Complete sequence of the IncPbeta plasmid R751: implications for evolution and organisation of the IncP backbone.

The broad host range IncP plasmids are of particular interest because of their ability to promote gene spread between diverse bacterial species. To facilitate study of these plasmids we have compiled the complete sequence of the IncPbeta plasmid R751. Comparison with the sequence of the IncPalpha plasmids confirms the conservation of the IncP backbone of replication, conjugative transfer and stable inheritance functions between the two branches of this family. As in the IncPalpha genome the DNA of this backbone appears to have been enriched for the GCCG/CGGC motifs characteristic of the genome of organisms with a high G+C content, such as P. aeruginosa, suggesting that IncPbeta plasmids have been subjected during their evolution to similar mutational and selective forces as IncPalpha plasmids and may have evolved in pseudomonad hosts. The IncP genome is consistently interrupted by insertion of phenotypic markers and/or transposable elements between oriV and trfA and between the tra and trb operons. The R751 genome reveals a family of repeated sequences in these regions which may form the basis of a hot spot for insertion of foreign DNA. Sequence analysis of the cryptic transposon Tn4321 revealed that it is not a member of the Tn21 family as we had proposed previously from an inspection of its ends. Rather it is a composite transposon defined by inverted repeats of a 1347 bp IS element belonging to a recently discovered family which is distributed throughout the prokaryotes. The central unique region of Tn4321 encodes two predicted proteins, one of which is a regulatory protein while the other is presumably responsible for an as yet unidentified phenotype. The most striking feature of the IncPalpha plasmids, the global regulation of replication and transfer by the KorA and KorB proteins encoded in the central control operon, is conserved between the two plasmids although there appear to be significant differences in the specificity of repressor-operator interactions. The importance of these global regulatory circuits is emphasised by the observation that the operator sequences for KorB are highly conserved even in contexts where the surrounding region, either a protein coding or intergenic sequence, has diverged considerably. There appears to be no equivalent of the parABCDE region which in the IncPalpha plasmids provides multimer resolution, lethality to plasmid-free segregants and active partitioning functions. However, we found that the continuous sector from co-ordinate 0 to 9100 bp, encoding the co-regulated klc and kle operons as well as the central control region, could confer a high degree of segregational stability on a low copy number test vector. Thus R751 appears to exhibit very clearly what was first revealed by study of the IncPalpha plasmids, namely a fully functional co-ordinately regulated set of replication, transfer and stable inheritance functions.

Unaccounted blood loss in operations using cardiopulmonary bypass.

Typically, blood loss after operations requiring cardiopulmonary bypass is estimated from the sum of blood on sponges and drapes, in the suction system reservoir, and in chest drainage bottles. Prime of the extracorporeal circuit is usually returned to the patient, but no accounting is made of blood remaining in the circuit. In 50 patients, we examined 25 bubble and 25 membrane oxygenator circuits after completion of cardiopulmonary bypass and after return of all prime to the patients. Saline solution was added to each circuit and recirculated for 3 minutes, after which the volume and hematocrit of the recirculated saline were determined. From these values, we estimated that 92.0 +/- 18.1 mL of red blood cells remained in the bubble oxygenators and 100 +/- 11.6 mL in the membrane oxygenators. This volume of red cells is equivalent to 250 mL of whole blood with 35% hematocrit or 30 grams of hemoglobin at 12 gm% concentration. We conclude that blood loss after operations requiring cardiopulmonary bypass is systematically underestimated by the approximately 250 mL left as red cells on the walls and filters of the extracorporeal circuit after return of all prime to the patient.

Plasmid pRTL1 controlling 1-chloroalkane degradation by Rhodococcus rhodochrous NCIMB13064.

Rhodococcus rhodochrous NCIMB13064 can dehalogenate and use a wide range of 1-haloalkanes as sole carbon and energy source. The 1-chloroalkane degradation phenotype may be lost by cells spontaneously or after treatment with Mitomycin C. Two laboratory derivatives of the original strain exhibited differing degrees of stability of the chloroalkane degradation marker. Plasmids of approximately 100 kbp (pRTL1) and 80 kbp (pRTL2) have been found in R. rhodochrous NCIMB13064. pRTL1 was shown to be carrying at least some genes for the dehalogenation of 1-chloroalkanes with short chain lengths (C3 to C9). However, no connection was found between the utilization of 1-chloroalkanes with longer chain lengths (C12 to C18) and the presence of pRTL1. Three separate events were observed to lead to the inability of NCIMB13064 to dehalogenate the short-chain 1-chloroalkanes; the complete loss of pRTL1, the integration of pRTL1 into the chromosome, or the deletion of a 20-kbp fragment in pRTL1. High-frequency transfer of the 1-chloroalkane degradation marker associated with pRTL1 has been demonstrated in bacterial crosses between different derivatives of R. rhodochrous NCIMB13064.

Removal of colored tattoos with the Q-switched alexandrite laser.

The Q-switched alexandrite laser at 760 nm and 50- to 100-ns pulse duration has been shown to effectively remove blue-black as well as green, red, and mauve in seven human subjects with colored tattoos. An average of 9.0 treatments were required to completely remove the green pigment at an average fluence of 5.80 J/cm2 compared with an average of 9.7 treatments at an average fluence of 6.10 J/cm2 for red and an average of 10 treatments at an average fluence of 6.20 J/cm2 for mauve. Orange and yellow were unaffected by the alexandrite laser over a range of fluences tested.

Role of the Alexandrite laser for removal of tattoos.

BACKGROUND AND OBJECTIVE: The development of the Alexandrite laser for the removal of blue-black tattoos is described. STUDY DESIGN/MATERIALS AND METHODS: The responses of an animal study, using professionally tattooed skin and a human study involving 22 (professional and nonprofessional) blue-black tattoos, to the Alexandrite laser are reported. RESULTS: Histopathologic evaluation of tattooed pig skin biopsies demonstrated the method of removal of dermal tattoo pigment. An average 11.6 treatments were required to remove completely the ten human blue-black professional tattoos compared to an average of 10.3 treatments to reach the same endpoint in six subjects with nonprofessional tattoos. CONCLUSION: Of significance was the fact that unlike the Q-switched Ruby and Nd:YAG lasers where punctate bleeding and tissue splattering have been reported to occur during laser tattoo removal, epidermal integrity was maintained during exposure of tattooed skin to the Q-switched Alexandrite laser at therapeutic fluences used.

Haloalkane degradation and assimilation by Rhodococcus rhodochrous NCIMB 13064.

The bacterium Rhodococcus rhodochrous NCIMB 13064, isolated from an industrial site, could use a wide range of 1-haloalkanes as sole carbon source but apparently utilized several different mechanisms simultaneously for assimilation of substrate. Catabolism of 1-chlorobutane occurred mainly by attack at the C-1 atom by a hydrolytic dehalogenase with the formation of butanol which was metabolized via butyric acid. The detection of small amounts of gamma-butyrolactone in the medium suggested that some oxygenase attack at C-4 also occurred, leading to the formation of 4-chlorobutyric acid which subsequently lactonized chemically to gamma-butyrolactone. Although 1-chlorobutane-grown cells exhibited little dehalogenase activity on 1-chloroalkanes with chain lengths above C10, the organism utilized such compounds as growth substrates with the release of chloride. Concomitantly, gamma-butyrolactone accumulated to 1 mM in the culture medium with 1-chlorohexadecane as substrate. Traces of 4-hydroxybutyric acid were also detected. It is suggested that attack on the long-chain chloroalkane is initiated by an oxygenase at the non-halogenated end of the molecule leading to the formation of an omega-chlorofatty acid. This is degraded by beta-oxidation to 4-chlorobutyric acid which is chemically lactonized to gamma-butyrolactone which is only slowly further catabolized via 4-hydroxybutyric acid and succinic acid. However, release of chloride into the medium during growth on long-chain chloroalkanes was insufficient to account for all the halogen present in the substrate. Analysis of the fatty acid composition of 1-chlorohexadecane-grown cells indicated that chlorofatty acids comprised 75% of the total fatty acid content with C14:0, C16:0, C16:1 and C18:1 acids predominating. Thus the incorporation of 16-chlorohexadecanoic acid, the product of oxygenase attack directly into cellular lipid represents a third route of chloroalkane assimilation. This pathway accounts at least in part for the incomplete mineralization of long-chain chloroalkane substrates. This is the first report of the coexistence of a dehalogenase and the ability to incorporate long-chain haloalkanes into the lipid fraction within a single organism and raises important questions regarding the biological treatment of haloalkane containing effluents.

Pulsed dye laser treatment of recalcitrant verrucae: a preliminary report.

Patients with recalcitrant warts on the fingers and hands, periungual, and other parts of the body including verrucae plana and plantar surfaces were treated using the pulsed dye laser at 585 nm, 450 usec, and a spotsize of 5 mm diameter. Of the 39 patients treated, 28 (72%) were cleared of their warts after an average of 1.68 treatments at fluences of 6.25-7.5 J/cm2. Seven (18%) patients had a reduction of between 80-95% of their warts after 1.3 treatments, and verrucae reduced by 50% in four of the 39 patients after one treatment. The average follow-up period of the 28 cases cleared of their warts has been 5 months. Of this group, those with periungual warts have been followed for up to 6.4 months, compared to 4.8 months for those with warts on other parts of their body, 4.0 months for those with finger and hand warts, and 2.0 months for plantar warts. Only one of the 28 patients has had a recurrence after 3 months of clearance.

EMLA for laser treatment of portwine stains in children.

Vascular-specific lasers with pulse durations of between 300 and 500 microseconds are the treatment of choice for portwine stains (PWS), particularly in children. Although the discomfort felt following laser irradiation from a single pulse is transient, these sensations are intensified when multiple pulses are delivered over a confined area of skin. Because 75-80% of PWS are located in the head and neck regions, laser treatment of these sites tends to be more painful than those located in most other parts of the body. A prospective double blind randomized study, assessing pain during laser irradiation on PWS skin occluded with either EMLA, placebo, or no cream (control) was performed in 73 PWS subjects between 5 and 16 years of age. Analysis of the data showed that the pain scores for EMLA-treated sites were the smallest, the control scores tended to be the highest, with the placebo scores in the middle. The differences between the treatments were significant (P < 0.0001).

Location of port-wine stains and the likelihood of ophthalmic and/or central nervous system complications.

Of 310 patients with port-wine stains, 68% had more than one dermatome involved; 85% had unilateral and 15% had a bilateral distribution of their port-wine stain. At the time of examination, 8% of all patients with trigeminal port-wine stains had evidence of eye and/or central nervous system (CNS) involvement. Extensive involvement, with port-wine stain over the trunk and extremities as well as the head and neck, was observed in 12%. Patients who did not have port-wine stains on the areas served by branches V1 and V2 of the trigeminal nerve had no signs or symptoms of eye and/or CNS involvement. Port-wine stains of the eyelids, bilateral distribution of the birthmark, and unilateral port-wine stains involving all three branches of the trigeminal nerve were associated with a significantly higher likelihood of having eye and/or CNS complications. Twenty-four percent of those with bilateral trigeminal nerve port-wine stains had eye and/or CNS involvement compared with 6% of those with unilateral lesions. All those who had eye and/or CNS complications had port-wine stain involvement of the eyelids; in 91% both upper and lower eyelids were involved, whereas in 9% only the lower eyelid was involved. None of those with upper eyelid port-wine stains alone had eye and/or CNS complications. In addition, 3 (75%) of the 4 subjects with seizures alone had bilateral port-wine stain involvement. A third group, these with unilateral V1, V2, and V3 port-wine stains, had eye and/or CNS complications in 3 (19%) of 16 subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Validity of the bulk-staining technique to separate artifactual from in vivo bone microdamage.

Frost's bulk-staining technique clearly separates artifactual cracking in bone from that caused by mechanical loading in vivo. One-centimeter sections of human rib were either bulk stained before preparation of a thin section (experimental) or stained after grinding to a thickness of 150 microns (control). Microdamage produced before preparation can be separated from that caused by the preparation itself. Moreover, the technique does not cause additional artifactual cracking through dehydration. This technique provides a valid way to test hypotheses about the role that microdamage creation or its repair plays in skeletal pathology.

A real-time anesthesia record keeping system using video.

A real-time, Automated Anesthesia Record Keeping (AARK) system was constructed using commercially available video hardware. This system can acquire all of the information that is presented visually to an anesthesiologist directly from monitor screens. The information is recorded on VHS format tape. Use of such a system avoids the need for digital processing while capturing all waveforms, trends, data messages and numerics. Since the data are stored directly to tape, subsequent data analysis can be accomplished. This system can be used with virtually any medical monitor.

Histologic comparison of the pulsed dye laser and copper vapor laser effects on pig skin.

Albino pig skin was exposed to the copper vapor (CVL) and flash-lamp pulsed dye (PDL) lasers at 578 nm with a 3 mm diameter spotsize over a range of fluences until purpura and whitening were first established. The total irradiation time was the parameter that was varied in order for the CVL to reach the desired fluence. The lowest fluence producing each clinical endpoint was designated the threshold fluence: 34 J/cm2 was required to produce purpura using the CVL compared to 7.5 J/cm2 with the PDL laser. Histologically, skin exposed to purpura fluences from the CVL revealed the presence of constricted, disrupted papillary dermal blood vessels with trapped RBC's within them which were unlike those exposed to PDL where the irradiated vessels were dilated and packed with masses of intravascular agglutinated RBC's. The whitening threshold fluences for the CVL and PDL lasers were 67 J/cm2 and 29 J/cm2, respectively. Streaming of epidermal cells and dermal collagen denaturation were observed in CVL irradiated skin, compared to occasional dyskeratotic epidermal cells and focal dermal collagen denaturation following PDL exposure. The mechanisms responsible for the clinical and histologic changes produced by the two laser systems are discussed.

The use of video in anesthesia record keeping.

Although formal comparisons of record keeping systems are difficult, the authors are persuaded that a video system provides substantial advantages over AARK in several (though not all) of its aims and at a substantially reduced cost. The video system might be viewed as a parallel rather than a replacement system in some cases. The authors have been struck by certain capabilities such a video system offers. Not the least of these is the advantage of having a multiple OR monitoring system for use by the floor supervisor to check all rooms easily. The implications of a video technology in clinical research also are important. At present, a clinical trial involving 40 cases is being organized and will require that physiologic parameters be recorded in detail. Plans to use the ubiquitous multichannel strip recorder for logging the parameters have been changed. Video will be used instead, since it is more than adequate to meet most research needs of the department and greatly simplifies the recording of such data at reduced cost.

Treatment of port-wine stains at 577 nm: clinical results.

A pulsed, dye laser at 577 nm was used to treat 65 patients with port-wine stains. Results clearly indicate that the laser parameters of dose, wavelength, and pulse duration contribute to the outcome of laser treatment of port-wine stains.

Radiocarbon dates on bones of extinct birds from Hawaii.

Bones from a stratified sedimentary deposit in the Puu Naio Cave site on Maui, Hawaiian Islands, reveal the late Holocene extinction of 19 species of birds. The age of the sediment and associated fauna was determined by direct radiocarbon dating (tandem particle accelerator-mass spectrometer; TAMS) of amino acids extracted from bones weighing as little as 450 mg. The 14C dates indicate that sediment has been accumulating in the lava tube for at least the last 7750 years, a suitable time frame for testing the hypothesis that Holocene extinction on islands began after human colonization. Despite growing evidence that a worldwide wave of extinctions coincided with human colonization of oceanic islands, little radiometric data have been available to date the extinction of most small fossil vertebrates on islands. The TAMS technique of dating purified collagen from the bones of small vertebrates could lead to vastly improved chronologies of extinction for oceanic islands where catastrophic mid- to late-Holocene extinction is expected or known to have occurred. Chronologies derived from nonarcheological sites that show continuous sedimentation, such as the Puu Naio Cave deposit, may also yield key evidence on the timing of earliest human settlement of Oceania.

Cardiopulmonary response to extracorporeal venous CO2 removal in awake spontaneously breathing dogs.

The ventilatory response to a reduction in mixed venous PCO2 has been reported to be a decrease in breathing even to the point of apnea with no change in arterial CO2 partial pressure (PaCO2), whereas a recent report in exercising dogs found a small but significant drop in PaCO2 (F. M. Bennett et al. J. Appl. Physiol. 56: 1335-1337, 1984). The purpose of the present study was to attempt to reconcile this discrepancy by carefully investigating the cardiopulmonary response to venous CO2 removal over the entire range from eupnea to the apneic threshold in awake, spontaneously breathing normoxic dogs. Six dogs with chronic tracheostomies were prepared with bilateral femoral arteriovenous shunts under general anesthesia. Following recovery, an extracorporeal venovenous bypass circuit, consisting of a roller pump and a silicone-membrane gas exchanger, was attached to the femoral venous cannulas. Cardiopulmonary responses were measured during removal of CO2 from the venous blood and during inhalation of low levels of CO2. Arterial PO2 was kept constant by adjusting inspired O2. The response to venous CO2 unloading was a reduction in PaCO2 and minute ventilation (VE). The slope of the response, delta VE/delta PaCO2, was the same as that observed during CO2 inhalation. This response continued linearly to the point of apnea without significant changes in cardiovascular function.(ABSTRACT TRUNCATED AT 250 WORDS)