RESUMEN
We report the five-year outcome of a randomised controlled trial which used radiostereometric analysis (RSA) to assess the influence of surface oxidised zirconium (OxZr, Oxinium) on polyethylene wear in vivo. A total of 120 patients, 85 women and 35 men with a mean age of 70 years (59 to 80) who were scheduled for primary cemented total hip arthroplasty were randomly allocated to four study groups. Patients were blinded to their group assignment and received either a conventional polyethylene (CPE) or a highly cross-linked (HXL) acetabular component of identical design. On the femoral side patients received a 28 mm head made of either cobalt-chromium (CoCr) or OxZr. The proximal head penetration (wear) was measured with repeated RSA examinations over five years. Clinical outcome was measured using the Harris hip score. There was no difference in polyethylene wear between the two head materials when used with either of the two types of acetabular component (p = 0.3 to 0.6). When comparing the two types of polyethylene there was a significant difference in favour of HXLPE, regardless of the head material used (p < 0.001). In conclusion, we found no advantage of OxZr over CoCr in terms of polyethylene wear after five years of follow-up. Our findings do not support laboratory results which have shown a reduced rate of wear with OxZr. They do however add to the evidence on the better resistance to wear of HXLPE over CPE.
Asunto(s)
Artroplastia de Reemplazo de Cadera/instrumentación , Cabeza Femoral/cirugía , Prótesis de Cadera , Circonio , Anciano , Anciano de 80 o más Años , Artroplastia de Reemplazo de Cadera/métodos , Cementación , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis de la Cadera/cirugía , Estudios Prospectivos , Diseño de Prótesis , Falla de Prótesis , Análisis Radioestereométrico/métodos , Resultado del TratamientoRESUMEN
In this prospective study we studied the effect of the inclination angle of the acetabular component on polyethylene wear and component migration in cemented acetabular sockets using radiostereometric analysis. A total of 120 patients received either a cemented Reflection All-Poly ultra-high-molecular-weight polyethylene or a cemented Reflection All-Poly highly cross-linked polyethylene acetabular component, combined with either cobalt-chrome or Oxinium femoral heads. Femoral head penetration and migration of the acetabular component were assessed with repeated radiostereometric analysis for two years. The inclination angle was measured on a standard post-operative anteroposterior pelvic radiograph. Linear regression analysis was used to determine the relationship between the inclination angle and femoral head penetration and migration of the acetabular component. We found no relationship between the inclination angle and penetration of the femoral head at two years' follow-up (p = 0.9). Similarly, our data failed to reveal any statistically significant correlation between inclination angle and migration of these cemented acetabular components (p = 0.07 to p = 0.9).
Asunto(s)
Acetábulo/patología , Artroplastia de Reemplazo de Cadera/métodos , Prótesis de Cadera , Falla de Prótesis/etiología , Acetábulo/diagnóstico por imagen , Acetábulo/cirugía , Anciano , Anciano de 80 o más Años , Cementación , Femenino , Cabeza Femoral/diagnóstico por imagen , Cabeza Femoral/patología , Migración de Cuerpo Extraño/etiología , Migración de Cuerpo Extraño/patología , Humanos , Masculino , Persona de Mediana Edad , Polietilenos , Estudios Prospectivos , Diseño de Prótesis , Análisis Radioestereométrico/métodosRESUMEN
Antibacterial chemicals in the mucus of fish such as lysozyme, lectins, peptides and proteases provide an efficient first line of defence against pathogens. This study shows that there are at least three antibacterial proteins in plaice skin mucus in addition to lysozyme. One of these proteins is responsible for approximately 74% of the antibacterial activity and is a 630 kDa protease complex designated KilC (bacterial killing metalloprotease C). Purified KilC kills the bacteria Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa efficiently. The protease activity of KilC is dependent upon the divalent cation Mg(2+) and shows pH dual optima of 5.0 and 8.0. The enzyme has a temperature optimum of 25 degrees C and is made up of at least five different sized peptides. Studies with protease inhibitors show that the catalytic site of KilC may be cysteine- or serine protease-like. KilC may kill bacterial cells by acting directly upon the bacteria or by producing low molecular weight bioactive compounds such as peptides.
Asunto(s)
Antibacterianos/aislamiento & purificación , Lenguado/inmunología , Metaloproteasas/aislamiento & purificación , Moco/enzimología , Piel/inmunología , Animales , Antibacterianos/inmunología , Acuicultura , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Metaloproteasas/inmunología , Metaloproteasas/farmacología , Pruebas de Sensibilidad Microbiana , Moco/inmunologíaRESUMEN
We performed a randomised, radiostereometric study comparing two different bone cements, one of which has been sparsely clinically documented. Randomisation of 60 total hip replacements (57 patients) into two groups of 30 was undertaken. All the patients were operated on using a cemented Charnley total hip replacement, the only difference between groups being the bone cement used to secure the femoral component. The two cements used were Palamed G and Palacos R with gentamicin. The patients were followed up with repeated clinical and radiostereometric examinations for two years to assess the micromovement of the femoral component and the clinical outcome. The mean subsidence was 0.18 mm and 0.21 mm, and the mean internal rotation was 1.7 degrees and 2.0 degrees at two years for the Palamed G and Palacos R with gentamicin bone cements, respectively. We found no statistically significant differences between the groups. Micromovement occurred between the femoral component and the cement, while the cement mantle was stable inside the bone. The Harris hip score improved from a mean of 38 points (14 to 54) and 36 (10 to 57) pre-operatively to a mean of 92 (77 to 100) and 91 (63 to 100) at two years in the Palamed G and Palacos R groups, respectively. No differences were found between the groups. Both bone cements provided good initial fixation of the femoral component and good clinical results at two years.
Asunto(s)
Antibacterianos/uso terapéutico , Artroplastia de Reemplazo de Cadera/métodos , Cementos para Huesos/uso terapéutico , Gentamicinas/uso terapéutico , Polimetil Metacrilato/uso terapéutico , Anciano , Femenino , Fémur/fisiología , Articulación de la Cadera/fisiopatología , Articulación de la Cadera/cirugía , Prótesis de Cadera , Humanos , Masculino , Persona de Mediana Edad , Movimiento , Radioisótopos , Rotación , Tantalio , Resultado del TratamientoRESUMEN
To examine the role of the renal nerves and sodium depletion for the acute antidiuretic response to bendroflumethiazide (BFTZ; 25 microg/hr) in rats with diabetes insipidus (DI), renal clearance experiments were performed in the following groups of conscious, chronically instrumented male Brattleboro rats with vasopressin-deficient DI: Control (n = 7), BFTZ (n = 9), BFTZ + sodium replacement (n = 7) and BFTZ + chronic bilateral renal denervation (n = 6). Urine flow rate and urinary sodium concentration were measured drop-by-drop with a sodium-sensitive electrode and by collection of urine in vials placed on an electronic balance. This allowed computer driven, servo-controlled, independent i.v. replacement of sodium and fluid losses, respectively. Mean arterial pressure, glomerular filtration rate (GFR) and proximal tubular water and sodium handling, assessed by lithium clearance (C(Li)), were stable in the control group. BFTZ produced a marked antidiuretic response (deltaV = -79%; deltaUrine osmolality = +218%) associated with decreases in GFR (-28%), C(Li) (-62%), free water clearance (-100%) and plasma Na (-5 mM). Fractional water reabsorption was increased by 19% in the proximal tubules and by 7% in segments beyond. Sodium replacement did not modify the fall in GFR or the antidiuresis, but partly prevented the increase in fractional proximal water reabsorption. Bilateral renal denervation did not affect the response to BFTZ. We conclude that the acute antidiuretic effect of BFTZ is independent of sodium balance and renal nerve activity and is elicited by a reduction in GFR accompanied by an increase in fractional water reabsorption in the proximal tubules and in the distal nephron.
Asunto(s)
Bendroflumetiazida/farmacología , Diabetes Insípida/tratamiento farmacológico , Diuresis/efectos de los fármacos , Riñón/inervación , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Sodio/metabolismo , Animales , Diabetes Insípida/metabolismo , Diuréticos , Tasa de Filtración Glomerular/efectos de los fármacos , Masculino , Ratas , Ratas BrattleboroRESUMEN
Five weeks after common bile duct ligation (CBL), Wistar rats had histologically verified liver cirrhosis with sodium retention but without ascites. Plasma concentrations of vasopressin and aldosterone were normal. Glomerular filtration rate was unchanged, although renal plasma flow was increased. A test dose of furosemide (7.5 mg/kg body wt iv) produced significantly greater diuretic (+59%) and natriuretic (+66%) responses in Wistar CBL rats than in sham-operated controls. Stereological examination of kidneys demonstrated a 47% increase in the volume of the inner stripe of the outer medulla, with a 55% increase in the volume of thick ascending limb of Henle's loop (TALH) epithelium in cirrhotic Wistar rats relative to controls. CBL produced a similar degree of liver cirrhosis in vasopressin-deficient Brattleboro rats. However, both functional and structural renal changes observed in cirrhotic Wistar rats were absent in vasopressin-deficient cirrhotic Brattleboro rats. These results suggest a permissive action of vasopressin for the adaptive changes in TALH in rats with experimental liver cirrhosis. Our results are consistent with the hypothesis that increased sodium chloride reabsorption in the TALH may contribute to the early sodium retention that precedes ascites formation in rats with secondary biliary liver cirrhosis.
Asunto(s)
Cirrosis Hepática Experimental/patología , Cirrosis Hepática Experimental/fisiopatología , Asa de la Nefrona/patología , Asa de la Nefrona/fisiopatología , Animales , Sangre/metabolismo , Agua Corporal/metabolismo , Electrólitos/metabolismo , Femenino , Hemodinámica , Riñón/patología , Túbulos Renales/metabolismo , Cirrosis Hepática Experimental/metabolismo , Asa de la Nefrona/metabolismo , Tamaño de los Órganos , Ratas , Ratas Brattleboro , Ratas Wistar , Circulación Renal , Sodio/metabolismoRESUMEN
BACKGROUND: That non-natriuretic doses of loop diuretics exert an antihypertensive action has been suggested, but not confirmed, by simultaneous measurements of the arterial pressure and sodium balance during therapy. OBJECTIVE: To examine the relationship between changes in arterial pressure and changes in sodium balance during furosemide treatment. DESIGN: Twenty hypertensive Dahl salt-sensitive rats fed a 4% NaCl diet were allocated to four groups and administered the following treatments: placebo once a day intraperitoneally, continuous infusion of 4 mg/day furosemide intraperitoneally, 4 mg furosemide once a day intraperitoneally and 12 mg furosemide once every third day intraperitoneally. METHODS: The mean arterial pressure (MAP) was measured continuously with radiotelemetry and the sodium balance was measured with the rats in metabolic cages. RESULTS: Administration of furosemide as a bolus injection once a day (P < 0.01) or once every third day (P < 0.05) lowered the MAP significantly compared with placebo, whereas continuous infusion of furosemide had no significant effect on the MAP (P < 0.07). Fast Fourier transformation analysis detected an acute antihypertensive action related to the temporary diuretic and natriuretic responses during the period 0-6 h after intraperitoneal bolus injections of 4 and 12 mg furosemide. None of the treatment regimens produced 24 h sodium or potassium losses. At the end of the study, the total body water, extracellular fluid volume, total body sodium and potassium were similar for rats in all groups. CONCLUSIONS: Furosemide has an acute antihypertensive action in Dahl salt-sensitive rats fed a 4% NaCl diet that is related to renal sodium and volume losses whereas the long-term antihypertensive effect is independent of changes in extracellular fluid volume, total body water, sodium and potassium.
Asunto(s)
Antihipertensivos/administración & dosificación , Presión Sanguínea/efectos de los fármacos , Diuréticos/administración & dosificación , Furosemida/administración & dosificación , Animales , Diuresis/efectos de los fármacos , Resistencia a Medicamentos , Frecuencia Cardíaca/efectos de los fármacos , Infusiones Parenterales , Inyecciones Intraperitoneales , Masculino , Natriuresis/efectos de los fármacos , Potasio/metabolismo , Ratas , Sodio/metabolismo , Sodio en la Dieta/toxicidad , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
Acute intracerebroventricular administration of the antihyperglycaemic agent metformin (0.25-1 mg) elicits sympathoinhibitory responses in spontaneously hypertensive rats. However, cardiovascular actions of chronic intracerebroventricular metformin administration are unknown. To define the dose-response relationship during chronic intracerebroventricular metformin administration, mean arterial pressure, heart rate, and locomotor activity were measured continuously by radiotelemetry in 40 normotensive rats. After a 10 day control period, an intracerebroventricular cannula was implanted and connected to an osmotic minipump which delivered metformin in the following doses: 0 [saline]. 0.01, 0.1, 1, and 10 mg/day. LD50 was 1.5 mg/day. Metformin, 1 mg/day attenuated the nocturnal, physiological increase in mean arterial pressure (-7.3 +/- 1.6% versus before metformin), produced behavioural changes and tended to increase locomotor activity. Lower doses of intracerebroventricular metformin (0.1 and 0.01 mg/day) did not affect mean arterial pressure, heart rate or locomotor activity. In conclusion, chronic intracerebroventricular administration of high dose metformin (1.0 mg/day) attenuates the nocturnal, physiological increase in mean arterial pressure. These findings are compatible with a toxic, sympathoinhibitory action of high doses of metformin intracerebroventricularly.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Hipoglucemiantes/farmacología , Metformina/farmacología , Actividad Motora/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/toxicidad , Inyecciones Intraventriculares , Masculino , Metformina/administración & dosificación , Metformina/toxicidad , Ratas , Ratas WistarRESUMEN
To test the hypothesis that the long-term antihypertensive action of furosemide is mediated by a renomedullary vasodepressor substance, we measured mean arterial pressure (MAP) by radiotelemetry in Dahl-S rats with either intact or bromoethylamine-induced (BEA, 100 mg/kg i.p.) lesion of the renal papilla and medulla. Seven days of recovery after BEA administration, the rats diet was changed from 1 to 4% NaCl, and during days 8 to 31, rats were randomized to daily treatment with placebo or furosemide (50 mg/kg p.o.). Then furosemide treatment was stopped and the rat food was changed to 1% NaCl diet. After a 10-day wash-out period, renal function was measured. BEA produced a rapid (within min) and sustained increase in MAP which was accelerated during 4% NaCl diet. Furosemide prevented 4% NaCl-induced hypertension in both rats with intact kidneys and in rats with BEA-induced renal papillary-medullary lesion. A significant decrease in renal plasma flow (-34%) and glomerular filtration rate (-40%) was observed in all BEA-treated rats independent of previous furosemide treatment. In response to an i.v. load of isotonic saline (10% body weight), rats with renal papillary-medullary lesion had an impaired ability to excrete sodium. Histological examination showed that BEA-treated rats had severe lesions of the renal papilla and medulla, with light-to-moderate changes in the renal cortex. It is concluded that the antihypertensive effect of furosemide is not mediated by a renomedullary vasodepressor substance. The accelerated NaCI-sensitive hypertension in rats with BEA-induced renal papillary-medullary lesion is related to an impaired ability to excrete excess NaCl.
Asunto(s)
Antihipertensivos/farmacología , Furosemida/farmacología , Hipertensión/tratamiento farmacológico , Médula Renal/patología , Animales , Peso Corporal , Modelos Animales de Enfermedad , Conducta de Ingestión de Líquido , Hipertensión/etiología , Hipertensión/fisiopatología , Pruebas de Función Renal , Médula Renal/fisiopatología , Masculino , Ratas , Ratas Endogámicas , Cloruro de SodioRESUMEN
The plasmid vectors described in this report are derived from the broad-host-range RK2 replicon and can be maintained in many gram-negative bacterial species. The complete nucleotide sequences of all of the cloning and expression vectors are known. Important characteristics of the cloning vectors are as follows: a size range of 4.8 to 7.1 kb, unique cloning sites, different antibiotic resistance markers for selection of plasmid-containing cells, oriT-mediated conjugative plasmid transfer, plasmid stabilization functions, and a means for a simple method for modification of plasmid copy number. Expression vectors were constructed by insertion of the inducible Pu or Pm promoter together with its regulatory gene xylR or xylS, respectively, from the TOL plasmid of Pseudomonas putida. One of these vectors was used in an analysis of the correlation between phosphoglucomutase activity and amylose accumulation in Escherichia coli. The experiments showed that amylose synthesis was only marginally affected by the level of basal expression from the Pm promoter of the Acetobacter xylinum phosphoglucomutase gene (celB). In contrast, amylose accumulation was strongly reduced when transcription from Pm was induced. CelB was also expressed with a very high induction ratio in Xanthomonas campestris. These experiments showed that the A. xylinum celB gene could not complement the role of the bifunctional X. campestris phosphoglucomutase-phosphomannomutase gene in xanthan biosynthesis. We believe that the vectors described here are useful for cloning experiments, gene expression, and physiological studies with a wide range of bacteria and presumably also for analysis of gene transfer in the environment.
Asunto(s)
Clonación Molecular/métodos , Vectores Genéticos , Factores R/genética , Replicón , Acetobacter/enzimología , Acetobacter/genética , Amilosa/biosíntesis , Proteínas Bacterianas/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Fosfoglucomutasa/biosíntesis , Fosfoglucomutasa/genética , Regiones Promotoras GenéticasRESUMEN
This study was designed to compare the renal effects of sedation with alphaxalone-alphadolone, etomidate, propofol, midazolam, fentanyl-fluanisone, and thiopental in rats. The sedative dose was defined as the highest dose that abolished the escape response without affecting the righting reflex. Female Wistar rats were chronically catheterized with a jugular vein catheter, and urine flow rate and renal clearances of inulin (glomerular filtration rate = GFR), sodium, and lithium (used as an index of proximal tubular function) were measured in the conscious, unrestrained state (n = 107 experiments). In a separate series (n = 70 experiments), the effect of sedative doses of each drug on the nociceptive threshold was tested with the tail-flick test. Responses in sedated animals were compared to responses in animals infused with the vehicle. Fentanyl-fluanisone and thiopental had hypoalgesic actions in sedating doses. Propofol, fentanyl-fluanisone, and thiopental reduced GFR by 20-30%. Urine flow rate was significantly decreased by propofol (-24%) and thiopental (-48%). Propofol and fentanyl-fluanisone reduced fractional lithium excretion by 9-13%. Only alphaxalone-alphadolone, etomidate, and midazolam produced sedation without affecting renal function in rats. Because midazolam produced the most consistent degree of sedation, we conclude that midazolam is the least confounding sedative agent for renal function studies in conscious rats.
Asunto(s)
Conducta Animal/efectos de los fármacos , Hipnóticos y Sedantes/farmacología , Riñón/efectos de los fármacos , Micción/efectos de los fármacos , Animales , Etomidato/farmacología , Femenino , Midazolam/farmacología , Pregnanodionas/farmacología , Propofol/farmacología , Ratas , Ratas WistarRESUMEN
The minimal replicon of the broad-host-range plasmid RK2 consists of a gene, trfA (trans-acting replication), encoding a protein required for initiation of plasmid replication. The TrfA protein binds to iterons in the cis-acting origin of vegetative replication (oriV), but the exact mechanism by which TrfA-mediated replication initiation takes place is not known. We report here the isolation and characterization of five mini RK2 trfA mutant plasmids with an elevated plasmid copy number, four in Pseudomonas aeruginosa and one in Azotobacter vinelandii. The mutations are localized between or downstream of previously reported Escherichia coli copy-up mutations in trfA, and one of the mutations has been described earlier as an independent copy-up isolate in E. coli. The five mutant plasmids were all moderately copy up in both E. coli and their host of origin, in spite of the use of isolation procedures which were expected to select efficiently in favor of plasmid mutants specifying high copy numbers. In contrast, previously described high copy-up mutants isolated in E. coli could not be established in P. aeruginosa and A. vinelandii. These high copy-up mutants were shown to induce cell killing in E. coli under conditions where the plasmid copy number was increased as a physiological response to reduced growth rate. We propose that the reason for this killing effect is that the copy number under these conditions exceeds an upper tolerance level specific for E. coli. By assuming that the corresponding tolerance level is lower in P. aeruginosa and A. vinelandii than in E. coli, and that the mechanism of copy number regulation is similar, the model can explain the phenotypes of all tested copy up mutants in these two hosts. Analogous studies were also performed in Salmonella typhimurium and Acetobacter xylinum. The data obtained in these studies indicate that the above model is probably generally true for gram-negative bacteria, and the results also indicate that the maximum tolerable copy number is surprisingly low in some hosts.
Asunto(s)
Bacterias/genética , Proteínas Bacterianas/genética , Replicación del ADN , Proteínas de Escherichia coli , Escherichia coli/genética , Mutagénesis , Plásmidos , Replicón , Ampicilina/farmacología , Azotobacter vinelandii/genética , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Proteínas Bacterianas/biosíntesis , Conjugación Genética , Escherichia coli/metabolismo , Pruebas de Sensibilidad Microbiana , Fenotipo , Pseudomonas aeruginosa/genética , Mapeo Restrictivo , Salmonella typhimurium/genética , Transformación BacterianaRESUMEN
The minimal replicon of the broad-host-range plasmid RK2 consists of the origin of vegetative replication (oriV) and a gene (trfA) encoding an essential replication protein that binds to short repeats in oriV. We report here the results of a DNA sequence analysis of seven unique mutants that are temperature sensitive for replication in Escherichia coli. The mutations (designated rts) were distributed throughout 40% of the downstream part of the trfA gene. Spontaneous revertants of the rts mutants were isolated, and further analysis of four such revertants demonstrated that the new phenotypes resulted from intragenic second-site copy up (cop) mutations. Subcloning experiments showed that all tested intragenic combinations of rts and cop mutations resulted in elimination or strong reduction of the temperature sensitivity of replication. This suppression was also observed under conditions where the mutant TrfA protein was provided in trans with respect to oriV, indicating that the reduction in temperature sensitivity could not be a TrfA protein dosage effect. The phenotypes of two of the cop mutants in Pseudomonas aeruginosa were analyzed; the results demonstrated that the mutants were either not functional or poorly functional in this host. The rts mutant plasmids were also reduced in their ability to replicate in P. aeruginosa, and the intragenic cop mutations did not improve the functionality of these mutants. The significance of the results is discussed in relation to current models of the mechanism of action of the TrfA protein.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos/genética , Replicón/genética , Supresión Genética , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Mapeo Cromosómico , Genes Reguladores/genética , Calor , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Plásmidos/genética , Pseudomonas aeruginosa/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
All reported natural isolates of the marine fish pathogen Vibrio salmonicida contain plasmids, and in another marine fish pathogen, Vibrio anguillarum, it has been shown that a plasmid is important for expression of virulence by the organism. To study the function of the plasmids in V. salmonicida, we developed a gene transfer system based on the plasmid RSF1010 replicon. The gene transfer system was used to construct a plasmid-free strain, and this strain was found to behave similarly to the wild type in a fish pathogenicity test based on intraperitoneal injection of the bacteria. We were unable to detect any other phenotypic differences between the two strains. It could therefore be concluded that at least in the V. salmonicida strain tested, extrachromosomal DNA is not required for expression of virulence.
Asunto(s)
Plásmidos , Transfección , Vibrio/genética , Animales , Mapeo Restrictivo , Salmón/microbiología , Serotipificación , Vibrio/clasificación , Vibrio/patogenicidad , Virulencia/genéticaRESUMEN
Two small plasmid RK2 derivatives, pSV6 and pSV16, were constructed and used for the isolation and characterization of trfA mutants temperature-sensitive (ts) for replication in Escherichia coli. Four of the mutants were examined for their ability to initiate replication from the RK2 replication origin in E. coli when present in cis with respect to the origin and in trans when present on a multicopy pBR322 replicon. Each of the mutant trfA genes exhibited temperature-sensitivity in supporting replication from the RK2 origin when present in cis, and the lowest nonpermissive temperature varied depending on the mutant. When the mutant trfA genes were present on the multicopy replicon (in trans), three of the four mutant genes could support replication of the RK2-oriV plasmid pSV16 at all temperatures tested. However, with the exception of one of the mutants, the activity was reduced when compared to wild-type. The increased activity in trans possibly is the result of the increased cellular level of the TrfA protein when compared with the in cis situation where the mutant trfA gene is at a much lower copy-number. Two of the mutants also were tested in cis for temperature sensitivity in Pseudomonas aeruginosa. One of the mutants did not exhibit temperature sensitivity under the conditions employed. The second mutant showed some temperature sensitivity but the nonpermissive temperature pattern was different than that found in E. coli.