TaqMan probes based on oligonucleotide-hairpin minor groove ligand conjugates

Hybridization of TaqMan probes derived from oligonucleotides containing fluorophores (fluorescein, FAM, or tetramethylrhodamine, (Tamra)), fluorescence quenchers (BHQ1 or BHQ2), and a conjugated hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues connected through an aminobutyric acid residue were proposed for discrimination of point mutations using the real time PCR technique. Identification of point A/C substitution was shown to be highly specific for hepatitis C virus subtypes 1a and 1b with two variants of the probe (5'-3'): ATTGAGCGGGTTTAp-BHQ2-MGB for subtype 1a and FAM-ATTGAGCGGGTTGAp-BHQ1-MGB for subtype 1b. Perfect duplexes (A.T and G.C pairs) increase fluorescence in the process of amplification, whereas imperfect duplexes (A.G and T.C pairs) induce no fluorescence changes. This phenomenon enables simultaneous genotyping of hepatitis C virus subtypes 1a and 1b.     

Infection ability of mycelium and spores of<Emphasis Type="Underline">Microdochium</Emphasis><Emphasis Type="Underline">nivale</Emphasis> (Fr) samuels hallett to<Emphasis Type="Underline">Lolium</Emphasis><Emphasis Type="Underline">Perenne</Emphasis> L.

Mycelium and spores ofMicrodochium nivale /Syn.Fusarium nivale/ were compared according to their ability to infect plants ofLolium perenne. The experiments were carried out according to the “cold chamber” method (Cormack, Lebeau 1956 modified by Pronczuk 1987). Between these two types of inoculum significant differences were found. The spore inoculum did not give any symptoms while the mycelial inoculum incited a severe disease in plants ofLolium perenne during one month of incubation.Under laboratory conditions it was found that the spore cultures ofMicrodochium nivale grew very slowly at 0 – 1°C, whereas their growth at 18 – 20°C was very fast. Growth of the mycelial cultures was not as profoundly affected by temperatures studied as the spore ones.It was concluded that to incite a disease the spore inoculum require longer incubation time than mycelial ones. The mycelial inoculum is more useful for screening of plants for resistsance.     

Pathogenicity of<Emphasis Type="Underline">Fusarium</Emphasis><Emphasis Type="Underline">SPP</Emphasis>. contributing to the stalk rot of Maize in Poland

During 1985–1989 a stalk rot of early maturity hybrids of maize was studied in Radzikow (Central Poland). It was found thatFusarium species were dominant on plants with stalk rot symptoms. Spectrum ofFusarium spp. had changed within the years. The most frequently isolated were:F.subglutinans,F.culmorum andF.crookwellense. When the disease developed early in the seasonF.graminearum was also present.Predominant species were examined for their pathogenicity according to the modified method of Molot, Simone (1967). Isolates ofF.graminearum andF.culmorum were found to be strong pathogens,F.crookwellense andF.subglutinans — moderate andF.oxysporum andF.eguiseti were the weak ones     

Endowing plants with tolerance to virus infection by their preliminary treatment with short interfering RNAs

RNA interference (RNAi) is one of the key defense mechanisms directed against virus infections in plants and other organisms. In this case in plants infected with viruses, short interfering RNAs (siRNAs) are formed from two-chain replicated forms of virus molecules of RNA. These siRNAs program one of the RNAi basic components, RNA-induced complex of genes silencing (RISC, RNA induced silencing complex) associated with sequence-specific removing virus RNA. Virus protein P19 is a suppressor of RNAi and is capable of trapping the siRNAs being formed before their binding with RISC. Here, it was shown that preliminary entering leaves of plants Nicotiana benthamiana Domin (before virus infecting) of siRNAs eluted from the complex P19/siRNA from the infected plant lowers development of infection symptoms induced by tomato bushy stunt virus (TBSV) in inoculated plants. Exogenous addition of suppressor-associated siRNAs to plants leads to not only lowering virus accumulation but also to survival of infected plants. Thus, it has been established that preliminary addition of virus siRNAs elevates plant tolerance to the virus infection by means of early programming RISC and activation of the defense action of RNAi.     

Alternative methods in toxicology tests: In vitro toxicity

Toxicity testing is required for new chemicals being introduced onto the market. The use of animals in evaluating chemical safety is costly and time consuming. Furthermore, there is the ethical need to develope alternative methods to reduce the required number of animals. The newinvitro assays offer numerous advantages such as speed, reproducibility and control of test conditions, and increased sensitivity. Although the dermal irritation assays might be substituted by theinvitro tests in the near future (Duffy, 1989), much work is required to evaluate organ toxicity withinvitro methods. We present data regarding the use of Balb/3T3 mice fibroblasts and primary rat hepatocytes as test systems forinvitro toxicity. The end-points we have analysed are total protein content, dye accumulation in lysosomes, reductase mytochondrial activity, intracellular content and leakage of enzymes into the medium.     

Solute Carriers in the Blood–Brain Barier: Safety in Abundance

Blood–brain barrier formed by brain capillary endothelial cells, being in contact with astrocytes endfeet and pericytes, separates extracellular fluid from plasma. Supply of necessary nutrients and removal of certain metabolites takes place due to the activity of transporting proteins from ABC (ATP binding cassette) and SLC (solute carrier) superfamilies. This review is focused on the SLC families involved in transport though the blood–brain barrier of energetic substrates (glucose, monocarboxylates, creatine), amino acids, neurotransmitters and their precursors, as well as organic ions. Members of SLC1, SLC2, SLC3/SLC7, SLC5, SLC6, SLC16, SLC22, SLC38, SLC44, SLC47 and SLCO (SLC21), whose presence in the blood–brain barriers has been demonstrated are characterized with a special emphasis put on polarity of transporters localization in a luminal (blood side) versus an abluminal (brain side) membrane.     

The Peptide-Mediated Interactions Between Human Osteoclast-Stimulating Factor and Its Partner Proteins in Osteoporosis: Which Binds to Which?

Human osteoclast-stimulating factor (hOSF) is an intracellular protein produced by osteoclasts that induces osteoclast formation and bone resorption in osteoporosis by recruiting multiple signaling complexes with its diverse biological partners through peptide-mediated interactions (PMIs). The protein contains a modular peptide-recognition domain of Src homology 3 (SH3), which can recognize and bind to the polyproline regions of its partner proteins, as well as two N-terminal polyproline segments, which can be recognized and bound by the SH3 domains of its partner proteins. Here, we attempted to elucidate the complicated PMIs between the different SH3 domains and different polyprolines of hOSF and its three known interacting partners, i.e. proto-oncogene tyrosine-protein kinase (c-Src), survival motor neuron (SMN) and Src-associated in mitosis, 68 kD (Sam68). A total of 29 peptide segments containing the SH3-binding motif PXXP were extracted from these partner proteins, which are potential binding sites of hOSF SH3 domain, while the c-Src kinase also possesses a SH3 domain that may recognize and bind the two polyproline peptides at hOSF N-terminus. Structural bioinformatics analysis identified a number of biologically functional PMI candidates between these SH3 domains and these polyproline peptides, which were then tested in vitro using fluorescence spectroscopy assays. Consequently, it is found that (i) hOSF SH3 domain exhibits strong binding potency to two Sam68 peptides ³⁶RQPPLPHR⁴³ (K _d = 13.7 μM) and ⁴²⁵APPARPVK⁴³² (K _d = 3.2 μM) as well as moderate affinity to three SMN peptides ¹⁹³FLPPPPPM²⁰⁰ (K _d = 56.2 μM), ²³⁵PFPSGPPI²⁴² (K _d = 28.4 μM) and ²⁴⁶PPPICPDS²⁵³ (K _d = 74.5 μM), but has only weak or no binding to c-Src peptides. Instead, a proline-rich region at hOSF N-terminal that contains two overlapping peptides (³KPPPKPVK¹⁰ and ⁶PKPVKPGQ¹³) can be bound tightly by c-Src SH3 domain with high and moderate affinity (K _d = 5.8 and 39.6 μM, respectively).     

Cultural characteristics and zearalenone production by strains of<Emphasis Type="Underline">Fusarium</Emphasis> from wheat

Conidia of the speciesFusarium culmorum /W.G.Sm./ Sacc. andFusarium graminearum Schwabe are characterized by variability in zearalenone production and dimensions depending on the substrate. The sporulation of isolates from some wheat eultivars have been deprived in vivo and in vitro in the first passage, but not their pathogenicity and toxic metabolites production. Nonsporulating strains produced lower quantités of zearalenone than sporulating ones. Liquid filtrates of such nonsporulating strains had a high phytotoxic effect on wheat caryopses. The crystalline toxin DAS /0,25 ug/ml/ had low phytotoxic effect on wheat caryopses.     

Mycotoxins and fungi accompanying wheat head fusariosis in Poland

The occurrence of wheat head fusariosis in various regions of Poland was observed in 1985 and 1986. The incidence of fusariosis was usually low — about 0,01% — only on South — East in some localities reached 5 – 20%. The most important species isolated from infected heads were :Fusarium culmorum F.grami nearurn,F.ni vale andF.avenaceum, in addition to whichF.crookwel1ense,F.eqiseti,F.subqluti nans andF.tricinctum (sensu Nelson et al.1983) were observed. Deoxynivalenol was present in 100% examined kernels subsamples at level 5–18 mg/kg and 3-acetyl deoxyn i val enol in 70% at level 1–3 mg/kg. The mycotoxins amount in chaff was 1,4 to 2,6 and 1,4 to 11 times higher (DON and 3AcD0N resp.) than in kernels.     

Contamination of freshly harvested maize kernels with<Emphasis Type="Underline">Fusarium</Emphasis> mycotoxins on Bihar state,India

Freshly harvested maize samples, collected from different fields of Bhagalpur during January-March, 1989, were analysed for the presence of Fusarium species and their toxins.F. moniliforme was most common followed byF. roseum,F. sporotrichioides,F. graminearum andF. equiseti. Different strains of these species produced zearalenone (11.2–28.2 μ/g), DON (0.3–2.9 μg/g) and T-2 (5.2–20.6 μg/g) toxins on mostrice medium. Fifteen per cent, out of 86 maize samples analysed, were found to be contaminated with various levels of above toxins, which occurred either alone or in groups. Toxin concentration in contaminated samples varied from 0.76–1.5 μg/g (ZEN), 0.41–202 μg/g (DON) and 0.55–2.92 μg/g (T-2).     

Widespread presence of "bacterial-like" PPP phosphatases in eukaryotes

Background

In eukaryotes, PPP (p rotein p hosphatase P) family is one of the two known protein phosphatase families specific for Ser and Thr. The role of PPP phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. Unlike eukaryotic PPP phosphatases, bacterial members of the family have broad substrate specificity or may even be Tyr-specific. Moreover, one group of bacterial PPPs are diadenosine tetraphosphatases, indicating that bacterial PPP phosphatases may not necessarily function as protein phosphatases.

Results

We describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the PPP family. These enzymes are more closely related to bacterial PPP phosphatases than to the known eukaryotic members of the family. One group, found exclusively in land plants, is most closely related to PPP phosphatases from some α-Proteobacteria, including Rhizobiales, Rhodobacterales and Rhodospirillaceae. This group is therefore termed Rhi zobiales / Rh odobacterales / Rh odospirillaceae-l ike ph osphatases, or Rhilphs. Phosphatases of the other group are found in Viridiplantae, Rhodophyta, Trypanosomatidae, Plasmodium and some fungi. They are structurally related to phosphatases from psychrophilic bacteria Shewanella and Colwellia, and are termed She wanella-l ike ph osphatases, or Shelphs. Phosphatases of the third group are distantly related to ApaH, bacterial diadenosine tetraphosphatases, and are termed A paH-l ike ph osphatases, or Alphs. Patchy distribution of Alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. Rhilphs, Shelphs and Alphs form PPP clades, as divergent from "conventional" eukaryotic PPP phosphatases as they are from each other and from major bacterial clades. In addition, comparison of primary structures revealed a previously unrecognised (I/L/V)D(S/T)G motif, conserved in all bacterial and "bacterial-like" eukaryotic PPPs, but not in "conventional" eukaryotic and archaeal PPPs.

Conclusions

Our findings demonstrate that many eukaryotes possess diverse "bacterial-like" PPP phosphatases, the enzymatic characteristics, physiological roles and precise evolutionary history of which have yet to be determined.

     

Fusariosis of spring barley cultivated in Lublin region

Diseases of spring barley in 1986–1988 seasons have been examined on barley plantations in Lublin region. Observations in eight weeks after sowing each year spring showed the occurrence of root rot and sheath rot in seedlings. As a result of mycological examination of infected seedlings 34 species of fungi were isolated:Fusarium spp. amounted up to 23% of all isolates. Each year,Fusarium culmorum andF.avenaceum were isolated, butF graminearum only in 1987. On all inspected fields there occurred plants with eye — spots or necrotic stripes on lower internodes. As a result of fungi isolation the colonies belonging to 30 species were identified from stems and roots of examined plants. There was about 35% of fusaria between isolates each year.Fusarium culmorum was most frequently isolated. This fungus both from stems with two mentioned kinds of symptoms and from roots was isolated.Fusarium avenaceum each year andFusarium graminearum in 1986 and 1988 were isolated. Mentioned there species were also isolated from kernels.     

Zearalenone formation by<Emphasis Type="Underline">Fusarium crookwellense</Emphasis> /Burgess,Nelson and Toussoun/ isolates from Poland and their pathogenicity towards cereals

Fusarium crookwellense /B.N. and T./ isolated from affected cereals in Poland formed zearalenone on wheat grain up to 602 mg/kg. Tested isolates have been found strong to severe pathogens of wheat, rye, triticale and barley seedlings and corn ears with pathogenicity similar to that ofF. culmorum andF. graminearum.     

Zearalenone production in wheat cultivars infected with the fungus<Emphasis Type="Underline">Fusarium</Emphasis><Emphasis Type="Underline">graminearum</Emphasis> Schwabe

In wheat plants of the eultivars “Danubia”, “Agra”, “Selekta” and “Jubilejna” the fungusFusarium graminsarum Schwabe produced toxic metabolite zearalenone/F-2/ which simultaneously influenced the development of plants characterized by a lower germinating capacity, a reduced growth rate and a higher production of side branches. The presence ofFusarium graminearum was confirmed only in infected plants after plating of organs (root, stem base, stem) and soil on agar medium. The mycotoxin production is dependent on the pathogen development in host plants. The F-2 level progressed from the root into the soil, stem base and stem. The highest F-2 production was identified in cultivar “Selekta” the lowest in cultivar “Danubia”. The highest F-2 level (in all wheat eultivars) was identified in the stem base.     

Nucleotide Correspondence between Protein-Coding Sequences of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains 26695 and J99

Comparison of ORFs between H. pylori strains 26695 and J99 showed that transitions (more than 3%) prevail over transversions (less than 1%). The predominance of transitions was explained by the high rates of cytosine replacement by thymine in the coding (3.5–5.3%) and noncoding (2.9–3.9%) DNA strands. The proportion of transversion-type correspondences (A → C, A → T, C → A, C → G, G → C, G → T, T → A, and T → G) did not exceed 0.84%. The highest proportion (28.3%) was observed for correspondences between C and T in ACGT-ATGT, the target site of active methyltransferase of H. pylori J99 (M.Hpy99XI). It was assumed that C → T mutations due to cytosine methylation-deamination are prevalent in H. pylori.     

The PAM domain,a multi-protein complex-associated module with an all-alpha-helix fold

Background

Multimeric protein complexes have a role in many cellular pathways and are highly interconnected with various other proteins. The characterization of their domain composition and organization provides useful information on the specific role of each region of their sequence.

Results

We identified a new module, the PAM domain (P CI/PINT a ssociated m odule), present in single subunits of well characterized multiprotein complexes, like the regulatory lid of the 26S proteasome, the COP-9 signalosome and the Sac3-Thp1 complex. This module is an around 200 residue long domain with a predicted TPR-like all-alpha-helical fold.

Conclusions

The occurrence of the PAM domain in specific subunits of multimeric protein complexes, together with the role of other all-alpha-helical folds in protein-protein interactions, suggest a function for this domain in mediating transient binding to diverse target proteins.

     

Global insight into the distribution of velvet-like B protein in <Emphasis Type="Italic">Cochliobolus</Emphasis> species and implication in pathogenicity and fungicide resistance

The Cochliobolus genus consist of over 55 species among which the 5 most devastating are Cochliobolus carbonum, Cochliobolus heterostrophus, Cochliobolus miyabeanus, Crocus sativus and Cochliobolus lunatus causing damages in sorghum, wheat, rice, maize, cassava and soybean estimated at over 10 billion USD per annum worldwide. The dynamic pathogenicity of Cochliobolus species and the plethora of infected hosts is determined by the evolution of virulence determinants such as the velvet-like B protein (VelB). Nonetheless, the knowledge on the distribution of Cochliobolus VelB and its implication in pathogenicity and fungicide resistance are often lacking. By scanning through the annotated genomes of C. lunatus, C. heterostrophus, C. carbonum, C. victoriae, C. sativus and C. miyabeanus, it is revealed that the numbers of ortholog VelB and cognates vary. By using the phylogenetic approach, it is established that the diversification rates among velvet-domain-containing proteins for phytopathogenic Cochliobolus species could impact differently on their oxidant and fungicide resistance potentials, ability to form appressoria-like structures and infection pegs during infection. This study provides new insights into the pathogenicity evolution of Cochliobolus species at the VelB locus which is relevant for designing effective strategies for durable management of Cochliobolus diseases.