Inhibitory Effect of Polyethylene Oxide and Polypropylene Oxide Triblock Copolymers on Aggregation and Fusion of Atherogenic Low Density Lipoproteins.

Triblock copolymers of poly(ethylene oxide) and poly(propylene oxide) (so-called pluronics) were shown to influence the aggregation and fusion of atherogenic low density lipoproteins (atLDL) and be able to inhibit these processes. The character of the influence and the degree of the stabilizing effect depended on the structure, relative hydrophobicity, and concentration of the copolymer. Pluronics L61, P85, and L64 characterized by the hydrophilic-lipophilic balance (HLB) value from 3 to 16 had the greatest ability to suppress the aggregation of atLDL. Pluronic L81 with the higher hydrophobicity (HLB = 2) partially inhibited atLDL aggregation at low concentrations but stimulated it at high concentrations. The influence of pluronics did not have a direct connection with their ability for micelle formation, but it was realized through individual macromolecules. We suppose that effects of pluronics could be due to their interaction with the lipid component of LDL and to a possible influence of these copolymers on the structure and hydrophilic-lipophilic characteristics of lipoproteins.

Subcellular localization and self-interaction of plant-specific Nt-4/1 protein.

The Nicotiana tabacum Nt-4/1 protein is a plant-specific protein of unknown function. Analysis of bacterially expressed Nt-4/1 protein in vitro revealed that the protein secondary structure is mostly alpha-helical and suggested that it could consist of three structural domains. Earlier studies of At-4/1, the Arabidopsis thaliana-encoded ortholog of Nt-4/1, demonstrated that GFP-fused At-4/1 was capable of polar localization in plant cells, association with plasmodesmata, and cell-to-cell transport. Together with the At-4/1 ability to interact with a plant virus movement protein, these data supported the hypothesis of the At-4/1 protein involvement in viral transport through plasmodesmata. Studies of the Nt-4/1-GFP fusion protein reported in this paper revealed that the protein was localized to cytoplasmic bodies, which were co-aligned with actin filaments and capable of actin-dependent intracellular movement. The Nt-4/1-GFP bodies, being non-membrane structures, were found in association with the plasma membrane, the tubular endoplasmic reticulum and endosome-like structures. Bimolecular fluorescence complementation experiments and inhibition of nuclear export showed that the Nt-4/1 protein was capable of nuclear-cytoplasmic transport. The nuclear export signal (NES) was identified in the Nt-4/1 protein by site-directed mutagenesis. The Nt-4/1 NES mutant was localized to the nucleoplasm forming spherical bodies. Immunogold labeling and electron microscopy of cytoplasmic Nt-4/1-containing bodies and nuclear structures containing the Nt-4/1 NES mutant revealed differences in their fine structure. In mammalian cells, Nt-4/1-GFP formed cytoplasmic spherical bodies similar to those found for the Nt-4/1 NES mutant in plant cell nuclei. Using dynamic laser light scattering and electron microscopy, the Nt-4/1 protein was found to form multimeric complexes in vitro.

The compound sternal flap for laryngeal reconstruction.

The authors present the successful transfer of a compound flap based on the sternocleidomastoid muscle in a patient who had undergone radiation therapy and a near-total laryngectomy. The surgical procedure, a one-stage reconstruction with a regional rotational flap, utilized a segment of the sternal periosteum and underlying anterior cortex attached to the sternal belly of the sternocleidomastoid muscle. The rigid wall provided by the flap over the temporary laryngeal endoprosthetic stent was an adequate replacement for the subtotally resected larynx, obviated the need for postoperative tracheostomy and nasogastric feeding, prevented collapse or stenosis of the airway, spared the voice, and provided an excellent functional result. This reliable flap presents a regional alternative to other local tissue transfers, such as epiglottic laryngoplasty, and more complicated microsurgical reconstructions.

Characterization of cucumber mosaic virus. VI. Generation of deletions in defective RNA 3s during passage in transgenic tobacco expressing the 3a gene.

Defective mutants of cucumber mosaic virus (CMV) RNA 3, containing deletions in the 3a gene were passaged in transgenic tobacco plants expressing the CMV 3a gene. After six passages, the various progeny RNA 3 were characterized. In all but one case, the size of the deletion increased. For the NheI-fs RNA 3 mutant of the Fny-strain of CMV (with an 8 nucleotide deletion), the deletion increased in the progeny viral RNA 3 to 570 nucleotides. For a similar frameshift mutant in RNA 3 of the M strain of CMV, either single RNA 3 species (with deletions of 579 or 627 nucleotides) or mixtures of RNA 3 deletion variants were observed in different plants. The DeltaE-H mutant (with a deletion of 202 nucleotides) of Fny-CMV RNA 3 underwent further deletion resulting in the loss of the entire 3a gene and flanking sequences. The DeltaKpnI mutant (deletion of 501 nucleotides) of Fny-CMV RNA 3 underwent a further deletion of 30 nucleotides. Except for the deletion progeny of the DeltaE-H RNA 3 mutant, the other defective RNA 3s all contained inframe deletions. It is proposed that the various deletions were created by different types of recombination and that packaging may be an important factor in the selection of particular defective RNA 3 variants.

Characterization of cucumber mosaic virus. V. Cell-to-cell movement requires capsid protein but not virions.

To ascertain the importance of amino-terminal proximal capsid protein (CP) sequences in cel-to-cell movement, virion formation, and stabilization, two CP mutants of cucumber mosaic virus (CMV) were generated by deletion of sequences encoding CP amino acids 15-40 (delta Sal-Nru) or 26-40 (delta Sac-Nru). Wildtype CMV and CMV containing delta Sac-Nru could infect systemically four host species, although symptoms induced by the two viruses usually were different CMV containing delta Sal-Nru could only infect Nicotiana benthamiana and N. clevelandii systemically, but only slowly, suggesting phloem-independent long-distance movement. A variant mutant designated delta Sal-Nru* could systemically infect N. tabacum as well as the above two Nicotiana species, rapidly, but could not systemically infect Cucurbita pepo. Virus particles could not be detected in plants infected by delta Sal-Nru, while delta Sal-Nru* and delta Sac-Nru formed particles of lower stabilities than for wildtype virus. The CPs of delta Sal-Nru and delta Sal-Nru* could bind RNA in vitro, although less strongly than delta Sac-Nru or wildtype CMV. These data indicate that amino-terminal proximal sequences of the CMV CP interact with viral RNA and are required for the formation of stable virions. Moreover, while the CP is necessary for cell-to-cell movement, the ability to form virions is not a prerequisite for cell-to-cell movement.

Characterization of cucumber mosaic virus. III. Localization of sequences in the movement protein controlling systemic infection in cucurbits.

Cucumber mosaic virus (CMV), generated from biologically active cDNA clones of Fny-CMV RNA 1 plus 2 and Sny-CMV RNA 3, derived from the Fny- and Sny-strains of CMV, was able to infect tobacco but not squash plants systemically. In squash, viral RNA, movement protein, and coat protein all accumulated in the inoculated cotyledons. The lack of systemic infection was associated with a reduced rate of cell-to-cell movement within the cotyledons. The restricted movement mapped to two sequence changes in the codons of amino acids 51 and 240 of the Sny-CMV 3a gene. These same sequence changes previously were shown to be associated with high levels of 3a protein accumulation and chronic vs acute, cyclic infection typical of Sny-CMV vs Fny-CMV [Gal-on et al. (1996). Virology 226, 354-361]. Fny-CMV, mutated in the codons of 3a gene amino acids 51 and 240, was still able to infect several solanaceous hosts (tobacco, tomato, and pepper) systemically, but did not elicit a typical CMV systemic infection on any of several cucurbit hosts (cucumber, melon, or squash). The significance of the location of amino acid positions 51 and 240 in the 3a movement protein is discussed.

Characterization of cucumber mosaic virus. II. Identification of movement protein sequences that influence its accumulation and systemic infection in tobacco.

Tobacco plants infected by the Sny strain of cucumber mosaic virus (CMV) showed chronic symptoms rather than cycles of acute disease followed by no symptoms, as seen for many strains such as Fny-CMV. The Sny-CMV 3a movement protein (MP) accumulated at 10 to 50 times the level of the Fny-CMV MP in the first few systemically infected leaves showing disease symptoms. The increased production of movement protein was not associated with an increase in synthesis of viral RNA and/or capsid protein. The correlation of high levels of accumulated MP and chronic infection in tobacco mapped to the 3a gene of Sny-CMV. Sequence analysis of the 3a gene of Sny-CMV showed that there were three nucleotide changes that altered amino acid sequences within the MP. Analysis of the kinetics of infection and level of MP accumulation for various CMV RNA 3 chimeric constructs and site-directed mutants of RNA 3 showed that chronic infection and high MP accumulation were associated with nucleotide sequence changes at two positions within codons 51 and 240 of the MP gene. These changes in the MP of Fny-CMV resulted in an altered subcellular distribution of the 3a protein, as also was observed for the Sny-CMV 3a protein. In both cases, considerably more 3a protein was associated with a fraction containing membranes. The potential relationship between membrane association and chronic infection is discussed.

Complementation of virus movement in transgenic tobacco expressing the cucumber mosaic virus 3a gene.

Tobacco plants were transformed with the cucumber mosaic cucumovirus (CMV) 3a gene and the in planta-expressed 3a protein was detected immunologically. The 3a protein was predominantly localized in a subcellular fraction corresponding to the cytosol. Two frameshift and four deletion mutants were created within the 3a open reading frame of CMV RNA 3. Five of these mutants, containing an N-terminal, large central, or C-terminal 70-amino-acid deletion could not infect nontransformed tobacco plants, but could infect the 3a transgenic tobacco plants, and generally accumulated to wild-type levels. The sixth mutant, lacking the C-terminal 43 amino acids of the 3a protein, was able to infect nontransformed tobacco plants. A delay in accumulation of viral RNA in both the inoculated and the systemically infected leaves was demonstrated for one of the mutants. Thus, the CMV 3a protein is a virus movement protein, the functions of which can be complemented in a transgenic plant. The CMV 3a transgenic plants were able to complement the long-distance movement of a pseudorecombinant cucumovirus defective for this function in tobacco, as well as the cell-to-cell, but not the long-distance, movement of two other related viruses. However, these transgenic plants were unable to complement the long-distance movement of viruses from several other taxonomic groups that could move cell to cell but not long distance in tobacco.

High-voltage electrical injury: a role for mandatory exploration of deep muscle compartments.

Patients sustaining high-voltage electrical burns involving the upper extremities were evaluated for deep muscle compartment necrosis by early surgical exploration. Following resuscitation, patients were taken to the operating room where mandatory exploration of volar and extensor forearm and hand compartments was done to evaluate muscle viability. It was observed that deep muscle layers sustained the greatest thermal injury and that extended fasciotomies were required to obviate compartment syndrome and the potential for continued ischemia. Although 10 emergency amputations were required for six patients, those who underwent extensive exploration of their forearms and hands did not require subsequent amputation for nonviable extremities during second-look operations. These observations support the role for early exploration with generous exposure of deep muscle compartments to access and treat the thermal injury that results from high-voltage burns.

Experimental and clinical use of pH monitoring of free tissue transfers.

No current method of flap monitoring is ideal for use in all types of free tissue transfers. No method provides objective, easily communicated data that is identical in all types of transfers. In particular, reliable monitoring of buried transfers has proved difficult with available methods. The rat anterior thigh flap based on the external iliac vascular pedicle was introduced by us as a model of deep free tissue transfer. Four sets of 10 flaps were raised in the following groups: Group A (control), Group B (arterial occlusion), Group C (venous occlusion), and Group D (arterial and venous occlusion). Postoperative muscle flap pH was measured with a micro-pH electrode (1.2 mm) and correlated with arterial blood gas. Results showed excellent correlation of flap and serum pH over time (mean flap pH, 7.28; mean serum pH, 7.30). Arterial occlusion produced a rapid drop in flap pH of 0.66 pH units at 1 hour. Venous occlusion pH drop was 0.27 pH units at 1 hour, 0.53 pH units at 3 hours. Arterial and venous occlusion produced a pH drop of 0.55 pH units at 1 hour. The most rapid rate of pH drop occurred immediately after vessel occlusion. We have used continuous pH monitoring in 21 free tissue transfers for up to 84 hours after surgery. PH values remained constant in each transfer (range, 7.20-7.50; grand mean, 7.35). There was one flap failure among the monitored group of flaps, which was predicted by pH drop before loss of Doppler pulse.(ABSTRACT TRUNCATED AT 250 WORDS)

The lateral arm flap for elbow coverage.

A technique for coverage of periolecranon (elbow) defects is presented. The technique uses the local tissues of the lateral upper arm as an axial flap and minimizes the donor defect for this type of reconstruction by obviating the need for a skin graft.

Reduction of tobacco mosaic virus accumulation in transgenic plants producing non-functional viral transport proteins.

Transgenic plants producing the 30K temperature-sensitive transport protein (TP) of tobacco mosaic virus (TMV) mutant Ni2519 (affecting cell-to-cell transport) were found to: (i) be susceptible to wild-type TMV U1 at 24 degrees C (a permissive temperature for Ni2519 TP), (ii) acquire a certain level of resistance to TMV U1 accumulation when maintained at 33 degrees C (a non-permissive temperature for Ni2519 TP) and (iii) lose the resistance to wild-type TMV after their transfer from 33 degrees C to 24 degrees C. It is suggested that reversible temperature-dependent conformational changes in Ni2519 TP are responsible for these phenomena and that production of a TP which is only partially functional in transgenic plants confers on these plants a resistance to the virus owing to reduction of the level of cell-to-cell transport. Transgenic tobacco plants producing the 32K TP of brome mosaic virus (BMV) acquired resistance to TMV U1 suggesting that BMV TP is partially functional in tobacco plants.

Biological activities of human interferon and 2'-5' oligoadenylates in plants.

Exogenous human interferon 2 (IFN) and 2'-5' oligoadenylates (2-5A) have been shown to cause at least a dual physiological effect in tobacco and wheat: (i) increased cytokinin activity and (ii) induced synthesis of numerous proteins, among which members of two groups of stress proteins have been identified, namely pathogenesis-related (PR) and heat shock (HS) proteins. These effects were observed only by low concentrations of these substances: IFN at 0.1-1 u/ml and 2-5A at 1-10 nM.

Production of the tobacco mosaic virus (TMV) transport protein in transgenic plants is essential but insufficient for complementing foreign virus transport: a need for the full-length TMV genome or some other TMV-encoded product.

We have reported previously that tobamoviruses enable the transport of red clover mottle comovirus (RCMV) in tobacco plants normally resistant to RCMV. Here we show that RCMV transport does not take place in transgenic tobacco plants (line To-4) producing the 30K transport protein of tobacco mosaic virus (TMV), whereas the transport of the TMV Ls1 mutant, the cell-to-cell movement of which is temperature sensitive, is complemented in these plants. However, RCMV transport is observed when these transgenic plants are infected with both RCMV and TMV Ls1 at the non-permissive temperature (33 degrees C). It is suggested that (i) the hypothetical modification of transgenic plant plasmodesmata by the TMV 30K transport protein can specifically mediate the cell-to-cell movement of the homologous virus (TMV), but is insufficient to mediate RCMV transport; (ii) the presence of the full-length TMV genome or a certain TMV-encoded product(s) besides the 30K protein is essential for complementation of the RCMV transport function. The possibility that line To-4 might provide enough 30K protein to complement TMV Ls1 but not RCMV cannot be ruled out. During double infection the mutant 30K protein may, in concert with the wild-type 30K protein, provide the transport function for RCMV.

Use of a reverse cross-finger flap as a vascularized vein graft carrier in ring avulsion injuries.

We describe the use of a reverse cross-finger pedicle flap, previously described by Atasoy, that carries veins used as vascularized grafts to restore venous drainage in ring avulsion injuries. In addition, vascularized soft tissue is provided to cover extensor tendon and exposed bone.

The vascularized fascia of the scalp.

The fascial layers of the temporal and occipital regions of the scalp were examined in 11 fresh cadavers. In the temporal region, three independently vascularized layers were isolated, all of which could be elevated on a single superficial temporal artery, but separated to remain independently vascularized from specific branches of this parent trunk. In the occipital area, the occipital vessels could be dissected to yield a long pedicle for an independent, fascial territory that could be transposed locally or elevated as a free flap and that will, in all likelihood, carry vascularized occipital bone. Realized and as yet unrealized uses of these ultrathin vascularized tissues remain boundless. Three representative cases are presented.