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L Rubino

Publications and source records attributed to L Rubino.

At least 19 recordsLinked to original sources
PubMed2001

The open reading frame 1-encoded ('36K') protein of Carnation Italian ringspot virus localizes to mitochondria.

The localization of the 36 kDa ('36K') protein encoded by open reading frame 1 of Carnation Italian ringspot virus was studied in infected cells and in cells transiently expressing the 36K protein fused to green fluorescent protein (GFP). Subcellular fractionation demonstrated that the 36K protein accumulated in fractions containing mostly mitochondria. Fluorescence microscopy of transiently transformed cells showed that the 36K-GFP fusion protein accumulated in structures which could be stained with the mitochondrial-specific dye MitoTracker. However, these structures were larger than normal mitochondria and were irregular in shape and distribution in the cytoplasm. Electron microscopy showed severe alterations of mitochondria, which were often clumped. The stroma was more electron-opaque, the cristae were irregularly shaped, the intermembrane space was enlarged and the outer membrane was covered with an electron-dense amorphous material whose nature could not be determined. The organelle-targeted 36K protein seems to promote the overgrowth of the mitochondrial outer membrane.

Cells, Cultured↗
PubMed2001

An experiential learning simulation exercise for healthcare management students.

Preparing competent administrators to work in today's dynamic healthcare environment is a challenging task for contemporary educators. Experiential exercises in the classroom can contribute significantly to student training. This paper reviews the benefits to students of actively participating in simulations and role-plays, as well as the challenges in running such exercises. A simulation designed specifically for healthcare administration students is presented with details and implementation instructions. The intent of this article is to impart to other educators the learning experiences in running this particular simulation to inspire dialogue and program improvement. Readers are encouraged to create simulation exercises with optimum relevance for their students. Information to obtain e-mail versions of the simulation is also provided.

Competency-Based Education↗
PubMed2000

Skin and wound infection by rapidly growing mycobacteria: an unexpected complication of liposuction and liposculpture. The Venezuelan Collaborative Infectious and Tropical Diseases Study Group.

OBJECTIVE: To describe 10 patients with skin and soft tissue infection caused by rapidly growing mycobacteria after cosmetic liposuction and liposculpture. DESIGN: Case series. SETTINGS: Eight private geographically separate surgical facilities from a single metropolitan area. PATIENTS: Eight patients with definite and 2 with presumptive cases of skin and soft tissue infection caused by rapidly growing mycobacteria after cosmetic surgery procedures during a 24-month period. Microorganisms were isolated from the purulent drainage obtained from wounds or fistulas in 8 cases and were identified as Mycobacterium fortuitum (3 cases) and Mycobacterium abscessus (5 cases) by routine microbiologic techniques. Acid-fast bacilli were observed on Ziehl-Neelsen-stained smears in the 2 remaining cases, but these ultimately failed to grow. In 2 of the surgical units, no apparent environmental predisposing factors were identified after thorough microbiologic environmental investigation. Clinically, all patients exhibited signs of inflammation, microabscesses, and purulent wound drainage within 24 months of abdominal and/or thigh liposuction or homologous fat tissue injection. INTERVENTION: A combined therapeutic approach including surgical drainage, debridment, and prolonged (>3 months) treatment with combined antimicrobial agents including clarithromycin was used in all cases. RESULTS: Nine of 10 patients responded to the combined therapeutic approach, and no evidence of infection was present during at least 12 months of follow-up. CONCLUSION: To our knowledge, this is the first series of patients with rapidly growing mycobacterial infections to be described after liposuction and liposculpture. Rapidly growing mycobacteria should be included in the differential diagnosis of skin and soft tissue infection after cosmetic surgery.

Abdomen↗
PubMed2000

Relationships between choline magnetic resonance spectroscopy, apparent diffusion coefficient and quantitative histopathology in human glioma.

This study sought to correlate quantitative presurgical proton magnetic resonance spectroscopic imaging (1H-MRSI) and diffusion imaging (DI) results with quantitative histopathological features of resected glioma tissue. The primary hypotheses were (1) glioma choline signal correlates with cell density, (2) glioma apparent diffusion coefficient (ADC) correlates inversely with cell density, (3) glioma choline signal correlates with cell proliferative index. Eighteen adult glioma patients were preoperatively imaged with 1H-MRSI and DI as part of clinically-indicated MRI evaluations. Cell density and proliferative index readings were made on surgical specimens obtained at surgery performed within 12 days of the radiologic scans. The resected tissue location was identified by comparing preoperative and postoperative MRI. The tumor to contralateral normalized choline signal ratio (nCho) and the ADC from resected tumor regions were measured from the preoperative imaging data. Counts of nuclei per high power field in 5-10 fields provided a quantitative measure of cell density. MIB-1 immunohistochemistry provided an index of the proportion of proliferating cells. There was a statistically significant inverse linear correlation between glioma ADC and cell density. There was also a statistically significant linear correlation between the glioma nCho and the cell density. The nCho measure did not significantly correlate with proliferative index. The results indicate that both ADC and spectroscopic choline measures are related to glioma cell density. Therefore they may prove useful for differentiating dense cellular neoplastic lesions from those that contain large proportions of acellular necrotic space.

Adult↗
PubMed2000

Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology.

Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells. To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection. The 36K protein was expressed in the yeast Saccharomyces cerevisiae in native form or fused to the green fluorescent protein. Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria. These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes. The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein. Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins.

Cell Membrane↗
PubMed1998

Membrane targeting sequences in tombusvirus infections.

Infection of Nicotiana benthamiana cells with cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) viruses results in the formation of conspicuous membranous bodies [multivesicular bodies (MVBs)], which develop from modified peroxisomes or mitochondria, respectively. The organelle targeting signal is located in the proteins of 33 kDa (CymRSV) or 36 kDa (CIRV) encoded by ORF 1, which contain an N-terminal hydrophilic portion followed by two predicted hydrophobic transmembrane segments. Biochemical analysis showed that the 33- and 36-kDa proteins are integral membrane proteins. By exchanging small portions of the ORF 1 sequence between the infectious full-length clones of the two viruses, hybrid constructs were obtained of which the in vitro synthesized RNA was inoculated to N. benthamiana plants and protoplasts. The structure of infectious clones suggested that both the N-terminal hydrophilic region and the transmembrane segments of the ORF 1-encoded proteins specify which organelle is involved in the synthesis of MVBs. Mutational analysis of the CIRV 36-kDa protein also suggested the presence of an internal mitochondrial targeting sequence similar to that found in several normal host proteins that are synthesized in the cytoplasm and transported to mitochondria. The CymRSV 33-kDa protein did not contain the obvious consensus signals thought to be characteristic of proteins targeted to peroxisomes, and an mitochondrial targeting sequence motif was not evident.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and complete nucleotide sequence of galinsoga mosaic virus genomic RNA.

The complete genomic sequence of galinsoga mosaic virus (GaMV) was determined. The genome consists of 3,803 nucleotides and has five open reading frames (ORFs). The 5' ORF (ORF 1) encodes a protein with predicted molecular mass of 23 kDa and readthrough of its amber stop codon probably yields a 82 kDa protein (ORF 2). ORFs 3 and 4 encode two polypeptides with molecular masses of 8 and 7 kDa, respectively. ORF 5 encodes the 36 kDa capsid protein. Amino acid sequence comparisons revealed that the nonstructural proteins encoded by ORFs 1, 3, and 4 were more similar to the corresponding gene products of tobacco necrosis virus, strain A, than to those of carmoviruses. Conversely, the coat protein was more similar to that of tombusviruses. The readthrough region of the viral replicase (ORF 2) had high sequence homology with that of carmo-, tombus-, and necroviruses. Computer analysis of the protein encoded by ORF 1 as well as of the corresponding product of turnip crinkle (TCV) and melon necrotic spot (MNSV) carmoviruses revealed the presence of a sequence with local hydrophobicity and hydrophobic moment characteristic of mitochondrial targeting sequence which may explain the origin of the carmovirus-induced multivesicular bodies from mitochondria.

Amino Acid Sequence↗
PubMed1997

Molecular analysis of the pothos latent virus genome.

Pothos latent virus (PoLV) is an isometric virus with a positive-sense, single-stranded RNA genome of 4415 nt. The genome contains five open reading frames (ORF), coding for five proteins with approximate molecular masses of 25, 84, 40, 27 and 14 kDa, respectively. In vitro synthesized PoLV RNA was infectious to Nicotiana benthamiana plants and protoplasts, but could not support replication of the defective interfering (DI) and satellite RNAs associated with Cymbidium ringspot tombusvirus. No DI RNA related to PoLV was generated after repeated passaging with infected sap. Mutagenesis studies defined the role of the ORF 4 product (27 kDa) as a movement protein, and the ORF 5 product (14 kDa) as being responsible for symptom severity. Moreover, it was shown that the coat protein (CP) is important in regulating synthesis of the 14 kDa protein, excess production of which is lethal to infected plants. CP mutants defective in capsid formation infected plants systemically and induced severe necrotic symptoms. Conversely, CP mutants able to form apparently normal virus particles induced symptoms indistinguishable from those elicited by wild-type virus.

Amino Acid Sequence↗
PubMed1996

Characterization, nucleotide sequence and genome organization of leek white stripe virus, a putative new species of the genus Necrovirus.

White stripe is a disease affecting leek in France with which an isometric virus c. 30 nm in diameter is associated. The most evident symptom is the presence of white stripes on the leaves extending to the stem. Attempts to demonstrate transmission through the soil by sowing or transplanting leek in contaminated soil were unsuccessful. The virus was transmitted by sap inoculation to a narrow range of herbaceous hosts, all of which were infected only locally. Virus purification was from infected leek tissues, where it accumulated in large amounts, as demonstrated by ultrastructural observations. RNA was extracted from purified virus preparations and cDNA clones were prepared. The complete nucleotide sequence of the viral RNA was determined: The genome is 3,662 nucleotides long and contains five open reading frames (ORFs). The first (ORF 1) encodes a putative translation product of M(r) 23,803 (p24) and read through of its amber stop codon results in a protein of M(r) 82,625 (p83) (ORF 2). ORF 3 and ORF 4 encode two small polypeptides of M(r) 11,280 (p11) and M(r) 6,261 (p6), respectively. ORF 5 encodes the capsid protein of M(r) 27,460 (p27). The genome organization and sequence alignments with the corresponding products of necroviruses suggest that the virus isolated from leek is a new species in the genus Necrovirus, for which the name of leek white stripe virus (LWSV) is proposed.

Amino Acid Sequence↗
PubMed1996

The 5'-terminal region of a tombusvirus genome determines the origin of multivesicular bodies.

Multivesicular bodies (MVB) are membranous cytoplasmic inclusions that are invariably associated with tombusvirus infections regardless of the virus species, the host, or the tissue examined. MVB are virus-induced structures since they are absent from tissues of healthy plants and are always present both in infected plants and protoplasts. MVB derive from peroxisomes in cells infected by a number of tombusviruses including cymbidium ringspot virus (CymRSV) and from mitochondria in cells infected by another tombusvirus, carnation Italian ringspot virus (CIRV). By using common restriction sites in full-length infectious clones, hybrid clones of these two viruses were constructed. In addition, a mutant of CIRV was prepared in which the protein encoded by the first open reading frame was shortened by 22 amino acids. All mutant transcripts were viable and infected Nicotiana benthamiana plants. Infected leaf tissue samples were collected, processed for thin sectioning, and observed in the electron microscope. The origin of MVB was shown to be under the control of the 5' region of the viral genome. A sequence as short as about 600 nucleotides in ORF 1 contained the determinants for formation of MVB from peroxisomes or mitochondria.

Base Sequence↗
PubMed1995

Characterization of resistance to cymbidium ringspot virus in transgenic plants expressing a full-length viral replicase gene.

Transgenic Nicotiana benthamiana plants expressing the replicase gene of cymbidium ringspot tombusvirus (CymRSV) were tested for the biological activity of the transgene. Protoplasts from one resistant and three susceptible transgenic lines were transfected with in vitro synthesized defective interfering (DI) RNA, a small subviral molecule which requires only the virus replicase for replication. Only protoplasts of susceptible transgenic lines were able to sustain the replication of DI RNA, whereas protoplasts of the resistant line were not. The transgene of the resistant line was recovered from transgenic cells, cloned, and shown to be intact and functional, which suggests that this is not a case of dysfunctional replicase conferring resistance to transgenic plants as a "dominant negative mutant." Plants of the transgenic line resistant to CymRSV were shown to be susceptible to two other tombusviruses, artichoke mottled crinkle and carnation Italian ringspot virus. Since the resistance is specific and inversely correlated to the level of expression of the transgene, it appears to be a form of RNA-mediated resistance.

Base Sequence↗
PubMed1995

Replication of cymbidium ringspot virus satellite RNA mutants.

Cymbidium ringspot tombusvirus (CyRSV) supports the replication of an RNA molecule known to be a satellite (sat-RNA). Sequence analysis of wild-type sat-RNA showed that the 3' terminus is heterogeneous and terminates with one, two, or three C residues. Transcripts from mutant clones which lacked up to eight nucleotides at the 3' end were biologically active and yielded progeny RNA that had the 3' end restored as in wild-type RNA. Deletions or substitutions at the 5' end produced nonviable molecules. Studies with mutants containing deletions in internal regions showed that replication was affected essentially by the location of deletions; viable mutants were much less encapsidated than the wild type, showing that size also may be important in the encapsidation and survival of CyRSV sat-RNA.

Base Sequence↗
PubMed1995

Molecular cloning and complete nucleotide sequence of carnation Italian ringspot tombusvirus genomic and defective interfering RNAs.

The complete sequences of genomic and defective interfering (DI) RNAs of carnation Italian ringspot tombusvirus (CIRV) were determined. The genome (4760 nt) has an organization identical to that reported for other tombusviruses except that the pre-readthrough domain of the viral replicase encoded by the 5'-proximal open reading frame (ORF) is larger. In particular, the N-terminal region of this protein differs from the corresponding region of the other members of the genus Tombusvirus and Carmovirus. Two DI RNAs were found in infected tissues whose sequences were completely derived from genomic RNA. The smaller molecule (474 nt) is contained completely within the larger molecule (656 nt), which suggests that it is derived from the larger one.

Base Sequence↗
PubMed1995

Sequence analysis of pothos latent virus genomic RNA.

The complete genomic sequence of pothos latent virus (PoLV) has been determined. The genome organization is very similar to that of tombusviruses. The genome is 4415 nucleotides long and contains five ORFs. The 5' ORF (ORF 1) encodes a protein with a predicted molecular mass of 25 kDa and readthrough of its amber stop codon results in an 84 kDa protein (ORF 2). ORF 3 encodes the 40 kDa capsid protein. Two nested ORFs (ORFs 4 and 5) in different reading frames encode 27 and 14 kDa proteins, respectively. Amino acid sequence alignments revealed significant similarities between the readthrough portion of ORF 2 and the coat protein (ORF 3) of PoLV and the corresponding proteins of several tombusviruses. Conversely, the predicted products of ORFs 1, 4 and 5 showed only limited similarity to the equivalent tombusvirus proteins. These results support the conclusion that PoLV is a new but atypical member of the family Tombusviridae.

Amino Acid Sequence↗
PubMed1994

Immunodetection of the 33 K/92 K polymerase proteins in cymbidium ringspot virus-infected and in transgenic plant tissue extracts.

An antiserum was raised against the 33 K protein encoded by the 5' proximal gene of cymbidium ringspot tombusvirus RNA. This antiserum reacts specifically with the 33 K and 92 K proteins, which constitute the viral replicase, in CyRSV-infected Nicotiana benthamiana plants and in transgenic plants transformed with the full-length replicase gene. In inoculated leaves of infected plants, synthesis of the 33 K/92 K proteins stops ten days after inoculation, whereas in newly produced systemically infected leaves there was continuous production of these proteins. In transgenic plants, both proteins were detected showing that readthrough of the termination codon of the 33 K protein does not depend on the presence of the replicating virus. The subcellular localization of the 33 K/92 K proteins is similar in infected and transgenic plants. No correlation was found between the level of expression of integrated virus gene and level of resistance to the challenging virus.

Amino Acid Sequence↗
PubMed1994

The nature of multimeric forms of cymbidium ringspot tombusvirus satellite RNA.

The multimeric forms of cymbidium ringspot tombusvirus (CyRSV) satellite (sat) RNA were analysed. Attempts to amplify the putative junction region of oligomers using the polymerase chain reaction (PCR) failed, indicating the absence of such structures. SatRNA-related species having the size double than the unit length were shown to be double-stranded monomers and not single-stranded dimers. Similarly, satRNA species of a size four times the unit length were shown to be constituted by aggregates of double-stranded monomers. The absence of single-stranded CyRSV satRNA oligomers indicates that the formation of multimers is not a step in the replication of this RNA molecule.

Blotting, Northern↗