In vivo uncoating and efficient expression of foreign mRNAs packaged in TMV-like particles.

The ribonucleocapsids of many plant viruses are extremely stable. The protein coat protects the RNA genome against degradation during the accumulation and spread of progeny virions. Chimeric single-stranded RNA molecules were transcribed in vitro from recombinant plasmids and later encapsidated, in vitro, into ribonucleoprotein particles (pseudoviruses) 60 nanometers long that resembled tobacco mosaic virus. Transcripts encoding an assayable enzyme, chloramphenicol acetyltransferase (CAT), were packaged into pseudovirus particles to assess the utility of this single-stranded RNA delivery system in a wide range of cell types. In all cases, packaged CAT messenger RNA was uncoated and transiently expressed. Significantly higher levels of CAT activity were detected with packaged than with naked CAT messenger RNA after inoculation of plant protoplasts in the presence of polyethylene glycol or abrasive inoculation of intact leaf surfaces. Structural events that lead to the uncoating and expression of CAT messenger RNA showed no cell specificity. This observation may support the view that the comparatively restricted host range of a true plant virus results from events that occur later during the infection cycle.

The mast cell and allergic diseases: role in pathogenesis and implications for therapy.

Mast cells have long been recognized for their role in the genesis of allergic inflammation; and more recently for their participation in innate and acquired immune responses. Mast cells reside within tissues including the skin and mucosal membranes, which interface with the external environment; as well as being found within vascularized tissues next to nerves, blood vessels and glandular structures. Mast cells have the capability of reacting both within minutes and over hours to specific stimuli, with local and systemic effects. Mast cells express the high affinity IgE receptor (FcepsilonRI) and upon aggregation of FcepsilonRI by allergen-specific IgE, mast cells release and generate biologically active preformed and newly synthesized mediators which are involved in many aspects of allergic inflammation. While mast cells have been well documented to be essential for acute allergic reactions, more recently the importance of mast cells in reacting through pattern recognition receptors in innate immune responses has become recognized. Moreover, as our molecular understanding of the mast cell has evolved, novel targets for modulation have been identified with promising therapeutic potential.

Prognostic Value of Occult Isolated Tumour Cells within Regional Lymph Nodes of Dogs with Malignant Mammary Tumours.

Canine mammary tumours (CMTs) are the most common type of neoplasm in bitches. As in women, the presence of metastasis in regional lymph nodes is an important prognostic factor in bitches with mammary carcinomas, but the clinical significance of occult isolated tumour cells (ITCs) within lymph nodes is still undefined in this species. The effectiveness of immunohistochemistry (IHC) in identifying occult ITCs and micrometastasis (MIC) was compared with that of the conventional haematoxylin and eosin staining technique. The relationship between tumour size, histological type, histological grade and the presence of metastasis was evaluated. The overall survival (OS) of female dogs with occult mammary carcinomas and ITCs within lymph nodes was analysed. Fragments of mammary carcinoma and regional lymph nodes of 59 female dogs were also evaluated. Histological sections of mammary carcinoma and lymph node samples were studied for tumour diagnosis and lymph node samples were tested by IHC using a pan-cytokeratin antibody. It was found that 35.2% of occult ITCs and 2.8% of hidden MIC were detected when IHC was used. There was a good correlation between the size of the tumour and metastasis to the lymph nodes (P = 0.77). ITCs were observed more frequently in the medullary region (60.7%) and metastases in the cortical region (44.4%). There was no significant difference in the OS between female dogs with occult ITCs and lymph nodes without ITCs. IHC can detect occult tumour cells in lymph nodes that are negative by histopathological examination. Female dogs with nodal ITCs do not have lower survival.

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein.

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.

Microbial forensics for natural and intentional incidents of infectious disease involving animals.

Microbial forensics is a relatively new scientific discipline dedicated to analysing microbiological evidence from a crime for attribution purposes. It builds on traditional microbiology and epidemiology but within a legal framework. Important motives for forensic investigations include interdiction of criminals, prosecution of justice, and ideally, deterrence of others from committing similar acts. Forensic capabilities in animal health should focus on building capacity for detection and reporting of increases in infectious disease morbidity and mortality among animals that might reflect a covert release of a pathogen. Suspicion should be raised when epidemiological patterns are different from those expected for the animal population and the pathogen in question. Existing capacities for the detection and reporting of epidemic and even endemic diseases should be an international priority for the prevention of catastrophic losses in animal and potentially in human life. The veterinary community needs to be more aware of the legal requirements related to forensic investigations so that veterinarians will be prepared to handle evidence properly within their own fields.

Differential cellular and subcellular expression of the human multifunctional apurinic/apyrimidinic endonuclease (APE/ref-1) DNA repair enzyme.

The multifunctional mammalian apurinic/apyrimidinic endonuclease (APE) is responsible for the repair of apurinic/apyrimidinic sites in DNA. In addition, this enzyme has been shown to function as a redox factor facilitating the DNA-binding capability of JUN and FOS, as well as numerous other transcription factors through the alteration of the transcription factor redox state. Biochemical studies of organ homogenates have shown that APE is present in the different tissues studied at similar concentrations. The present study examines the immunohistochemical distribution of APE in several organs and demonstrates new and unexpected patterns of cellular and subcellular localization of this enzyme. In the hippocampus, the APE protein was highly expressed in neurons of the dentate gyrus and regions CA3 and CA4, and unexpectedly, the staining was primarily cytoplasmic. AP endonuclease immunoreactivity in the cerebellum was found in the granule and Purkinje cells, both cytoplasmic and nuclear. APE staining of the hypoglossal nucleus of the brainstem, where motor neurons that control tongue movement reside, showed reactivity in the cytoplasmic Nissl substance. Skin, liver, and duodenum demonstrated nuclear staining; however, in the duodenum, only the enterocyte nuclei of the proximal villus and the crypts of Lieberkuhn were stained, with no staining of the distal villus. These results suggest that APE has different regulatory and functional roles in different cells and organs of the body. This study shows the importance of correlating in vitro findings in tissue culture cells with the organism as a whole. The cytoplasmic staining seen in parts of the brain and in liver suggests that there may be additional functions for the APE yet to be described.

Differential cellular expression of the human MSH2 repair enzyme in small and large intestine.

The human MSH2 (hMSH2) protein is responsible for the initial recognition of mismatched nucleotides during the postreplication mismatch repair process. Loss of hMSH2 function has been demonstrated to lead to the accumulation of replication errors, resulting in a mutator phenotype, which may be responsible for the multiple mutations required for multi-stage carcinogenesis. Alterations of the hMSH2 gene has been linked to approximately 60% of hereditary nonpolyposis colon cancer cases. Colon tumors in hereditary nonpolyposis colon cancer patients originate within benign preneoplastic adenomas and display replication errors in the form of microsatellite instability. The aim of this study was to investigate the cellular expression of the hMSH2 protein in cells of the large and small intestines. Using antibody specific for hMSH2, we have determined that this protein is highly expressed in cells of the crypts of Lieberkühn that are undergoing rapid renewal in both the ileum and colon. Proliferative perifibroblasts in the colon also showed significant presence of the hMSH2 protein. These results confirm the hypothesis that hMSH2 is expressed in highly proliferative cells of the gut, and mutations in this gene could, therefore, be expected to expedite the progression of adenoma to carcinoma in this tissue.

Bright focused ion beam sources based on laser-cooled atoms.

Nanoscale focused ion beams (FIBs) represent one of the most useful tools in nanotechnology, enabling nanofabrication via milling and gas-assisted deposition, microscopy and microanalysis, and selective, spatially resolved doping of materials. Recently, a new type of FIB source has emerged, which uses ionization of laser cooled neutral atoms to produce the ion beam. The extremely cold temperatures attainable with laser cooling (in the range of 100 µK or below) result in a beam of ions with a very small transverse velocity distribution. This corresponds to a source with extremely high brightness that rivals or may even exceed the brightness of the industry standard Ga+ liquid metal ion source. In this review we discuss the context of ion beam technology in which these new ion sources can play a role, their principles of operation, and some examples of recent demonstrations. The field is relatively new, so only a few applications have been demonstrated, most notably low energy ion microscopy with Li ions. Nevertheless, a number of promising new approaches have been proposed and/or demonstrated, suggesting that a rapid evolution of this type of source is likely in the near future.

The origin of multiple polypeptides of molecular weight below 110 000 encoded by tobacco mosaic virus RNA in the messenger-dependent rabbit reticulocyte lysate.

Multiple polypeptides encoded by tobacco mosaic virus (TMV) RNA in the messenger-dependent rabbit reticulocyte lysate are not attributable to contaminating 3'-coterminal RNA fragments, multiple leaky termination codons or endonuclease activity opening-up legitimate or spurious internal initiation sites. Quantitative analysis of polypeptides encoded over a range of added RNA concentrations from 0.09 microgram X ml-1 to 180 micrograms X ml-1 compared with those synthesized in response to size-fractionated RNAs from a crude virus preparation, or with RNA extracted from the alkali-stable fraction of TMV suggest that apart from four legitimate virus-coded products of apparent Mr approx. 165 000, 110 000, 30 000 and 17 500 all other polypeptides arise from the overlapping 5'-proximal cistrons either by (i) site-selective endonucleolytic cleavage, (ii) sense codon misreading, or (iii) specific regions of secondary structure on TMV RNA which impede ribosome translocation.

Translation initiation factor-dependent extracts from Saccharomyces cerevisiae.

Translation initiation factor 4A- and 4E-dependent extracts were developed from Saccharomyces cerevisiae and used to study factor requirements for translation of individual mRNAs in vitro. Whereas all mRNAs tested required eIF-4A, mRNAs devoid of secondary structure in their 5' untranslated region did not require exogenous eIF-4E for translation. The latter included alfalfa mosaic virus RNA4, mRNA containing the untranslated region of tobacco mosaic virus RNA and mRNA containing part of the untranslated region of poliovirus RNA. Furthermore, initiation of translation on mRNAs containing part of the untranslated region of poliovirus RNA is most likely internal.

Coordinate gene expression of luteinizing hormone-releasing hormone (LHRH) and the LHRH-receptor after prolactin stimulation in the rat Nb2 T-cell line: implications for a role in immunomodulation and cell cycle gene expression.

PRL has been shown to induce a number of genes after the stimulation of quiescent Nb2 T-cells, including c-fos, c-myc, ornithine decarboxylase, interferon regulatory factor-1, and others. One of these genes, LHRH, has not previously been reported to respond in this manner, although we and others have reported its presence in rat and human T- and B-cells. Furthermore, recent evidence suggests that LHRH functions as an immunoregulator in a cytokine-like manner. Using the rat immature T-cell line Nb2, we present data showing for the first time that 1) the LHRH gene is regulated by PRL at various times during the cell cycle; 2) an alternatively spliced LHRH messenger RNA exists in Nb2 cells and may produce a new truncated GnRH-associated peptide (alternatively called PIF for PRL-inhibiting factor); 3) the LHRH receptor is expressed in lymphocytes in a manner similar to the LHRH gene after PRL addition, and its complementary DNA sequence is identical to that of the pituitary receptor; 5) the SH gene, found on the opposite strand of the LHRH gene, is expressed in lymphocytes at the same time and in the same manner as the LHRH gene; 6) the LHRH messenger RNA has a very short half-life in these cells; and 7) the lymphocyte LHRH transcription start site is essentially the same as the hypothalamic site. These data strengthen the relationship between PRL and LHRH expression in the immune system and further support our contention that LHRH is an important immunoregulator, on par with other known cytokines.

Primary gastric rupture in 20 horses from Midwestern Brazil: case report / Ruptura gástrica primária em 20 equinos no Centro-Oeste do Brasil: relato de caso

An 11-year (2007-2018) survey of epidemiological, clinical and pathological findings of horses with primary gastric rupture (PGR) was conducted. Twenty horses presented PGR, nine (45%) horses were clinically evaluated, eleven (55%) were sent dead, and all animals were necropsied. PGR contributed to a prevalence of 0.31% (9/2,868) of all equid attendances, 1.83% (9/491) of colic cases, and 4.1% (20/487) of all equid necropsies. Highly fermentable feed (n=7), gastric impaction (n=4), and perforating gastric ulcer (n=1) were the main causes of PGR; whilst eight horses presented idiopathic gastric rupture. Clinically evaluated horses were tachycardic, tachypneic, febrile, dehydrated, with increased abdominal tension, abnormal mucous membranes and reduced to absent intestinal borborygmi. Improper dietary management, such as the ingestion of low-quality roughage and highly fermentable feedstuffs were detected as the main factors associated with PGR in Midwestern Brazil. It is important to raise awareness in horse owners about proper feed management to minimize PGR.(AU)

Foi realizado um levantamento de 11 anos (2007-2018) dos achados epidemiológicos, clínicos e patológicos de equinos com ruptura gástrica primária (RGP). Vinte equinos apresentaram RGP, dos quais nove (45%) foram avaliados clinicamente e 11 (55%) foram enviados mortos, sendo todos os animais necropsiados. A RGP contribuiu com prevalência de 0,31% de todos os atendimentos de equídeos (9/2.868), 1,83% (9/491) dos casos de cólica, e 4,1% (20/487) das necropsias em equídeos. Alimentos altamente fermentáveis (n=7), compactação gástrica (n=4) e perfuração de úlcera gástrica (n=1) foram as principais causas de RGP, enquanto oito equinos tiveram ruptura gástrica idiopática. Os equinos avaliados clinicamente apresentaram-se taquicárdicos, taquipneicos, febris, desidratados, com mucosas anormais, aumento da tensão abdominal e motilidade intestinal reduzida. O manejo inadequado da dieta, como a ingestão de forragens de baixa qualidade e alimentos altamente fermentáveis, foi o principal fator de risco associado à RGP no Centro-Oeste do Brasil. É importante aumentar a conscientização dos proprietários de equinos sobre o manejo alimentar adequado para minimizar a RGP.(AU)

Chromosomal insertion of phenazine-1-carboxylic acid biosynthetic pathway enhances efficacy of damping-off disease control by Pseudomonas fluorescens.

A disarmed Tn5 vector (pUT::Ptac-phzABCDEFG) was used to introduce a single copy of the genes responsible for phenazine-1-carboxylic acid (PCA) biosynthesis into the chromosome of a plant-growth-promoting rhizobacterium Pseudomonas fluorescens. The PCA gene cluster was modified for expression under a constitutive Ptac promoter and lacked the phzIR regulators. PCA-producing variants significantly improved the ability of the wild-type P. fluorescens to reduce damping-off disease of pea seedlings caused by Pythium ultimum, even under conditions of heavy soil infestation. Under conditions of oxygen limitation that are typical of the rhizosphere, PCA production per cell in vitro was greater than that recorded in fast-growing, nutrient-rich cultures. Similarly, when the in vitro nutrient supply was limited, P fluorescens::phz variants that produced the most PCA effectively competed against P. ultimum by suppressing mycelial development. Soil-based bioassays confirmed that the level of PCA biosynthesis correlated directly with the efficacy of biological control and the persistence of inocula in soil microcosms. They also showed that soil pretreatment with bacteria provides a suitable method for plant protection by reducing infection, effectively decontaminating the soil. These data demonstrate that the insertion of a single chromosomal copy of the genes for a novel antifungal compound, PCA, enhances the ecological fitness of a natural isolate already adapted to the rhizosphere and capable of suppressing fungal disease.

The 5'-leader sequence of tobacco mosaic virus RNA mediates initiation-factor-4E-independent, but still initiation-factor-4A-dependent translation in yeast extracts.

Messenger RNAs encoding chloramphenicol acetyltransferase (CAT) with or without the 5'-leader sequence of tobacco mosaic virus (TMV) RNA were synthesized in vitro and translated in Saccharomyces cerevisiae extracts dependent on eukaryotic initiation factors eIF-4E or eIF-4A. The 5'-leader sequence of TMV RNA renders translation of CAT mRNA eIF-4E-independent but still 4A-dependent.

Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro.

Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5'-terminal 67-nucleotide sequence (omega') derived from the untranslated 5'-leader (omega) of tobacco mosaic virus (TMV) RNA. Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC). The omega' sequence enhanced expression of each mRNA in almost every translation system. While MDL was the least responsive to omega', this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega'. The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega' sequence. These findings extend the utility of omega' as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.

Production of a functional single chain antibody attached to the surface of a plant virus.

A potato virus X (PVX) vector was used to express a single chain antibody fragment (scFv) against the herbicide diuron, as a fusion to the viral coat protein. The modified virus accumulated in inoculated Nicotiana clevelandii plants and assembled to give virus particles carrying the antibody fragment. Electron microscopy was used to show that virus particles from infected leaf sap were specifically trapped on grids coated with a diuron-BSA conjugate. The results demonstrate that the PVX vector can be used as a presentation system for functional scFv.

Choroidal and cerebral blood flow in baboons measured by the external monitoring of radioactive inert gases.

Quantitative measurements of blood flow using xenon-133 to measure cerebral blood flow and krypton-85 to measure choroidal blood flow were made in 12 anesthetized baboons (Papio anubis). Analysis of the clearance curves of krypton from the eye and studies of the diffusion of krypton in the eye show that the inert gas-clearance method measures choroidal blood flow only. The mean choroidal blood flow at normocapnia in this group of baboons is 463 +/- 44 ml./100 mg./min., and the mean cerebral blood flow is 46 +/- 5 ml./100 mg./min. (mean +/- 1 S.D.).

The response of the choroidal and cerebral circulations to changing arterial PCO2 and acetazolamide in the baboon.

Having established control values for choroidal and cerebral blood flow in twelve baboons, the response of both circulations to changing arterial PCO2 and intravenous acetazolamide was studied. The blood flow in both circulations varied directly with the PACO2, the magnitude of the response being very similar. There was a 3.6 percent change in both choroidal and cerebral blood flow per millimeter of mercury change in PACO2. Intravenous acetazolamide (25 mg./kg.) produced an increase in flow lasting approximately 50 minutes in both cerebral and choroidal circulations.

Single-strand conformation polymorphism analysis of RT-PCR products of UK isolates of barley yellow mosaic virus.

Single-strand conformation polymorphism (SSCP) analysis of the bipartite genomes of several UK isolates of barley yellow mosaic virus (Ba YMV) was done using fragments of cDNA amplified by RT-PCR. Isolates differed in their SSCP patterns in several regions, but in no case was the pattern able to distinguish between common and resistance-breaking strains. In regions where the nucleotide sequences of UK isolates had been determined, there was no simple relationship between numbers of nucleotide differences and SSCP patterns: differences of only 2 or 3 nucleotides (nt) gave different SSCP patterns, whereas differences of as many as 29 nt did not. Although SSCP analysis has some potential as a rapid and sensitive tool for distinguishing virus isolates, differences detected do not necessarily relate to biological properties and the results are highly dependent on gel conditions.

Molecular characterisation of UK isolates of barley yellow mosaic bymovirus.

Several isolates of barley yellow mosaic virus (BaYMV) from different sites in the UK, including some that were virulent on European resistant winter barley cultivars (resistance-breaking strain: BaYMV-2) and some that were not, were examined by RT-PCR, restriction mapping and sequencing of selected parts of the virus genome. Nucleotide and predicted amino acid sequences were determined for the 5'-terminal region, part of the NIa coding region and the coat protein coding region on RNA 1 and an area at the N-terminus of the 70-kDa protein coding region on RNA 2. The sequences differed from those previously reported for a BaYMV isolate from Japan and for two German isolates, one of which was of the BaYMV-2 strain. There were no strain-specific amino acid differences and the few, non-consecutive, nucleotide differences detected were probably not significant and were insufficient to develop a rapid diagnostic test to distinguish BaYMV-2 from other isolates. Restriction mapping of RNA 2 cDNA again showed no consistent strain-related differences. The differences previously reported between the two German isolates are probably not strain-related.