Africans. We found neither African allele associated with the DYS199 T allele in any of the Native American or native Siberian populations. However, we did find DYS287 YAP+ individuals who harbored the DYS199 C allele in one Native American population, the Mixe, and in one Asian group, the Tibetans. A correlation of these Y chromosome alleles in Native Americans with those of the DYS1 locus, as detected by the p49a/p49f (p49a,f) probes on TaqI-digested genomic DNA, revealed a complete association of DYS1 alleles (p49a,f haplotypes) 13, 18, 66, 67 and 69 with the DYS199 T allele, while DYS1 alleles 8 and 63 were associated with both the DYS199 C and T allele. Received: 18 November 1996 / Accepted: 19 May 1997     

Astrovirus MLB1 is not associated with diarrhea in a cohort of Indian children

Astroviruses are a known cause of human diarrhea. Recently the highly divergent astrovirus MLB1 (MLB1) was identified in a stool sample from a patient with diarrhea. It has subsequently been detected in stool from individuals with and without diarrhea. To determine whether MLB1 is associated with diarrhea, we conducted a case control study of MLB1. In parallel, the prevalence of the classic human astroviruses (HAstVs) was also determined in the same case control cohort. 400 cases and 400 paired controls from a longitudinal birth cohort in Vellore, India were analyzed by RT-PCR. While HAstVs were associated with diarrhea (p = 0.029) in this cohort, MLB1 was not; 14 of the controls and 4 cases were positive for MLB1. Furthermore, MLB1 viral load did not differ significantly between the cases and controls. The role of MLB1 in human health still remains unknown and future studies are needed.     

Fruit Mineral Contents of Six Wild Species of the North Andean Patagonia, Argentina

The fruit mineral contents (K, Ca, Ba, Br, Zn, Co, Cr, Fe, Na, Rb, Cs, and Sr) of four native and two exotic naturalized shrubs growing in different areas of the Andean Patagonian region of Argentina were investigated. Native species Berberis darwinii, Berberis microphylla (Berberidaceae), Aristotelia chilensis (Elaeocarpaceae) and Ribes magellanicum (Saxifragaceae) produce small berries while the fruits of the exotic species Rosa rubiginosa and Rosa canina (Rosaceae) are aggregates of aquenes. They are used to prepare jams, tea, liquors, and ice creams. Native shrub fruits had higher content of Br, Zn, Co, Cr, Fe, Mo, and Na than those of the exotic naturalized species. Rosa species showed the highest contents of Ca and Ba in their fruits (the mean content doubled those of the native plant fruits). The fruit nutrient content found in the studied species was similar or higher than other values reported for fruits of temperate and tropical species in the world.     

Comparative analysis of response to phenotypic and marker-assisted selection for multiple lateral branching in cucumber (Cucumis sativus L.)

Yield increase in processing cucumber ( Cucumis sativus L.) is positively correlated with an increase in number of fruit-bearing branches. Multiple lateral branching (MLB) is a metric trait controlled by at least five effective factors. Breeding efficacy might be improved through marker-assisted selection (MAS) for MLB. Experiments were designed to independently confirm previously determined linkage of molecular markers (L18-2-H19A SNP, CSWTAAA01 SSR, CSWCT13 SSR, W7-2 RAPD and BC-551 RAPD) to MLB, and to determine their utility in MAS. These markers were present in significantly higher frequency than expected (1, presence:3, absence; p < 0.001) in BC(2) plants selected based on a high MLB phenotype (BC(2)PHE). However, markers that were considered selectively neutral fit the expected segregation of donor parent DNA in BC(2) progeny. Markers linked to MLB were used in MAS of BC(1) and BC(2) plants to produce BC(2)MAS, and BC(3)MAS progeny. Means for MLB in MAS populations were compared with backcross populations developed through phenotypic selection (BC(2)PHE, BC(3)PHE) and by random mating where no selection had been applied (BC(2)RND, BC(3)RND). Statistical analysis showed no significant differences ( p < 0.001) between means of phenotypic (BC(2)PHE = 3.02, BC(3)PHE = 3.29) and marker-aided selection (BC(2)MAS = 3.12, BC(3)MAS = 3.11) for MLB. However, both phenotypic and MAS population means were significantly higher than the random control (BC(2)RND = 2.27, BC(3)RND = 2.41) for MLB. Thus, given the observed response to selection and the rapid life-cycle of cucumber (4 months), markers linked to MLB when used in MAS will most likely be effective tools in cucumber improvement.