Predictive value of selected geriatric parameters for postoperative outcomes in older patients with rectal cancer - A multicenter cohort study.

INTRODUCTION: Older patients have a higher risk for complications after rectal cancer surgery. Although screening for geriatric impairments may improve risk prediction in this group, it has not been studied previously. METHODS: We retrospectively investigated patients ≥70 years with elective surgery for non-metastatic rectal cancer between 2014 and 2018 in nine Dutch hospitals. The predictive value of six geriatric parameters in combination with standard preoperative predictors was studied for postoperative complications, delirium, and length of stay (LOS) using logistic regression analyses. The geriatric parameters included the four VMS-questionnaire items pertaining to functional impairment, fall risk, delirium risk, and malnutrition, as well as mobility problems and polypharmacy. Standard predictors included age, sex, body mass index, American Society of Anesthesiologists (ASA)-classification, comorbidities, tumor stage, and neoadjuvant therapy. Changes in model performance were evaluated by comparing Area Under the Curve (AUC) of the regression models with and without geriatric parameters. RESULTS: We included 575 patients (median age 75 years; 32% female). None of the geriatric parameters improved risk prediction for complications or LOS. The addition of delirium risk to the standard preoperative prediction model improved model performance for predicting postoperative delirium (AUC 0.75 vs 0.65, p = 0.03). CONCLUSIONS: Geriatric parameters did not improve risk prediction for postoperative complications or LOS in older patients with rectal cancer. Delirium risk screening using the VMS-questionnaire improved risk prediction for delirium. Older patients undergoing rectal cancer surgery are a pre-selected group with few impairments. Geriatric screening may have additional value earlier in the care pathway before treatment decisions are made.

Alterations in Sub-Axonal Architecture Between Normal Aging and Parkinson's Diseased Human Brains Using Label-Free Cryogenic X-ray Nanotomography.

Gaining insight to pathologically relevant processes in continuous volumes of unstained brain tissue is important for a better understanding of neurological diseases. Many pathological processes in neurodegenerative disorders affect myelinated axons, which are a critical part of the neuronal circuitry. Cryo ptychographic X-ray computed tomography in the multi-keV energy range is an emerging technology providing phase contrast at high sensitivity, allowing label-free and non-destructive three dimensional imaging of large continuous volumes of tissue, currently spanning up to 400,000 µm3. This aspect makes the technique especially attractive for imaging complex biological material, especially neuronal tissues, in combination with downstream optical or electron microscopy techniques. A further advantage is that dehydration, additional contrast staining, and destructive sectioning/milling are not required for imaging. We have developed a pipeline for cryo ptychographic X-ray tomography of relatively large, hydrated and unstained biological tissue volumes beyond what is typical for the X-ray imaging, using human brain tissue and combining the technique with complementary methods. We present four imaged volumes of a Parkinson's diseased human brain and five volumes from a non-diseased control human brain using cryo ptychographic X-ray tomography. In both cases, we distinguish neuromelanin-containing neurons, lipid and melanic pigment, blood vessels and red blood cells, and nuclei of other brain cells. In the diseased sample, we observed several swellings containing dense granular material resembling clustered vesicles between the myelin sheaths arising from the cytoplasm of the parent oligodendrocyte, rather than the axoplasm. We further investigated the pathological relevance of such swollen axons in adjacent tissue sections by immunofluorescence microscopy for phosphorylated alpha-synuclein combined with multispectral imaging. Since cryo ptychographic X-ray tomography is non-destructive, the large dataset volumes were used to guide further investigation of such swollen axons by correlative electron microscopy and immunogold labeling post X-ray imaging, a possibility demonstrated for the first time. Interestingly, we find that protein antigenicity and ultrastructure of the tissue are preserved after the X-ray measurement. As many pathological processes in neurodegeneration affect myelinated axons, our work sets an unprecedented foundation for studies addressing axonal integrity and disease-related changes in unstained brain tissues.

The Prognostic Value of a Geriatric Risk Score for Older Patients with Colorectal Cancer.

INTRODUCTION: VMS is a Dutch risk assessment tool for hospitalized older adults that includes a short evaluation of four geriatric domains: risk for delirium, risk for undernutrition, risk for physical impairments, and fall risk. We investigated whether the information derived from this tool has prognostic value for outcomes of colorectal surgery. METHODS: All consecutive patients over age 70 years who underwent elective colorectal cancer surgery in three Dutch hospitals (2014-2016) were studied. The presence of risk was scored prior to surgery and per geriatric domain as either 0 (risk absent) or 1 (risk present). The total number of geriatric risk factors was summed. The primary outcome was long-term survival. Secondary outcomes were postoperative complications, including delirium. Cox proportional hazards models were used to evaluate the sumscore and risk factors associated with overall survival. RESULTS: Five hundred fifty patients were included. Median age was 76.5 years, and median follow-up was 870 days. Patients with intermediate (1-2) or high (3-4) sumscore were independently associated with lower overall survival, with hazard ratio (HR) of 1.9 [95% confidence interval (CI) 1.1-3.5; p = 0.03] and 8.7 (95% CI 4.0-19.2; p < 0.001), respectively. Sumscores were also associated with postoperative complications (intermediate sumscore OR 1.8; 95% CI 1.2-2.7; high sumscore OR 2.4; 95% CI 1.02-5.5). CONCLUSIONS: This easy-to-use geriatric sumscore has strong associations with long-term outcome and morbidity after colorectal cancer surgery. This information may be included in risk models for morbidity and mortality and can be used in shared decision-making.

The blood clotting Factor XIIIa forms unique complexes with amyloid-beta (Aß) and colocalizes with deposited Aß in cerebral amyloid angiopathy.

AIMS: Cerebral amyloid angiopathy (CAA) is a key pathological hallmark of Alzheimer's disease (AD) characterized by accumulation of amyloid-beta (Aß) protein in blood vessel walls. CAA impairs vessel functioning, affects blood brain barrier integrity and accelerates cognitive decline of AD patients. Unfortunately, mechanisms underlying Aß deposition in the vessel wall remain largely unknown. Factor XIIIa (FXIIIa) is a blood-derived transglutaminase crucial in blood coagulation by cross-linking fibrin molecules. Evidence is mounting that blood-derived factors are present in CAA and may play a role in protein deposition in the vessel wall. We therefore investigated whether FXIIIa is present in CAA and if FXIIIa cross-link activity affects Aß aggregation. METHODS: Using immunohistochemistry, we investigated the distribution of FXIIIa, its activator thrombin and in situ FXIIIa activity in CAA in post-mortem AD tissue. We used surface plasmon resonance and Western blot analysis to study binding of FXIIIa to Aß and the formation of FXIIIa-Aß complexes, respectively. In addition, we studied cytotoxicity of FXIIIa-Aß complexes to cerebrovascular cells. RESULTS: FXIIIa, thrombin and in situ FXIIIa activity colocalize with the Aß deposition in CAA. Furthermore, FXIIIa binds to Aß with a higher binding affinity for Aß1-42 compared with Aß1-40 . Moreover, highly stable FXIIIa-Aß complexes are formed independently of FXIIIa cross-linking activity that protected cerebrovascular cells from Aß-induced toxicity in vitro. CONCLUSIONS: Our data showed that FXIIIa colocalizes with Aß in CAA and that FXIIIa forms unique protein complexes with Aß that might play an important role in Aß deposition and persistence in the vessel wall.

Anti-inflammatory effect by lentiviral-mediated overexpression of IL-10 or IL-1 receptor antagonist in rat glial cells and macrophages.

Neuroinflammation, as defined by activation of local glial cells and production of various inflammatory mediators, is an important feature of many neurological disorders. Expression of pro-inflammatory mediators produced by glial cells in the central nervous system (CNS) is considered to contribute to the neuropathology observed in those diseases. To diminish the production or action of pro-inflammatory mediators, we have used lentiviral (LV) vector-mediated encoding rat interleukin-10 (rIL-10) or rat interleukin-1 receptor antagonist (rIL-1ra) to direct the local, long-term expression of these anti-inflammatory cytokines in the CNS. We have shown that cultured macrophages or astroglia transduced with LV-rIL-10 or LV-rIL-1ra produced far less tumor necrosis factor (TNF)alpha or IL-6, respectively in response to pro-inflammatory stimuli. Moreover, intracerebroventricular (i.c.v.) administration of LV-rIL-10 or LV-rIL-1ra resulted in transduction of glial cells and macrophages and, subsequently reduced TNFalpha, IL-6 and inducible nitric oxide synthase (iNOS) expression in various brain regions induced by inflammatory stimuli, whereas peripheral expression of these mediators remained unaffected. In addition, expression levels of the anti-inflammatory cytokines IL-4 and transforming growth factor-beta were not altered in either brain or pituitary gland. Furthermore, i.c.v. administration of LV-rIL-10 or LV-rIL-1ra given during the remission phase of chronic-relapsing experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, improved the clinical outcome of the relapse phase. Thus, local application of LV vectors expressing anti-inflammatory cytokines could be of therapeutic interest to counteract pro-inflammatory processes in the brain without interfering with the peripheral production of inflammatory mediators.

Renal function as a predictor of irinotecan-induced neutropenia.

Although approximately half of the administered dose of irinotecan is recovered in urine, scarce data are available on the association of renal function with irinotecan pharmacokinetics and toxicity. Here, these relationships are investigated in 187 patients treated with irinotecan in a three-weekly schedule. No significant effects on irinotecan pharmacokinetics were found in these patients. However, in 131 patients treated with the registered dose, categorized renal function was related to hematological toxicity. The incidence of grade 3-4 neutropenia decreased as function of creatinine clearance, particularly in nonsmoking patients (P < 0.01). Patients with slower creatinine clearance (35-66 ml/min) had a four-times higher risk of grade 3-4 neutropenia (58% vs. 14%; P < 0.001). This study suggests that pretreatment renal function values are associated with irinotecan-induced neutropenia. A confirmatory analysis is warranted to determine whether measures of renal function should be incorporated in future attempts toward individualized treatment with irinotecan.

Changed distribution pattern of the constitutive rather than the inducible HSP70 chaperone in neuromelanin-containing neurones of the Parkinsonian midbrain.

Aberrant protein aggregation has been recognized as an important factor in the degeneration of melanized dopaminergic neurones in Parkinson's disease (PD). The constitutive (HSP73) and (heat)-inducible (HSP72) proteins of the heat shock 70 family form a major defence system against pathological protein aggregation. However, the distribution patterns of these chaperones in nigral neuromelanin-laden neurones are largely unknown. The present study determined the distribution of HSP72 and HSP73 in control and Parkinsonian substantia nigra, using immunohistochemistry. In the neuromelanin-laden neurones of controls, HSP72 was nondetectable, whereas HSP73 was weakly expressed in both the cytosol and the nucleus. Surprisingly, in PD subjects, marked nuclear HSP73, but not HSP72 immunoreactivity was observed, while cytosolic immunoreactivity of the two chaperones resembled the labelling pattern observed in controls. Furthermore, HSP73 immunoreactivity was observed in a subset of the Lewy bodies (LBs) detected in the substantia nigra of PD subjects, whereas only few of these LBs were labelled with HSP72. Interestingly, HSP72 and to a lesser extent HSP73 immunoreactivity was much stronger in nonmelanized neurones as compared with melanized neurones in this area. Thus, we conclude that the distribution pattern of HSP73 rather than HSP72 is changed in the nigral neuromelanin-laden neurones of PD subjects as compared with control subjects. The impaired ability of aged, dopaminergic neurones to express high levels of chaperones, may contribute to the preferential vulnerability of the latter cells in PD.

Expression of NAD(P)H:quinone oxidoreductase in the normal and Parkinsonian substantia nigra.

Dopamine (DA) autooxidation, and consequent formation of neurotoxic DA-derived quinones and reactive oxygen species, has been implicated in dopaminergic cell death and, hence, in the pathogenesis of Parkinson's disease (PD). Stimulation of pathways involved in the detoxication of DA-quinones in the brain is hypothesized to be an effective means to limit oxidative stress and to confer neuroprotection in PD. In this respect, the inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) is of particular interest as it is directly implicated in the detoxication of DA-quinones and, in addition, has broad spectrum anti-oxidant properties. To study the potential pathophysiological role of NQO1 in PD, the cellular expression of NQO1 was examined in the mesencephalon of PD patients and age-matched controls. In the substantia nigra pars compacta (SNpc), NQO1 was found to be expressed in astroglial and endothelial cells and, albeit less frequently, also in dopaminergic neurons. Moreover, while overt NQO1 immunoreactivity was absent in the surrounding nervous tissue, in the Parkinsonian SNpc a marked increase in the astroglial and neuronal expression of NQO1 was consistently observed.

Composition of alphavirus-like replication complexes: involvement of virus and host encoded proteins.

The RNA-dependent RNA polymerases (RdRp's) of plus-strand RNA viruses contain one or more viral proteins. These proteins contain polymerase (POL) motifs corresponding to the active sites of the polymerase proteins. Additionally, domains corresponding to RNA helicase (HEL), methyltransferase (MT) or proteinase activity of the RdRp may be present. Comparison of available sequence data showed that each class of domain can be subdivided into two or three subclasses, and resulted in the classification of plus-strand RNA viruses into three supergroups. Here, we review our current knowledge on the composition of the RdRp's of alpha-like viruses from supergroup III. The strategy for the expression of viral replicase proteins, their stoichiometry in the enzyme complex, their mutual interactions and their possible functions in RNA synthesis are discussed. Moreover, the review covers host proteins that have been identified in viral RdRp's and their possible role in RNA synthesis.

Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA.

On entering a host cell, positive-strand RNA virus genomes have to serve as messenger for the translation of viral proteins. Efficient translation of cellular messengers requires interactions between initiation factors bound to the 5'-cap structure and the poly(A) binding protein bound to the 3'-poly(A) tail. Initiation of infection with the tripartite RNA genomes of alfalfa mosaic virus (AMV) and viruses from the genus Ilarvirus requires binding of a few molecules of coat protein (CP) to the 3' end of the nonpolyadenylated viral RNAs. Moreover, infection with the genomic RNAs can be initiated by addition of the subgenomic messenger for CP, RNA 4. We report here that extension of the AMV RNAs with a poly(A) tail of 40 to 80 A-residues permitted initiation of infection independently of CP or RNA 4 in the inoculum. Specifically, polyadenylation of RNA 1 relieved an apparent bottleneck in the translation of the viral RNAs. Translation of RNA 4 in plant protoplasts was autocatalytically stimulated by its encoded CP. Mutations that interfered with CP binding to the 3' end of viral RNAs reduced translation of RNA 4 to undetectable levels. Possibly, CP of AMV and ilarviruses stimulates translation of viral RNAs by acting as a functional analogue of poly(A) binding protein or other cellular proteins.

Sequence comparison and secondary structure analysis of the 3' noncoding region of flavivirus genomes reveals multiple pseudoknots.

Sequences of 191 flavivirus RNAs belonging to four sero-groups were used to predict the secondary structure of the 3' noncoding region (3' NCR) directly upstream of the conserved terminal hairpin. In mosquito-borne flavivirus RNAs (n = 164) a characteristic structure element was identified that includes a phylogenetically well-supported pseudoknot. This element is repeated in the dengue and Japanese encephalitis RNAs and centers around the conserved sequences CS2 and RCS2. In yellow fever virus RNAs that contain one CS2 motif, only one copy of this pseudoknotted structure was found. The conserved pseudoknotted element is absent from the 3' NCR of tick-borne virus RNAs, which altogether adopt a secondary structure that is very different from that of mosquito-borne virus RNAs. The strong conservation of the pseudoknot in mosquito-borne flavivirus RNAs implies a stronger relationship between these viruses than concluded from previous secondary structure analyses. The role of the (tandem) pseudoknots in flavivirus replication is discussed.

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport.

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.

Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants.

Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In addition, the mutations were introduced in the MP gene of AMV RNA 3 and the mutant RNAs were used to infect tobacco plants. Ten mutants were affected in one or more of the following functions of MP: the formation of tubular structures on the surface of protoplasts, association with the endoplasmic reticulum (ER) of suspension cells and epidermal cells, targeting to punctate structures in the cell wall of epidermis cells, movement from transfected cells to adjacent cells in epidermis tissue, cell-to-cell movement, or long-distance movement in plants. The mutations point to functional domains of the MP and support the proposed order of events in AMV transport. Studies with several inhibitors indicate that actin or microtubule components of the cytoskeleton are not involved in tubule formation by AMV MP. Evidence was obtained that tubular structures on the surface of transfected protoplasts contain ER- or plasmalemma-derived material.

Induction of a hypersensitive response by chimeric helicase sequences of tobamoviruses U1 and Ob in N-carrying tobacco.

Recently, the helicase domain of the Tobacco mosaic virus (TMV)-U1 replicase proteins (designated MOREHEL:U1) was identified as the elicitor of the N gene-mediated hypersensitive response (HR) in tobacco. In this study, we used agroinfiltration to express the equivalent MOREHEL domain of the non-HR-inducing tobamovirus strain TMV-Ob. It appeared that this MOREHEL:Ob sequence did not elicit a HR in N gene-carrying tobacco. Both MOREHEL sequences were divided into eight subdomains, and chimeras of MOREHEL sequences from U1 and Ob were constructed. Expression of these chimeric MOREHEL sequences revealed that, in the TMV-U1 MOREHEL sequence, at least four domains involved in full HR induction were present. The presence of at least three of these four domains seems a minimal requirement for HR induction. Two additional domains may play a minor role in HR induction. To study the elicitor function of the chimeras during the TMV life cycle, chimeric MOREHEL domains were introduced into full-length TMV cDNA clones. These constructs, however, were unable to establish an infection in Nicotiana benthamiana or Nicotiana tabacum plants.

Engineering of alfalfa mosaic virus RNA 3 into an expression vector.

RNA 3 of alfalfa mosaic virus (AMV) encodes the 5'-proximal movement protein (MP) gene and the 3'-proximal coat protein (CP) gene which is expressed from a subgenomic RNA. Several strategies were explored to use this RNA as a vector for expression of the green fluorescent protein (GFP) in Nicotiana tabaccum plants expressing the viral polymerase proteins P1 and P2 (P12 plants). Insertion of a subgenomic promoter (sgp)-GFP cassette between the CP gene and the 3'-untranslated region (UTR) interfered with RNA accumulation in protoplasts, indicating that cis-acting sequences required for replication were disrupted. When GFP was fused to the N-terminus of MP or CP, the chimeric RNAs accumulated in protoplasts but cell-to-cell movement in plants was blocked. Insertion of a GFP-sgp cassette immediately upstream of the CP gene caused a hypersensitive host response. However, insertion of a GFP-sgp cassette upstream of the MP gene did not affect symptom formation and yielded a vector that expressed GFP in inoculated but not in the systemic leaves of both P12 tobacco and non-transgenic N. benthamina plants. When the size of the GFP gene was reduced from 700 to 300 nucleotides, virus infection was observed in the non-inoculated leaves. Analysis of the progeny of some chimera revealed novel data on replication, encapsidation and recombination of AMV RNA 3.

Role of the 3'-untranslated regions of alfalfa mosaic virus RNAs in the formation of a transiently expressed replicase in plants and in the assembly of virions.

Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration of Nicotiana benthamiana, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3'-untranslated regions (UTRs) of both RNAs deleted (R1Delta/2Delta construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1Delta/2Delta construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1Delta/2 (3'-UTR of RNA 1 deleted), R1/2Delta (3'-UTR of RNA 2 deleted), or R1Delta/2Delta plus R3. This demonstrates that the 3'-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3'-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3'-termini of AMV RNAs is not required for assembly of virus particles.

A single administration of interleukin-1 or amphetamine induces long-lasting increases in evoked noradrenaline release in the hypothalamus and sensitization of ACTH and corticosterone responses in rats.

Single administration of the cytokine interleukin-1beta (IL-1) or the psychostimulant amphetamine causes long-term sensitization of the hypothalamus pituitary adrenal (HPA) axis, i.e. enhanced adrenocorticotropine hormone (ACTH) and corticosterone responses weeks later. HPA responses to these stimuli involve activation of hypothalamic corticotropin-releasing hormone (CRH) neurons by noradrenergic projections to the paraventricular nucleus (PVN). In search of the underlying mechanisms, we studied the temporal pattern of HPA sensitization in relation to (1) the reactivity of noradrenergic projections to the PVN and (2) altered secretagogue production in hypothalamic CRH neurons. Single exposure to IL-1 or amphetamine induced cross-sensitization of ACTH and corticosterone responses 11 and 22 days later, but not after 42 days. Amphetamine-induced HPA sensitization was not accompanied by increased costorage of arginine vasopressin (AVP) in CRH terminals, as found previously after IL-1 pretreatment. The reactivity of noradrenergic terminals was assessed by measuring the electrically evoked release of [3H]-noradrenaline from superfused PVN slices. Single administration of amphetamine and IL-1 induced a long-lasting (up to 22 days) increase (up to 165%) of evoked noradrenaline release. This indicates that single exposure to psychostimulants or to cytokines can induce a long-lasting increase in stimulus-secretion coupling in brainstem noradrenergic neurons that innervate the PVN. This common, long-lasting functional change may underlie, at least in part, IL-1- and amphetamine-induced HPA cross-sensitization. In addition, increased AVP signalling by hypothalamic CRH neurons appears to play a role in IL-1-induced, but not in amphetamine-induced, HPA sensitization.

Recognition of cis-acting sequences in RNA 3 of Prunus necrotic ringspot virus by the replicase of Alfalfa mosaic virus.

Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera ALFAMOVIRUS: and ILARVIRUS:, respectively, of the family BROMOVIRIDAE: Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3' termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. CIS:-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV-PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.

Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane.

Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity. A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction. In P2, the sequence of the N-terminal 241 aa was required for the interaction. In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies. While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell. As Brome mosaic virus replication complexes have been found to be associated with the endoplasmic reticulum (M. A. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908-8916, 1996), viruses in the family Bromoviridae apparently select different cellular membranes for the assembly of their replication complexes.

Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance.

After a hypersensitive response to invading pathogens, plants show elevated accumulation of salicylic acid (SA), induced expression of plant defense genes, and systemic acquired resistance (SAR) to further infection by a broad range of pathogens. There is compelling evidence that SA plays a crucial role in triggering SAR. We have transformed tobacco with two bacterial genes coding for enzymes that convert chorismate into SA by a two-step process. When the two enzymes were targeted to the chloroplasts, the transgenic (CSA, constitutive SA biosynthesis) plants showed a 500- to 1,000-fold increased accumulation of SA and SA glucoside compared to control plants. Defense genes, particularly those encoding acidic pathogenesis-related (PR) proteins, were constitutively expressed in CSA plants. This expression did not affect the plant phenotype, but the CSA plants showed a resistance to viral and fungal infection resembling SAR in nontransgenic plants.