Effects of an Oral-Pharyngeal Motor Training Programme on Children with Obstructive Sleep Apnea Syndrome in Hong Kong: A Retrospective Pilot Study.

BACKGROUND: This study aimed to investigate the effects of an oropharyngeal motor training programme on children with Obstructive Sleep Apnea Syndrome (OSAS) in Hong Kong. METHODS: In this retrospective study, we reviewed the outcomes of 10 children with OSAS who had received an oropharyngeal motor training programme in Occupational Therapy Department of an acute hospital in Hong Kong over a 1-year programme. Each participant attended an individual oropharyngeal motor training programme plus a follow-up session after 2 months. The training programme consisted of 10 individual mobilization exercises involving the orofacial and pharyngeal area for 45 minutes. Each exercise had to be repeated for 10 times. Three outcome measures were chosen to study the effectiveness of the training programme including tongue strength, tongue endurance level and orofacial function. Tongue strength and tongue endurance level were assessed using the Iowa Oral Pressure Instrument (IOPI). The Nordic Orofacial Test-Screening (NOT-S) Assessment was used to assess the orofacial function. Seven out of 10 participants completed the training programme and attended the follow-up session after two months. RESULTS: The tongue strength and the scores of NOT-S of the 7 participants were found to have significant improvement after training. However, there was no significant difference in tongue endurance level. CONCLUSION: The findings of this study support the role of occupational therapist in oromotor training modalities to improve the respiratory function for children with OSAS in Hong Kong. Copyright © 2017, Hong Kong Occupational Therapy Association. Published by Elsevier (Singapore) Pte Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

USR-VS: a web server for large-scale prospective virtual screening using ultrafast shape recognition techniques.

Ligand-based Virtual Screening (VS) methods aim at identifying molecules with a similar activity profile across phenotypic and macromolecular targets to that of a query molecule used as search template. VS using 3D similarity methods have the advantage of biasing this search toward active molecules with innovative chemical scaffolds, which are highly sought after in drug design to provide novel leads with improved properties over the query molecule (e.g. patentable, of lower toxicity or increased potency). Ultrafast Shape Recognition (USR) has demonstrated excellent performance in the discovery of molecules with previously-unknown phenotypic or target activity, with retrospective studies suggesting that its pharmacophoric extension (USRCAT) should obtain even better hit rates once it is used prospectively. Here we present USR-VS (http://usr.marseille.inserm.fr/), the first web server using these two validated ligand-based 3D methods for large-scale prospective VS. In about 2 s, 93.9 million 3D conformers, expanded from 23.1 million purchasable molecules, are screened and the 100 most similar molecules among them in terms of 3D shape and pharmacophoric properties are shown. USR-VS functionality also provides interactive visualization of the similarity of the query molecule against the hit molecules as well as vendor information to purchase selected hits in order to be experimentally tested.

Novel CLN1 mutation with atypical juvenile neuronal ceroid lipofuscinosis.

We detected a novel CLN1 gene mutation (p.Arg151X, heterogenous) in a 12-year-old boy. Low level of palmitoyl protein thioesterase and granular inclusion pattern in lymphocytes were also consistent with infantile Neuronal ceroid lipofuscinosis (INCL). However, the clinical phenotype was that of atypical juvenile neuronal ceroid lipofuscinosis (JNCL) and consisted of progressive visual loss from the age of 8 years. His visual acuity was 6/60 in both eyes at first presentation, 6/36 one month later, then 6/6 (right eye), and 6/60 (left eye) 6 months later. However, after 4 months, visual acuity dropped to 6/60 in both eyes and at last follow-up, it was 6/60 (right eye) and 3/60 (left eye). Visual hallucinations were also reported. Persistent normal fundi findings, normal electroretinogram (ERG), and delayed visual evoked potentials (VEP) were suggestive of non-retinal adolescence form/atypical JNCL. Visual loss in JNCL is secondary to retinal dystrophy. Our observations suggest that JNCL should be considered in any children presenting with bilateral progressive visual loss even with normal fundi and/or delayed VEP. Electron microscopy of buffy coat and palmitoyl protein thioesterase enzyme study are useful tools in diagnosis. Pertinent issues regarding clinical symptomatology, ophthalmologic findings, and laboratory results are discussed.

Cardiovascular Protective Effects of Adjunctive Alternative Medicine (Salvia miltiorrhiza and Pueraria lobata) in High-Risk Hypertension.

Introduction. Hypertension in association with diabetes (DM), renal impairment (RI), and left ventricular hypertrophy (LVH) increases the risk of future cardiovascular events. We hypothesize, traditional herbal medicines Danshen and Gegen (D&G) have beneficial effects on atherogenesis in these high-risk hypertensive subjects. Subjects and Methods. 90 asymptomatic hypertensive subjects associated with LVH (63.3%), DM (62.2%), or RI (30%) were randomized to receive D&G herbal capsules 1 gm/day, 2 gm/day, or identical placebo capsules in double-blind and parallel fashion for 12 months. Brachial flow-mediated dilation (endothelium-dependent dilation, FMD) and carotid intima-media thickness (IMT) were measured by ultrasound. All data were analyzed using the Statistical Package for Social Sciences in Windows 16.0. Results. Their mean age was 55 ± 8 years, and 74.4% were male. After 12 months of adjunctive therapies and compared with baseline, there were no significant changes in blood pressure, heart rate, hematological, glucose, and creatinine profiles in both placebo and D&G groups. FMD improved significantly during D&G (P = 0.0001) and less so after placebo treatment (P = 0.001). There was a mild but significant decrease in carotid IMT after D&G (P < 0.001) but no significant changes after placebo. A trend of better improvement in FMD after higher versus lower D&G dosages was seen. D&G were well tolerated, with no significant adverse events or blood biochemistry changes. Conclusion. D&G adjunctive treatment was well tolerated and significantly improved atherogenesis in high-risk hypertensive patients, with potential in primary atherosclerosis prevention.

Dystonia during feeding as an early sign of dopa-responsive dystonia.

A 21/2-month-old girl presented with feeding difficulties of 8 weeks' duration. She cried, vomited, arched, and became rigid during every feeding. She was suspected of having gastroesophageal reflux disease. Dystonia and developmental delay became apparent at age 8 months. Nasogastric tube feeding and gastrostomy with Nissen's fundoplication were performed at age 7 and 12 months, respectively. She was treated with baclofen, trihexyphenidyl, and antireflux therapy, without benefit. She became severely developmentally delayed with marked head lag, dystonia, and rigidity. Levodopa therapy was initiated at age 21 months. She manifested dramatic improvement over the next year. Dystonia, rigidity, and retching disappeared soon after treatment. She experienced good catch-up in development. She exhibited poor head control and an inability to reach out at age 21 months, but bottom shuffling was observed at age 26 months, and walking and speaking three-word sentences at age 2 years and 10 months. Pertinent issues relating to signs, pathophysiology, genetics, and biochemical defects are discussed briefly.

Test-retest reliability and criterion validity of the Chinese version of CBCL, TRF, and YSR.

BACKGROUND: Psychometric properties of the Chinese version of CBCL, TRF, and YSR were understudied. This study aimed at examining their test-retest reliability and criterion validity. METHODS: Three Chinese community and clinic samples were recruited in Hong Kong. The parents, teachers, and youths respectively completed the CBCL, TRF, and YSR. RESULTS: The Chinese CBCL, TRF, and YSR were test-retest reliable and valid. However, there was score/case attenuation at retest. CBCL and TRF appeared to screen externalizing and ADHD problems better, while YSR screened internalizing problems better. CONCLUSIONS: Clinicians should be cautious about score/case attenuation at retest while using CBCL, TRF, and YSR to chart patients' progress. They should also recognize their different strengths in screening various disorders.

Fiber depolymerization.

Depolymerization is, by definition, a crucial process in the reversible assembly of various biopolymers. It may also be an important factor in the pathology of sickle cell disease. If sickle hemoglobin fibers fail to depolymerize fully during passage through the lungs then they will reintroduce aggregates into the systemic circulation and eliminate or shorten the protective delay (nucleation) time for the subsequent growth of fibers. We study how depolymerization depends on the rates of end- and side-depolymerization, k(end) and k(side), which are, respectively, the rates at which fiber length is lost at each end and the rate at which new breaks appear per unit fiber length. We present both an analytic mean field theory and supporting simulations showing that the characteristic fiber depolymerization time tau= square root 1/k(end)k(side) depends on both rates, but not on the fiber length L, in a large intermediate regime 1 << k(side)L(2)/k(end) << (L/d)(2), with d the fiber diameter. We present new experimental data which confirms that both mechanisms are important and shows how the rate of side depolymerization depends strongly on the concentration of CO, acting as a proxy for oxygen. Our theory remains rather general and could be applied to the depolymerization of an entire class of linear aggregates, not just sickle hemoglobin fibers.

Dopamine receptor D4 (DRD4) gene in Han Chinese children with attention-deficit/hyperactivity disorder (ADHD): increased prevalence of the 2-repeat allele.

There is an increased prevalence of the 7-repeat (7R) allele of the dopamine receptor D4 (DRD4) gene in attention-deficit/hyperactivity disorder (ADHD). However, the population prevalence of the 7R allele varies considerably across ethnicity and is very low in Asians. To test whether this 7R allele/ADHD association still held in a Chinese clinical sample, 32 Han Chinese children with a confirmed ADHD diagnosis and normal IQ who were methylphenidate-responders were genotyped. None of them had a DRD4 7R allele. Instead, we observed a significantly increased prevalence of the 2-repeat (2R) allele in this clinical sample (33%) compared to ethnically-matched controls (20%) (chi(2)(1d.f.) = 5.90, P = 0.015). This approximately 1.65-fold increase of the 2R allele in our probands is close to the observed increase of the 7R allele in European-ancestry ADHD children. Recent genetic studies have indicated that the 2R allele in Asians is likely derived from the 7R allele. Further, available biochemical data indicate that both the 2R and 7R protein have blunted responses to dopamine compared to the 4R protein. Based on these results, we propose that the observed increased prevalence of the 2R allele in our Han Chinese ADHD probands is still consistent with the 7R allele hypothesis of ADHD in European-ancestry children. Recent studies have suggested that any variant from the conserved ancestral 4R allele might potentially alter biochemistry/phenotype. We hypothesize that an increased frequency of any non-4R allele may define the association of the DRD4 gene with ADHD that holds across ethnicity. The present findings, however, obtained with a small ADHD sample size, should be replicated.

Micro-tensile bond strengths to sclerotic dentin using a self-etching and a total-etching technique.

OBJECTIVES: To evaluate the in vitro regional micro-tensile bond strengths of a self-etching/self-priming adhesive system to sclerotic dentin, in the absence or presence of phosphoric acid conditioning. METHODS: Naturally-occurring, non-carious cervical lesions on extracted premolars were hand-cleaned with a slurry of pumice and chlorhexidine, then bonded without further cavity preparation. One group was bonded using Clearfil Liner Bond 2V (Kuraray Co. Ltd., Osaka, Japan). The other group was first conditioned with K-etchant; 40% phosphoric acid gel (Kuraray) prior to the application of the self-etching primer. Artificially prepared wedge-shaped cavities were also made in sound premolars and bonded with the two methods as controls. Resin composite build-ups were made using Clearfil Protect Liner F and AP-X resin composite (Kuraray). After storage in water for 24h, the teeth were sectioned into 0.7 x 0.7 mm composite-dentin beams along the occlusal and gingival walls, and at the apex of the lesions. The use of two conditioning methods, two substrate types, and three different locations yielded 10-14 beams for each of the 12 groups. After testing for the microTBS, representative beams that were stressed to failure were examined with SEM. Remaining fractured beams were demineralized and processed for TEM examination. Statistical analysis was performed using a three-way ANOVA and Student-Newman-Keuls tests. RESULTS: Regardless of the conditioning methods, bond strengths to sound dentin were significantly higher than to sclerotic dentin (P< 0.05). With sclerotic dentin, there was no significant difference for the conditioning methods used, except that K-etchant significantly improved the bond strength at the gingival aspect of the lesions. Fractographic analysis revealed that the self-etching primer could not etch beyond the surface hypermineralized layer of sclerotic dentin. Interfacial failure occurred along the surface of the mineralized intermicrobial matrix and/or hypermineralized layer. With the use of phosphoric acid, a hybrid layer was only seen when the surface layers were thin. Incompletely removed sclerotic casts were evident in both groups. SIGNIFICANCE: Removal of the surface layers of sclerotic dentin and/or conditioning with stronger acids may be beneficial to obtain stronger bonding to sclerotic dentin.

Cancer incidence in the United Farmworkers of America (UFW), 1987-1997.

BACKGROUND: The purpose of this study was to evaluate cancer incidence in the membership a largely Hispanic farmworker labor union in California, the United Farmworkers of America (UFW) and to examine cancer-site specific distributions as well as histology and stage of cancer at diagnosis in this group. METHODS: An electronic record linkage was conducted between a membership roster of the UFW and the database of the California Cancer Registry, the population-based cancer registry in California for the years 1987-1997. Based upon the results of the linkage, morbidity odds ratios were calculated using the distribution of cancer in the California Hispanic population as the reference to determine if risk of specific cancers was higher or lower in the UFW. Time since first joining the union was evaluated, as was the proportional distribution of histologic subtypes and stage at diagnosis, again comparing the experience of the UFW membership to the California Hispanic population. RESULTS: Several types of cancer were elevated in the UFW membership in comparison to the California Hispanic population. Morbidity odds ratios and 95% confidence limits were elevated for leukemia (O.R. = 1.59: 95% C.I. = 1.07-2.37), stomach cancer (O.R. = 1.69: 95% C.I. = 1.24-2.27), uterine cervix cancer (O.R. = 1.63: 95% C.I. = 1.11-2.44) and uterine corpus cancer (O.R. = 1.68: 95% C.I. = 1.05-2.67). Brain cancer was also elevated although not significantly so (O.R. = 1.57: 95% C.I. = 0.96-2.53). CONCLUSIONS: Risk of leukemia, stomach, cervix and uterine cancers was elevated in California farmworkers. The histologic distribution of leukemia and brain cancers within the UFW membership did not differ from the distribution in the general California population although small numbers of cancers in the UFW hindered interpretation of these results. Members of the UFW experienced later stage of disease at diagnosis in comparison to California Hispanics for most major cancer sites but not for breast cancer.

Molecular analysis of the pRA2 partitioning region: ParB autoregulates parAB transcription and forms a nucleoprotein complex with the plasmid partition site, parS.

The partitioning locus (par) of plasmid pRA2 belongs to a recently discovered subgroup of plasmid partitioning systems that are evolutionarily distinct from the P1, F and R1/NR1 prototypes. The pRA2 par region was effective in stabilizing both pRA2 and F mini-replicons. Analysis of the nucleotide sequence revealed three potential coding regions that were designated parA, parB and parC. Through mutagenesis, parA and parB were found to be essential for partitioning function, whereas parC did not appear to be required. Using transcriptional reporter systems, it was demonstrated in vivo that ParB repressed par promoter activity by 60-fold and that ParA had little effect on transcriptional activity. Primer extension analysis revealed that the par transcriptional start point was located 47 nucleotides upstream of the parA translational start codon. Based on this information, putative -10 and -35 transcriptional signals were identified, and their subsequent deletion resulted in a dramatic reduction in promoter activity. The par promoter region was also demonstrated to exert incompatibility towards a plasmid with an active pRA2 par system. Nested deletions in this region allowed the incompatibility determinant, designated parS, to be localized. Recombinant ParA and ParB proteins were overexpressed and purified by affinity chromatography. Through in vitro binding experiments, purified ParB was shown to interact specifically with the par promoter region. DNase I footprinting revealed that ParB not only binds to the conserved sequence 5'-TCA AA(T/C) (G/C)CT CAA (A/T)A, which is present in three copies in the par promoter region, but also binds to the pRA2 partitioning site, parS. It appears that ParB has a dual role in pRA2 partitioning, being responsible for both the regulation of par transcription and the formation of a partition nucleoprotein complex at parS.

An information-based sequence distance and its application to whole mitochondrial genome phylogeny.

MOTIVATION: Traditional sequence distances require an alignment and therefore are not directly applicable to the problem of whole genome phylogeny where events such as rearrangements make full length alignments impossible. We present a sequence distance that works on unaligned sequences using the information theoretical concept of Kolmogorov complexity and a program to estimate this distance. RESULTS: We establish the mathematical foundations of our distance and illustrate its use by constructing a phylogeny of the Eutherian orders using complete unaligned mitochondrial genomes. This phylogeny is consistent with the commonly accepted one for the Eutherians. A second, larger mammalian dataset is also analyzed, yielding a phylogeny generally consistent with the commonly accepted one for the mammals. AVAILABILITY: The program to estimate our sequence distance, is available at http://www.cs.cityu.edu.hk/~cssamk/gencomp/GenCompress1.htm. The distance matrices used to generate our phylogenies are available at http://www.math.uwaterloo.ca/~mli/distance.html.

Cytochrome P450 2D6 genotyping and association with tardive dyskinesia in Chinese schizophrenic patients.

BACKGROUND: The discovery of Cytochrome P450 2D6 (CYP 2D6) polymorphism is implicated in individual differences in drug metabolism rate. Mutation with defective alleles is associated with reduced metabolism of many anti-psychotic drugs metabolized by CYP 2D6. This may contribute to the development of tardive dyskinesia (TD) in patients with prolonged exposure to anti-psychotic drugs. METHODS: In this controlled study, the genotype of CYP 2D6*10 alleles, movement disorders and clinical characteristics in 38 Chinese schizophrenic patients with TD were compared with 38 age- and sex-matched schizophrenia patients without TD. RESULTS: There was no significant correlation between CYP 2D6*10 genotypes and TD in men. However, a significant increase in the frequency of CYP 2D6*10 allele was found in female patients with TD. CONCLUSIONS: The sex differences in CYP 2D6 genotyping and vulnerability to develop TD suggest that a biological predisposition that affects pharmacokinetics may be more significant in women, whereas other factors may be more important in men.

An ultrastructural study of the application of dentine adhesives to acid-conditioned sclerotic dentine.

OBJECTIVE: This in vitro study examined the ultrastructure of resin-infiltrated sclerotic dentine following the application of a self-etching primer, with or without the adjunctive use of phosphoric acid pre-conditioning. MATERIALS AND METHODS: Non-carious, natural cervical sclerotic lesions were hand-cleaned with a slurry of pumice and chlorhexidine and bonded without further cavity preparation. One group was bonded using Clearfil Liner Bond 2V (Kuraray Co. Ltd, Osaka, Japan) alone. Specimens from the other group were pre-conditioned with K-etchant (40% phosphoric acid gel, Kuraray) prior to the application of the same self-etching primer. Artificially prepared wedge-shaped lesions were also made in sound bicuspids, bonded using the two methods, and used as controls for the two groups. For SEM examination, each specimen was cryofractured into two halves through a pre-formed slit made on the lingual surface, after the respective conditioning treatment. Different locations within the lesions were examined after rinsing of the phosphoric acid/self-etching primer and specimen dehydration. For TEM investigation, specimens were bonded with the adhesive. Both demineralised and undemineralised ultrathin sections were prepared from the occlusal, gingival and deepest part of the wedge-shaped bonded lesions following specimen fixation, dehydration and resin embedding. RESULTS: A hypermineralised surface layer was present on the surface of etched sclerotic dentine. This layer was thicker in the deepest part of the natural lesions, where bacterial colonisation of the lesion surface was also apparent. Both treatment protocols were unable to effectively dissolve sclerotic casts that occluded the dentinal tubules. Depending upon the thickness of the surface layers at different locations in the natural lesion, self-etching primer treatment alone resulted in reduction of the thickness of the authentic hybrid layer (i.e. hybridised intertubular dentine). This was also true of phosphoric acid pre-conditioning along the deepest part of the natural lesions. Within this region, intertubular dentine completely devoid of an authentic hybrid layer could be seen in both treatment groups. Resin tags were also sparsely observed in such regions. CONCLUSIONS: Adhesive strategies that rely mostly on micromechanical retention alone may be compromised by the sporadic absence of the hybrid layer and resin tags in sclerotic dentine. Based on the ultrastructural features presented, it is further speculated that adaptive strategies such as removal of the surface layers and extended etching time may not be completely effective in improving bonding efficacy in highly sclerotic dentine. Interdisciplinary research should be continued to develop alternative procedures for bonding resins equally well to sound and sclerotic dentine.

Nonideality and the nucleation of sickle hemoglobin.

The homogeneous and heterogeneous nucleation kinetics of sickle hemoglobin (HbS) have been studied for various degrees of solution crowding by substitution of cross-linked hemoglobin A, amounting to 50% of the total hemoglobin. By cross-linking hemoglobin A, hybrid formation between hemoglobin A and hemoglobin S was prevented, thus simplifying the analysis of the results. Polymerization was induced by laser photolysis, and homogeneous nucleation kinetics were determined by observation of the stochastic behavior of the onset of light scattering. Heterogeneous nucleation was determined by observing the exponential growth of the progress curves, monitored by light scattering. At concentrations between 4 and 5 mM tetramer (i.e., approximately 30 g/dl), the substitution of 50% HbA for HbS slows the reaction by a factor of 10(3) to 10(4). Using scaled particle theory to account for the crowding of HbA, the observed decrease in the homogeneous nucleation rate was accurately predicted, with no variation of parameters required. Heterogeneous nucleation, on the other hand, is not well described in the present formulation, and the theory for this process appears to require modification of the way in which nonideality is introduced. Nonetheless, the accuracy of the homogeneous nucleation description suggests that such an approach may be useful for other assembly processes that occur in a crowded intracellular milieu.

Bonding of a self-etching primer to non-carious cervical sclerotic dentin: interfacial ultrastructure and microtensile bond strength evaluation.

PURPOSE: The objectives of this study were 1) to examine the ultrastructural features of the resin-sclerotic dentin interface following the application of Clearfil Liner Bond II sigma to natural cervical wedge-shaped lesions, and 2) to evaluate the regional tensile bond strength of this self-etching primer at different locations on natural and artificially-created cervical lesions. MATERIALS AND METHODS: Deep cervical natural lesions were bonded using the self-etching primer. Micromorphology of the bonded interface at different locations within the lesions were examined using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and scanning transmission electron microscopy/energy dispersive x-ray analysis (STEM/EDX). Ultrastructural features were further compared with the use of the same self-etching primer on artificial lesions created in sound cervical dentin. A nontrimming technique was used to evaluate the regional tensile bond strength from the occlusal, gingival, and the deepest central part of both natural and artificial cervical lesions. Beams with a mean area of 0.46 +/- 0.03 mm2 were prepared and were pulled to failure using a Bencor Multi-T testing device attached to an Instron universal tester. Bond strength results were evaluated using a two-way ANOVA design. RESULTS: A hypermineralized layer devoid of intact, banded collagen was invariably present on the surface of the natural lesions. Depending upon its thickness at different locations of the lesion, the action of a self-etching primer may be limited to this surface layer alone, producing a hybridized hypermineralized surface layer. Penetration of the self-etching primer into the underlying sclerotic dentin produced a hybridized complex containing a hybridized hypermineralized surface layer as well as a subsurface layer of hybridized intertubular dentin. Bacterial colonization of the lesion surface resulted in the formation of an additional zone of hybridized intermicrobial matrix over the surface of the lesions. Dentinal tubules remained blocked with sclerotic casts, and resin tags were rarely observed. Regional tensile bond strength results showed that the overall bond strength to natural sclerotic dentin was about 20% lower than sound cervical dentin, but was independent of the different locations within the lesions from which bond strength was evaluated. CONCLUSION: There were four factors that may have influenced the overall decrease in bond strength in natural cervical sclerotic lesions: a) the presence of a hybridized intermicrobial matrix together with entrapped bacteria may have weakened the bonds, b) inability of a self-etching primer to etch through a thick, hypermineralized surface layer, c) presence of a layer of possibly remineralized, denatured collagen at the base of the hypermineralized surface layer, and d) retention of acid-resistant sclerotic casts that obliterate the tubular lumina and prevent effective resin tag formation.

Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer.

The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.

Changes in the functional properties of bovine hemoglobin induced by covalent modification with polyethylene glycol.

Polyethylene glycol conjugation to proteins and peptides (PEGylation) has been shown to promote increased retention time in the circulation as well as to blunt immune or allergic reactions. PEGylated bovine hemoglobin (PEG-Hb) is being explored in human clinical trials as an oxygen delivering agent for the sensitization of solid tumors to radiation therapy. In this study the functional properties of PEG-Hb were compared to those of bovine hemoglobin (Hb), the mutant human hemoglobin Rothchild and bovine hemoglobin crosslinked between the beta chains. The rate of heme transfer from Hb to serum albumin at pH 9.0 was greatly increased by PEGylation, suggesting destabilization of the heme-globin linkage and of the bonds between alpha beta dimers. Measurement of oxygen binding equilibrium showed that the oxygen affinity of Hb became unusually dependent on temperature and Hb concentration after PEGylation. Evidence is presented to suggest that PEGylation of lysine beta-81 at the entrance to the central cavity of the Hb tetramer might be responsible for these observations. The alterations of the functional properties of Hb induced by PEGylation are consistent with the beneficial effects of PEG-Hb in exchange transfusion and radiation sensitization models of human conditions.