Construction and properties of a gene-silencing vector based on Poplar mosaic virus (genus Carlavirus).

A gene-silencing vector based on a full-length genomic clone of Poplar mosaic virus (PopMV) was constructed, with coat protein and movement protein genes removed, and containing instead, the coding sequence for green fluorescent protein (GFP). This paper demonstrates that the PopMV-derived gene-silencing vector was able to silence GFP expression in GFP transgenic Nicotiana benthamiana plants. The full-length genome of an Oxford isolate of PopMV (PV275) was cloned and sequenced. A full-length PopMV clone, under transcriptional control of the 35SCaMV promoter was then constructed, and the clone was able to replicate locally in Nicotiana species. Several autonomous plant RNA and DNA viruses have been converted into vectors and implemented for virus-induced gene-silencing (VIGS) of transgenes and endogenous genes [Burton, R., Gibeaut, D., Bacic, A., Findlay, K., Roberts, K., Hamilton, A., Baulcombe, D., Fincher, G., 2000. Virus-induced silencing of a plant cellulose synthase gene. Plant Cell 12, 691-706; Dalmay, T., Horsefield, R., Braunstein, T.H., Baulcombe, D.C., 2001. SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis. EMBO J. 20, 2069-2077; Gossele, V., Fache, I., Meulewaeter, F., Cornelissen, M., Metzlaff, M., 2002. SVISS--a novel transient gene silencing system for gene function discovery and validation in tobacco plants. Plant J. 32, 859-866; Holzberg, S., Brosio, P., Gross, C., Pogue, G.P., 2002. Barley stripe mosaic virus-induced gene silencing in a monocot plant. Plant J. 30, 315-327; Ratcliff, F., Martin-Hernandez, A., Baulcombe, D., 2000. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J. 25, 237-245; Ruiz, M., Vionnet, O., Baulcombe, D., 1998. Initiation and maintenance of virus-induced gene silencing. Plant Cell 10, 937-946]. The use of a virus that naturally infects trees as a gene-silencing vector has not been demonstrated before. The ability to systemically silence a plant transgene following the production of a gene-silencing signal from a locally replicating viral-construct derived from a carlavirus has not to our knowledge been shown before.

Alignment of trans-tibial prostheses based on roll-over shape principles.

The authors examined the roll-over shape alignment hypothesis, which states that prosthetic feet are aligned by matching their roll-over shapes with an "ideal" shape. The "ideal" shape was considered to be the roll-over shape of the able-bodied foot-ankle system. An alignment algorithm and computational alignment system were developed to set trans-tibial alignments based on this hypothesis. Three prosthetic feet with considerably different roll-over shapes were either aligned using the alignment system or not aligned (i.e. used previous foot's alignment), and then were aligned by a team of prosthetists. No significant differences were found between roll-over shapes aligned by the computational alignment system and those based on standard clinical techniques (p = 0.944). Significant differences were found between the "no alignment" shapes and the prosthetist alignment shapes (p = 0.006), and between the "no alignment" shapes and the computational alignment system shapes (p = 0.024). The results of the experiment support the hypothesis that the goal of alignment is to match the prosthetic foot's roll-over shape, as closely as possible, with an "ideal" shape. The hypothesis is also supported by its ability to explain the results of previous studies. Using an "ideal" roll-over shape or surface as a goal for prosthetic alignment could lead to a priori alignment, eliminating the need for alignment hardware in some cases. Being able to build the alignment into a prosthesis without special hardware could be beneficial in low-income countries and in the fabrication of lightweight prostheses for the elderly.

Characterisation of the culturable heterotrophic bacterial community in a small eutrophic lake (Priest Pot).

The community composition and structure of planktonic heterotrophic bacteria (903 isolates) sampled from a small eutrophic lake in northern England (Priest Pot) was studied with respect to season (four samples) and depth (to 3.1 m). Bacteria (887) were isolated on tryptic soy broth agar and identified to 48 genera using fatty acid methyl ester analysis. The two most abundant genera isolated were Aeromonas and Pseudomonas which, respectively, dominated the middle to bottom depths in August and all depths in February. The structure of the sampled community was described using: species richness, Simpson's index and the Shannon-Wiener index. All three indices detected a number of significant differences with depth demonstrating stratification. The greatest stratification of the bacterial community was observed in August when bacterial counts correlated strongly and negatively with diversity. Using structural measures was found to be preferable to the use of species frequencies in the analysis of perturbation and succession in community structure. Insensitivity to one or more of eight antibiotics was observed in 71% (61/86) of the isolates tested particularly in Gram-negative genera. Bacteriocinogeny and lysogeny was observed in 36% (32/90) of isolates. Using sensitive indicator strains, two of 10 producing strains produced virus, while the others produced bacteriocins.

Below knee prosthetic socket designs and suspension systems.

The prosthetic socket must act as a customized connection between the residual limb's surrounding tissues and the prosthetic components. The socket must be designed to control weight bearing, suspension, and ambulation stability. When making a below-the-knee socket, the prosthetist attempts to maximize loading and minimize displacements, such as vertical, transverse, or rotational. This article discusses the engineering designs or shapes of the two basic forms of below-the-knee prosthetic sockets used today.

The prevalence and spatial distribution of viruses in natural populations of Brassica oleracea.

We report a survey of four viruses (beet western yellows luteovirus (BWYV), cauliflower mosaic caulimovirus (CaMV), turnip mosaic potyvirus (TuMV), turnip yellow mosaic tymovirus (TYMV)) in five natural populations of Brassica oleracea in Dorset (UK). All four viruses were common; 43% of plants were infected with BWYV, 60% with CaMV, 43% with TuMV and 18% with TYMV. For each virus there were significant differences in the proportion of infected plants among populations, which were not completely explained by differences in the age of plants. Multiple virus infections were prevalent, with 54% of plants having two or more virus types. There were statistically significant associations between pairs of viruses. The CaMV was positively associated with the other three viruses, and BWYV was also positively associated with TuMV. There was no detectable association between BWYV and TYMV, whereas TuMV and TYMV were negatively associated. We suggest these associations result from BWYV, CaMV and TuMV having aphid vectors in common, as aphids are attracted to plants that already have a virus infection. Infected plants were distributed randomly or were very weakly aggregated within populations. The implications of widespread multiple virus infections in natural plant populations are discussed with respect to the release of transgenic plants expressing virus-derived genes.

Withdrawal after narcotic therapy: a survey of neonatal and pediatric clinicians.

Pharmacists at the 1995 American College of Clinical Pharmacy Pediatric Practice and Research Network meeting volunteered to act as coordinators at their sites and survey pediatric and neonatal nurses, pharmacists, and physicians regarding dependency in neonatal and pediatric patients after therapeutic administration of narcotics. Thirteen (60%) of 21 coordinators returned 244 surveys. Primary symptoms of withdrawal reported by clinicians were agitation (100%), irritability (100%), inconsolability (100%), crying (99%), tremors (98%), high heart rate (98%), fidgets (98%), high blood pressure (97%), less sleep (96%), and sweating (94%). Most clinicians considered narcotic withdrawal to be a problem (74%) that should be treated (87%). A dependency scale is being developed and will include symptoms reported by more than 75% of respondents.

Inhibition of myeloperoxidase release from rat polymorphonuclear leukocytes by a series of azachalcone derivatives.

A series of azachalcones was evaluated for ability to affect secretion of myeloperoxidase by rat polymorphonuclear leukocytes stimulated by fMLP. The compounds were found to interfere with cellular uptake of extracellular calcium. Structure-activity relationships are discussed.

The status of implant training in oral and maxillofacial surgery residency programs.

PURPOSE: The results of a survey concerning the status of oral and maxillofacial surgery resident training in oral implantology are discussed. RESULTS: The results demonstrate that oral and maxillofacial surgery residents are exposed to a variety of implant types and systems, with most implant surgery being performed in association with intravenous sedation. The number of implants placed by each resident during training varied greatly. CONCLUSION: Most program directors believe that the implantology training of oral and maxillofacial surgery residents is superior or comparable to that of periodontal graduate students. However, this survey suggests that certain aspects of residency training in implantology, such as the performance of sinus lift procedures and participation in implant research, may require more emphasis.

Coexistence of stuttering and disordered phonology in young children.

The purpose of the present study was to assess differences in stuttering, phonological, and diadochokinetic behaviors in young children who exhibit both stuttering and disordered phonology and children who exhibit only one of the disorders. Subjects were 21 male children (aged 4 to 6 years), representing three groups of seven children each: (a) stuttering and normal phonological abilities (S+NP), (b) stuttering and disordered phonology (S+DP), and (c) normal fluency and disordered phonology (NF+DP). Stuttering behavior was assessed during a 30-minute conversational speech task; phonological behavior was assessed during a 162 item picture-naming task; and diadochokinetic abilities were assessed during bi- and multisyllable productions. Results indicated that the S+DP group produced significantly more sound prolongations and significantly fewer iterations per whole-word repetition than the S+NP group. However, there were no differences between the two groups in other stuttering indices. Moreover, no differences were noted between the S+DP and NF+DP groups in phonological behavior. Diadochokinetic rates did not differ among the three groups. The possibility of two types of stuttering, one occurring with and one without disordered phonology, is discussed.

The emerging phonological system of an autistic child.

This case report provides a detailed phonological investigation of the speech of an 8-year-old autistic boy. Three approaches were used for elicitation of speech: delayed imitation, object naming, and a connected speech sample. Phonetic inventory analysis revealed that stops, nasals, and glides were generally present, whereas fricatives, affricates, and the liquid /r/ were absent. There were also positional restrictions on the use of specific sounds. This information, together with a phonological process analysis, revealed: (a) the existence of several phonological processes that are common in normal development; (b) the persistence of several phonological processes, e.g., velar fronting, beyond the expected age; (c) the occurrence of some unusual sound changes, e.g., extensive glottal replacement and segment coalescence; (d) evidence of "chronological mismatch" (Grunwell, 1981); and (e) restricted use of contrasts (Ingram, 1976). The subject's use of phonological processes resulted in extensive homonymy, which, together with process interactions and the use of jargon, resulted in severely reduced intelligibility. This child appeared to be acquiring his phonological system in at least a partly unique way, showing some typical patterns as well as some patterns that rarely appear in normally developing children.

Biochemical changes associated with the symptomatic human intervertebral disk.

Significant changes in disk biochemistry were evident in the nuclei pulposi of 25 symptomatic disks that demonstrated abnormal diskography. Compared with the results from ten normal disk from young subjects (mean age, ten years) and from ten asymptomatic disks from adult patients (mean age, 33 years), the symptomatic disks demonstrated significantly greater collagen content than the young and asymptomatic adult subjects, respectively. Significantly fewer total glycosaminoglycans (GAGs) and significantly less water content were also evident for the diseased disks. The mean pH of the nucleus pulposus from 23 patients was also significantly reduced for the symptomatic disks as compared with the mean pH measured for four asymptomatic adult subjects. Although significantly fewer total GAGs were observed for the symptomatic disks, there were no significant differences in the percent composition of hyaluronic acid (HA), chondroitin sulfate (CS), or keratan sulfate (KS) among the normal and symptomatic disks. Significant changes in disk biochemistry are associated with the symptomatic disk, and these changes could contribute to the development of the painful disk syndrome.

Lexical acquisition and acquisition of initial voiceless stops.

The interaction between lexical acquisition and acquisition of initial voiceless stops was studied in two normally developing children, aged 1;9 and 1;10, by acoustically examining the token-by-token accuracy of initial voiceless stop targets in different lexical items. Production accuracy was also examined as it related to the frequency of usage of different words, as well as the time when they entered the children's lexicons. Fewer than half of the words in the children's lexicons had tokens representing the emergence of accurate voiceless stop production prior to the session at which the voicing contrast was achieved. These words were primarily 'old' words that had been in the children's lexicons from the beginning of data collection, as opposed to 'new' words, first produced in later recording sessions. Findings are discussed in reference to the 'lexical diffusion' model of sound change and within the framework of nonlinear underspecification theory.

The identification, cloning, and sequence analysis of the coat protein coding region of a birch isolate (I2) of cherry leaf roll nepovirus.

The coat protein gene of RNA-2 of cherry leaf roll nepovirus (CLRV) birch isolate I2 was cloned, identified, and sequenced. Transcripts derived from cDNA to the coat protein gene made a polypeptide of M(r) 51.5 k when translated in vitro. The predicted amino acid sequence of the coat protein showed little identity with nepoviruses having small RNA-2s. It did, however, have 27% sequence identity with the coat protein of tomato ringspot nepovirus which, like CLRV, has a relatively large RNA-2.

The nucleotide sequence of RNA-2 of raspberry ringspot nepovirus.

The nucleotide sequence of raspberry ringspot nepovirus (RRV) RNA-2 consists of 3928 nucleotides and a poly(A) tract at the 3' end. RNA-2 contains one open reading frame which encodes a polypeptide of M(r) 123508 (123K). Edman degradation located the N terminus of the coat protein 514 residues from the C-terminal end of the 123K protein, which suggests that the coat protein is released from the polyprotein by cleavage of a C-A bond. The RRV coat protein has some sequence similarities with the coat proteins of other nepoviruses, but is no more like any one nepovirus than another. In contrast, the portion of the 123K protein to the N-terminal side of the coat protein is similar in sequence to the corresponding parts of the polyproteins of tomato black ring and grapevine chrome mosaic nepoviruses, though not to those of other nepoviruses.

Fibrillar collagen-biphasic calcium phosphate composite as a bone graft substitute for spinal fusion.

The osteoconductive capacity of fibrillar collagen-biphasic calcium phosphate composition was compared to autogenous bone in a canine spinal fusion model. All animals underwent a standard intervertebral body fusion (L2-L4) with rigid internal fixation and received either autogenous bone alone or a mixture of the ceramic and autogenous bone (3:1) as the graft material. Animals were followed for 12 months and the quality of fusion in each animal assessed by biomechanical testing and histological analysis. The fused L2-L4 segment of each dog was embedded in bone cement and mounted in a specially designed mechanical tester for testing in flexion, extension, and side bending. Overall, the mean rigidity of the fusion mass was not significantly different between the two groups [10.5 +/- 4.1 (SD) for autogenous bone vs. 11.3 +/- 1.7 for the ceramic plus autogenous bone, p greater than 0.05]. Similar findings were obtained for mean bending moment, compressive load, angular deformation, and energy absorbed for the two groups. Histological analysis was performed on transverse nondecalcified specimens. Quantitation of bone ingrowth using back-scattered electron imaging disclosed no significant differences in the amount of new bone formed at the graft site between autogenous bone and the ceramic plus autogenous bone recipients (23.4 +/- 10% vs. 25.8 +/- 8.8%) when correction for the autogenous bone volumes was performed. Light microscopic analysis of toluidine blue-stained transverse sections demonstrated new bone growth around and through the ceramic bone graft material. These results suggest that use of a collagen-biphasic calcium phosphate ceramic and autogenous bone mixture (3:1) provides a suitable osteoconductive alternative to the use of autogenous bone and results in the formation of a mechanically competent fusion mass not significantly different from that obtained with autogenous bone alone.

Partial nucleotide sequence of poplar mosaic virus RNA confirms its classification as a carlavirus.

The nucleotide sequence of the 3'-proximal 1328 nucleotides of poplar mosaic virus (PMV) was determined and shown to contain two large open reading frames (ORFs). The ORF nearer to the 3' terminus of the RNA is capable of encoding a polypeptide of 14K with a 'zinc-finger' motif, and is homologous to sequences in corresponding positions in five other carlaviruses. The other ORF encodes a protein of 36K which includes two sequences of amino acids identified in tryptic digests as virion capsid protein, and has amino acid sequences in common with both carlaviruses and potexviruses.

Antiretroviral activity of mechanism-based irreversible inhibitors of S-adenosylhomocysteine hydrolase.

S-Adenosylhomocysteine hydrolase (AdoHcy-nase) is a key enzyme in transmethylation reactions. The objective of the present study was to examine the potential antiretroviral activities of novel mechanism-based irreversible AdoHcy-nase inhibitors. (Z)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (ZDDFA), (E)-4',5'-didehydro-5'-deoxy-5'-fluoroadenosine (EDDFA), (Z)-4',5'-didehydro-5'-deoxy-5'-chloroadenosine (ZDDCA) and 5'-deoxy-5'-acetylenic adenosine (DAA) inhibited AdoHcy-nase activity with Ki values of 0.55, 1.04, greater than 10.0 and 3.30 microM, respectively. These four compounds were tested for antiviral activity in vitro against Moloney leukemia virus (MoLV) in the XC-plaque assay. MoLV replication in murine fibroblasts (SC-1) was inhibited by ZDDFA, EDDFA and DAA with IC50 values of 0.05, 0.25 and 3.30 micrograms/ml, respectively. ZDDCA did not inhibit MoLV infection at the concentrations tested. Antiviral activity correlated with the ability of the individual compounds to maintain sustained elevations in intracellular S-adenosylhomocysteine (AdoHcy) concentrations in the SC-1 cells. ZDDFA, the most potent inhibitor of AdoHcy-nase and MoLV was also the most active in maintaining sustained elevations in intracellular AdoHcy levels. The antiviral activity of ZDDFA was also examined in murine C3H1OT1/2 fibroblasts which constitutively produce MoLV. Pretreatment with ZDDFA (1.0 microgram/ml) for 24 hr inhibited virus production by 88%. Similar to the SC-1 cells, and concomitant with enzyme inhibition, there was a 300-fold increase in AdoHcy levels in ZDDFA (1.0 microgram/ml) treated C3H1OT1/2 cells. Incorporation of a [3H]methyl group from tritiated S-adenosylmethionine into total RNA in C3H1OT1/2 cells was inhibited by ZDDFA without affecting cell viability. These results suggest that mechanism-based inhibitors of AdoHcy-nase, such as ZDDFA, may have potential as antiretroviral agents.

1,10-bis(guanidino)decane inhibits N-methyl-D-aspartate responses in vitro and in vivo.

This study evaluated the interaction of the arcaine analog 1,10-bis(guanidino)decane (BG10) with the NMDA receptor. BG10 inhibited [3H]dizocilpine binding to well-washed rat brain membranes with an apparent affinity of 1.3 microM. The inhibition was not competitive with respect to glutamate or glycine, but was significantly altered by spermidine. However, unlike arcaine, BG10 slowed the dissociation of [3H]dizocilpine. BG10 also inhibited [3H]glycine binding at similar concentrations. BG10 inhibited N-methyl-D-aspartate (NMDA) and glycine-induced increases in intracellular Ca++ in cultured rat brain neurons monitored using the fluorescent dye, fura-2 (IC50 3 microM). This inhibition was not competitive with NMDA or glycine and could not be reversed by either spermidine or arcaine. BG10 also noncompetitively inhibited NMDA-stimulated cyclic GMP production in cerebellar slices from mouse brain. Finally, BG10 administered i.p. reduced harmaline-stimulated, NMDA-dependent cyclic GMP accumulation in mouse cerebellum in vivo (ED50 12.3 mg/kg). These data demonstrate that BG10 is a novel and effective NMDA receptor antagonist in vitro and in vivo.

Synthesis and DNA-binding properties of polyamine analogues.

The synthesis of a series of novel polyamine analogues is reported. The DNA binding of these compounds and a variety of other polyamines were compared with their IC50 values against HeLa cell. There seemed to be no apparent correlation between the DNA binding and toxicity against HeLa cells.

Transgenic plants and insect cells expressing the coat protein of arabis mosaic virus produce empty virus-like particles.

The 3' end of the RNA-2 of arabis mosaic virus (ArMV) was cloned and sequenced. The N-terminal amino acid sequence of the virion coat protein was determined by Edman degradation and the corresponding coding region identified. This gene was modified at the 5' and 3' ends by use of mismatched primers in the polymerase chain reaction (PCR), in order to facilitate the cloning of the gene, and to provide it with a methionine initiation codon. The modified cloned gene was expressed in transgenic plants, recombinant baculovirus-infected insect cells and bacteria. Both the insect cells and the plants expressing the modified coat protein gene contained empty virus-like particles (VLPs) similar to the empty virus shells found in plants infected with ArMV. These VLPs were not detected in the Escherichia coli expressing the coat protein. Analysis of the primary amino acid sequence in the ArMV coat protein revealed extensive regions of identity with that of grapevine fanleaf virus. Patterns in these identities may reflect a three-domain organization of the proteins.