Predictable multiple site root coverage using an acellular dermal matrix allograft.

BACKGROUND: The primary aim of this randomized, controlled, blinded clinical investigation was to determine if orientation of an acellular dermal matrix (ADM) allograft, basement membrane side against the tooth or connective tissue side against the tooth, affected the percent root coverage. Additional aims were to: 1) compare results of this study with results obtained from other root coverage studies; 2) determine if multiple additional sites could be successfully covered with the same surgery; 3) determine the effect of the procedure on keratinized tissue; and 4) evaluate the amount of creeping attachment obtained. METHODS: Ten patients with 2 Miller Class I or II buccal recession defects > or =3 mm were treated with a coronally positioned flap plus ADM and followed for 12 months. Test sites received ADM with the basement membrane side against the root (AB), while the control sites received the connective tissue side against the root (AC). Multiple additional recession sites were treated with the same flap procedure. RESULTS: Mean baseline recession for the AB sites was 4.2 mm and for the AC sites, 3.7 mm. Mean root coverage of 95% was obtained for both AB and AC sites. Sixty-eight additional Class I or II AB and AC sites obtained about 93% root coverage. The mean increase in keratinized tissue for both treatments was 0.80 mm. No additional root coverage was gained due to creeping attachment between 2 and 12 months. CONCLUSIONS: Treatment with ADM was an effective and predictable procedure for root coverage. The orientation of the material did not affect the treatment outcome for any of the parameters tested.

A comparison of porous and non-porous teflon membranes plus demineralized freeze-dried bone allograft in the treatment of class II buccal/lingual furcation defects: a clinical reentry study.

BACKGROUND: The aim of this 9-month reentry study was to compare the regenerative healing using porous (P) and non-porous (NP) teflon barrier membranes plus demineralized freeze dried bone allografts (DFDBA) in Class II buccal/lingual furcation defects. METHODS: Twenty-four patients, 13 males and 11 females, ages 38 to 75 (mean 54 +/- 10), were included in this study. Each patient had adult periodontitis and one Class II furcation defect measuring > or = 3 mm open horizontal probing depth. Twelve patients were randomly selected to receive the NP treatment and 12 received the P membrane. All defects received a DFDBA graft. Measurements were performed by a masked examiner. RESULTS: No statistically significant differences (P>0.05) were found between NP and P groups at any time with respect to any open or closed measure. Improvement in mean open horizontal probing depth was significant for both the NP (2.33 +/- 0.78 mm) and P (2.75 +/- 0.75 mm) groups. Mean clinical attachment level gains at 9 months were significant for both NP (1.50 +/- 1.62 mm) and P (2.50 +/- 2.11 mm) groups. Seventeen of 24 defects had an intrabony component and > or = 50% fill was obtained in 100% of these defects. CONCLUSIONS: The results of this 9-month reentry study comparing the use of porous and non-porous barrier membranes with a DFDBA graft indicate that there were no statistically significant differences between groups. Both groups showed a statistically significant improvement following the treatment of Class II furcation defects in humans.

Potato leafroll virus protein P1 contains a serine proteinase domain.

The multi-domain potato leafroll virus replicase protein P1 was expressed in insect cells from the polyhedrin promoter of Autographa californica nucleopolyhedrovirus. Using antisera raised against P1, it was shown that P1 was cleaved near the VPg in insect cells in a manner similar to that in plant cells, to produce a approximately 27 kDa C-terminal fragment. Furthermore, it was shown that the proposed serine proteinase-like domain within P1 is responsible for this processing and that this can occur in a trans (intermolecular) reaction. Four conserved residues within the serine proteinase domain that are essential for catalysis have been identified, consistent with the proposal that this domain comprises a serine proteinase.

Aphid Acquisition and Cellular Transport of Potato leafroll virus-like Particles Lacking P5 Readthrough Protein.

ABSTRACT Lepidopteran cells (Spodoptera frugiperda) produced isometric virus-like particles (VLP) when infected with a recombinant baculovirus Ac61 that contained the Potato leafroll virus (PLRV) coat protein gene modified with an N-terminal histidine tag (P3-6H). Cells infected with AcFL, a recombinant baculovirus that expressed cDNA copies of the PLRV genome RNA, did not produce virus-like particles (VLP). In cell lines doubly infected with Ac61 and AcFL, VLP were formed that contained PLRV-RNA packaged in P3-6H coat protein (FL). Both the P3-6H and the FL particles were morphologically indistinguishable from particles of PLRV despite the fact that they lacked the P5 readthrough protein present in wild-type PLRV. When aphids (Myzus persicae) were fed on, or injected with, purified PLRV, or VLP of either type (FL or P3-6H) and examined by electron microscopy, no differences were observed among treatments for particle endocytosis, transcellular transport, or exocytosis at the aphid midgut or accessory salivary glands. Particles were observed in the salivary canals and in the salivary duct leading out of the aphid. These results suggest that P5 readthrough protein of PLRV may not be essential for cellular transport of virus through aphid vectors.

Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus.

DNA encoding the coat protein (P3) of a Scottish isolate of potato leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost cystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co- sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density gradient fractions contained protein which reacted with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a protein, with an Mr of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.

Ribozymes that cleave potato leafroll virus RNA within the coat protein and polymerase genes.

Two ribozymes were synthesized which were designed to cleave potato leafroll virus (PLRV) positive strand RNA within the regions known to encode the viral coat protein and the predicted RNA polymerase gene. DNA sequences encoding the ribozymes were inserted into the Escherichia coli plasmids pTz18R and pTz19R under the control of the bacteriophage T7 promoter and enzymically active RNA molecules generated by transcription by T7 RNA polymerase in vitro. Each ribozyme cleaved its cognate site in RNA derived from either cDNA or PLRV particles. Ribozyme cleavage sites within the polymerase gene and coat protein gene were determined and shown to be at the predicted sequence immediately downstream from a GUC motif. An altered version of the ribozyme which recognized the sequence in the coat protein gene was isolated in which a single adenosine residue in the enzymic loop of the ribozyme was deleted. This mutated ribozyme was unable to cleave RNA molecules containing the coat protein ribozyme target site.

Mutational analysis of the Bradyrhizobium japonicum common nod genes and further nod box-linked genomic DNA regions.

By insertional and deletional marker replacement mutagenesis the common nod region of Bradyrhizobium japonicum was examined for the presence of additional, essential nodulation genes. An open reading frame located in the 800 bp large intergenic region between nodD1 and nodA did not appear to be essential for nodulation of soybean. Furthermore, a strain with a deletion of the nodI- and nodJ-like genes downstream of nodC had a Nod+ phenotype. A mutant with a 1.7 kb deletion immediately downstream of nodD1 considerably delayed the onset of nodulation. This region carried a second copy of nodD (nodD2). A nodD1-nodD2 double mutant had a similar phenotype to the nodD2 mutant. Using a 22-mer oligonucleotide probe partially identical to the nod box sequence, a total of six hybridizing regions were identified in B. japonicum genomic DNA and isolated from a cosmid library. Sequencing of the hybridizing regions revealed that at least three of them represented true nod box sequences whereas the others showed considerable deviations from the consensus sequence. One of the three nod box sequences was the one known to be associated with nodA, whereas the other two were located 60 to 70 kb away from nif cluster I. A deletion of one of these two sequences plus adjacent DNA material (mutant delta 308) led to a reduced nodulation on Vigna radiata but not on soybean. Thus, this region is probably involved in the determination of host specificity.

Identification of two classes of Rhizobium phaseoli genes required for melanin synthesis, one of which is required for nitrogen fixation and activates the transcription of the other.

The symbiotic plasmid pRP2JI of Rhizobium phaseoli strain 8002 was shown to contain two separate regions of DNA which are required and sufficient for the synthesis of the pigment melanin. One of these regions containing the class II mel gene(s) was located to other genes involved in nodulation and in nitrogen fixation. Mutations in this region abolished both the ability to synthesize melanin and to fix nitrogen in Phaseolus bean root nodules. Mutations in the other, unlinked region, containing class I mel gene(s), also abolished melanin synthesis but did not affect symbiotic nitrogen fixation. Transcriptional fusions between the class I mel gene and the Escherichia coli lacZ gene were constructed and it was demonstrated that the class II mel gene(s) activated their transcription in free-living culture. Further, strains containing the cloned regulatory class II gene(s) synthesized melanin when growing in minimal medium, in contrast to wild-type strains which became pigmented only in complete medium containing yeast extract and tryptone. It was shown by hybridization experiments that the regulatory mel gene was closely linked to or may correspond to the regulatory nifA gene; a fragment of R. phaseoli DNA which included the class II gene(s) of R. phaseoli hybridized to a previously identified nifA-like gene of R. leguminosarum, the species that nodulates peas.

Cloning of the nodulation (nod) genes of Rhizobium phaseoli and their homology to R. leguminosarum nod DNA.

In Rhizobium phaseoli strain 8002, a large indigenous plasmid, pRP2JI, had previously been shown to carry many of the genes necessary for the induction of nitrogen-fixing nodules on Phaseolus beans. A cosmid clone library was constructed using DNA from strain 8002. From this library, two overlapping recombinant plasmids (pIJ1097 and pIJ1098) were isolated which spanned about 43 kb of pRP2JI DNA. These plasmids could restore nodulation to some, but not all nodulation-deficient strains of R. phaseoli, indicating that the nodulation genes were not clustered within one small region of pRP2JI. The cloned R. phaseoli nodulation region shared extensive DNA homology with the nodulation genes of R. leguminosarum, and on the basis of DNA hybridization, the nitrogenase genes were found to be within 10 kb of the R. phaseoli nodulation genes. Close to the nodulation genes of R. phaseoli was located a sequence that was repeated on pRP2JI but which was not present elsewhere in the genome of strain 8002.

Appearance of a novel form of plant glutamine synthetase during nodule development in Phaseolus vulgaris L.

The activities of glutamine synthetase (GS), nitrogenase and leghaemoglobin were measured during nodule development in Phaseolus vulgaris infected with wild-type or two non-fixing (Fix(-)) mutants of Rhizobium phaseoli. The large increase in GS activity which was observed during nodulation with the wild-type rhizobial strain occurred concomitantly with the detection and increase in activity of nitrogenase and the amount of leghaemoglobin. Moreover, this increase in GS was found to be due entirely to the appearance of a novel form of the enzyme (GSn1) in the nodule. The activity of the form (GSn2) similar to the root enzyme (GSr) remained constant throughout the experiment. In nodules produced by infection with the two mutant strains of Rhizobium phaseoli (JL15 and JL19) only trace amounts of GSn1 and leghaemoglobin were detected.

Stability and blood level determinations of cefaclor, a new oral cephalosporin antibiotic.

Cefaclor solutions in pH 2.5 and 4.5 buffers contained at least 90% of their initial activity after 72 h at 4 degrees C. Samples in pH 6.0, 7.0, and 8.0 buffers contained 70, 46, and 34%, respectively, of their initial activity after 72 h at 4 degrees C. After 72 h at 25 degrees C, samples prepared with pH 2.5, 4.5, 6.0, 7.0, and 8.0 buffers contained 95, 69, 16, 5, and 3%, respectively, of their initial activity. After 72 h at 37 degrees C, cefaclor solutions in pH 2.5 buffer contained 80% of the initial activity, whereas samples prepared in pH 4.5, 6.0, 7.0, and 8.0 buffers contained less than 20%. Laboratory-prepared plasma and serum samples showed an 8% loss in activity when incubated for 6 h at 4 degrees C, a 51% loss when incubated for 6 h at 25 degrees C, and a 48% loss when incubated for 2 h at 37 degrees C. Clinical samples demonstrated a similar stability pattern. Degradation rates for cefaclor in commercially prepared serum increased from 4- to 10-fold in comparison to rates obtained when samples were made in human serum freshly prepared in our laboratory. Consequently, serum standards should be made in freshly prepared human serum.

Stability of parenteral solutions of tobramycin sulfate.

The stability and physical compatibility of tobramycin sulfate in commonly used intravenous fluids were evaluated; in addition, pH values were obtained on 1 mg/ml tobramycin solutions, and microbiological potencies were obtained on 1 mg/ml and 0.2 mg/ml concentrations for 24- and 48-hour periods at 25 C. Most solutions were stable for 48 hours at room temperature. However, it is recommended that solutions of tobramycin sulfate be discarded after 24 hours to minimize the potential for inadvertent introduction of microorganisms during manipulations in the hospital environment.

Factors influencing the microbiological assay of tobramycin.

Tobramycin belongs to the aminoglycoside class of antibiotics. Assay procedures were investigated to determine the most suitable methods for the assay of tobramycin in blood, urine, and pharmaceutical preparations. The agar-plate assay was the method of choice for determinations in urine and serum. The turbidimetric assay was selected for the assay of pharmaceutical preparations. An extraction technique is described that minimizes problems encountered in the estimation of tobramycin in urine. Sample diluents, pH, and molarity were important and were variable depending on assay methods. Evidence is presented to show binding of tobramycin to alpha-cellulose susceptibility discs. Iron and sodium ions demonstrated potential inhibition of tobramycin in all assay methods tested.