Purification, crystallization, and X-ray diffraction studies of lactotransferrin from buffalo colostrum.

Lactotransferrin is an iron-binding protein. It has been purified from buffalo colostrum. The purified lactotransferrin has been crystallized in 10% ethanol solution. The crystals are orthorhombic and the space group is P2(1)2(1)2(1) with unit cell dimensions a = 161.70 A, b = 155.75 A, c = 113.48 A. The asymmetric unit contains three molecules of the protein with a solvent content of about 59%. The crystals were stable in the X-ray beam and diffract beyond 3.5 A resolution. The native data have been collected and the structure determination is in progress.     

Viral replication is required for induction of ocular immunopathology by herpes simplex virus.

Corneal infection of BALB/c mice with herpes simplex virus type 1 results in a chronic inflammatory response in the stroma termed herpetic stromal keratitis (HSK). This disease is considered to be immunopathological and mediated primarily by CD4+ T cells of the type 1 cytokine profile. However, the nature of the antigens, virus or host derived, which drive the inflammatory response remains in doubt. In this study, the relevance of infection with replicating virus for the subsequent development of HSK was evaluated with immunocompetent mice as well as with SCID mice reconstituted with herpes simplex virus-immune CD4+ T cells. In the corneas of immunocompetent mice, infectious virus, viral antigen, and mRNA expression were detectable for only a brief period of time (< or = 7 days postinfection), and all were undetectable by the time clinical lesions were evident (10 to 15 days). Viral replication, however, was necessary for the development of HSK in both models, since infection with UV-inactivated virus or with mutant viruses which were incapable of multiple rounds of replication in vivo failed to induce HSK. The inactivated and mutant viral preparations did, however, stimulate T-cell immune responses in immunocompetent mice. The results are discussed in terms of possible involvement of host antigens exposed in response to transient progeny virion replication in the immune-privileged cornea.     

Crystallization and preliminary X-ray investigation of a sarcoplasmic calcium-binding protein from amphioxus

Crystals of a sarcoplasmic Ca(2+)-binding protein from the protochordate amphioxus have been grown from solutions of ammonium sulfate. The crystals are orthorhombic, space group C222(1), with unit cell axes a = 59.6(1) A, b = 81.3(1) A and c = 82.4(1) A. There is one molecule in the asymmetric unit. The crystals diffract beyond 2.5 A and show less than 20% decline in diffraction intensities after a three day exposure to X-rays from a laboratory rotating anode source.     

Evidence for autophosphorylation of hyaluronate binding protein and its enhanced phosphorylation in rat histiocytoma.

This report documents for the first time the in vitro autophosphorylation of purified 68 kDa hyaluronate binding protein in presence of [32P] ATP. The rate of phosphorylation is proportional to the concentration of protein and to the time of incubation up to 5 min. By both phosphoamino acid and western blot analysis with antiphosphotyrosine antibodies, we have confirmed that the phosphorylation occurs at tyrosine residues. Immunoprecipitation with anti HA binding protein antibody shows a 5 fold increase in the phosphorylation in macrophage histiocytoma compared to normal macrophage. Supplementing hyaluronate with hyaluronate binding protein in the medium is further shown to enhance total protein phosphorylation in rat histiocytoma.     

Hydrophobic surfaces in oligosaccharides: linear dextrins are amphiphilic chains.

Polysaccharide chains are usually considered to be highly hydrophilic, since they have no obvious nonpolar moieties in them. Yet, it is possible to realise conformations in these chains wherein all the hydroxy groups are disposed in one side or face of the chain and the hydrogens disposed in the other. We experimentally demonstrate that such an amphiphilic surface is present in linear oligomeric dextrins, i.e., alpha-1,4-linked D-glucosides, but not in alpha-1,6-D-glucosides (dextrans) or in beta-1,4-D-glucosides (cellulose). This amphiphilicity is generated as a consequence of the stereochemical constraints, which vary with the structure of the sugar and with the type of linkage. Oligosaccharide chains that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in heteromolecular recognition process than as homomolecular self-aggregation.     

Effect of long term starvation on the biochemical composition of the muscles and hepatopancreas in the crab Menippe rumphii (Fabricius) (Crustacea: Brachyura)

During the process of long term starvation both muscles and hepatopancreas are affected in their biochemical composition at different rates. During early days of starvation an increase in the muscular and hepatopancreatic glycogen is observed. At the same time a simultaneous decrease in the muscular lipid content is also observed. At a slightly later period a decrease in the hepatopancreatic lipid content is also noticed. This amount of decrease is slow in the early days of starvation and rapid in later days. Decrease in the muscular and hepatopancreatic protein content is observed when there is not an adequate quantity of hepatopancreatic lipid to be consumed.     

DIFFERENTIAL SENSITIVITY OF “DOUBLE” AND “SINGLE” FLOWERS OF NIGELLA DAMASCENA (RANUNCULACEAE) TO EMASCULATION AND TO GA3

In Nigella damascena “double” flower is inherited as a single-gene recessive to “single” flower. Early emasculation of “double” flowers greatly inhibits gynoecium development and, to a lesser degree, development of sepals and bracts. No comparable inhibition of gynoecia was induced in “single” flowers though some sepal and bract inhibition was detected. A differential nutritional requirement for organ initiation on cultured flower apices was also detected. Apices of both varieties initiated stamens and carpels if kinetin was added to the basal medium. However, in the absence of kinetin, GA₃ was required for organ initiation in “doubles” but completely inhibited stamen initiation in “singles.” Both these differential effects are thought to reflect rather different patterns of gibberellin metabolism in the two genetic strains.     

Spatiotemporal spread of Plasmodium falciparum mutations for resistance to sulfadoxine-pyrimethamine across Africa, 1990–2020

BackgroundSulfadoxine-pyrimethamine (SP) is recommended in Africa in several antimalarial preventive regimens including Intermittent Preventive Treatment in pregnant women (IPTp), Intermittent Preventive Treatment in infants (IPTi) and Seasonal Malaria Chemoprevention (SMC). The effectiveness of SP-based preventive treatments are threatened in areas where Plasmodium falciparum resistance to SP is high. The prevalence of mutations in the dihydropteroate synthase gene (pfdhps) can be used to monitor SP effectiveness. IPTi-SP is recommended only in areas where the prevalence of the pfdhps540E mutation is below 50%. It has also been suggested that IPTp-SP does not have a protective effect in areas where the pfdhps581G mutation, exceeds 10%. However, pfdhps mutation prevalence data in Africa are extremely heterogenous and scattered, with data completely missing from many areas.Methods and findingsThe WWARN SP Molecular Surveyor database was designed to summarize dihydrofolate reductase (pfdhfr) and pfdhps gene mutation prevalence data. In this paper, pfdhps mutation prevalence data was used to generate continuous spatiotemporal surface maps of the estimated prevalence of the SP resistance markers pfdhps437G, pfdhps540E, and pfdhps581G in Africa from 1990 to 2020 using a geostatistical model, with a Bayesian inference framework to estimate uncertainty. The maps of estimated prevalence show an expansion of the pfdhps437G mutations across the entire continent over the last three decades. The pfdhps540E mutation emerged from limited foci in East Africa to currently exceeding 50% estimated prevalence in most of East and South East Africa. pfdhps540E distribution is expanding at low or moderate prevalence in central Africa and a predicted focus in West Africa. Although the pfdhps581G mutation spread from one focus in East Africa in 2000, to exceeding 10% estimated prevalence in several foci in 2010, the predicted distribution of the marker did not expand in 2020, however our analysis indicated high uncertainty in areas where pfdhps581G is present. Uncertainty was higher in spatial regions where the prevalence of a marker is intermediate or where prevalence is changing over time.ConclusionsThe WWARN SP Molecular Surveyor database and a set of continuous spatiotemporal surface maps were built to provide users with standardized, current information on resistance marker distribution and prevalence estimates. According to the maps, the high prevalence of pfdhps540E mutation was to date restricted to East and South East Africa, which is reassuring for continued use of IPTi and SMC in West Africa, but continuous monitoring is needed as the pfdhps540E distribution is expanding. Several foci where pfdhps581G prevalence exceeded 10% were identified. More data on the pfdhps581G distribution in these areas needs to be collected to guide IPTp-SP recommendations. Prevalence and uncertainty maps can be utilized together to strategically identify sites where increased surveillance can be most informative. This study combines a molecular marker database and predictive modelling to highlight areas of concern, which can be used to support decisions in public health, highlight knowledge gaps in certain regions, and guide future research.     

Diclofenac-induced stimulation of SMCT1 (SLC5A8) in a heterologous expression system: A RPE specific phenomenon

SMCT1 is a Na⁺-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na⁺-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, β-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.     

Global gene expression in rice blast pathogen Magnaporthe oryzae treated with a natural rice soil isolate

The rhizospheric microbiome is comprised of many microbes, some of which reduce the virulence of their phytopathogenic neighbors; however, the mechanisms underlying these interactions are largely unknown. Rice soil isolate Pseudomonas chlororaphis EA105 strongly inhibits Magnaporthe oryzae’s in vitro growth by restricting fungal diameter as well as inhibiting the formation of the appressorium, required for penetration. We were interested in elucidating M. oryzae’s response to EA105 treatment, and utilized a microarray approach to obtain a global perspective of EA105 elicited changes in this pathogen. Based on this analysis, three genes of interest were knocked out in M. oryzae 70-15, and their sensitivity to EA105 treatment as well as their ability to infect rice was determined. Priming rice plants with EA105 prior to M. oryzae infection decreased lesion size, and the mutants were tested to see if this effect was retained. A null 70-15 mutant in a trichothecene biosynthesis gene showed less susceptibility to bacterial treatment, forming more appressoria than the parental type 70-15. A similar pattern was seen in a null mutant for a stress-inducible protein, MGG_03098. In addition, when this mutant was inoculated onto the leaves of EA105-primed rice plants, lesions were reduced to a greater extent than in 70-15, implicating the lack of this gene with an increased ISR response in rice. Understanding the global effect of biocontrol bacteria on phytopathogens is a key for developing successful and lasting solutions to crop loss caused by plant diseases and has the potential to greatly increase food supply.