Development and evaluation of a novel pre-operative surgery school and behavioural change intervention for patients undergoing elective major surgery: Fit-4-Surgery School.

Group pre-operative education has usually been limited to conditioning expectations and providing education. Prehabilitation has highlighted modifiable lifestyle factors that are amenable to change and may improve clinical outcomes. We instituted a pre-operative 'Fit-4-Surgery School' for patients scheduled for major surgery, to educate and promote healthy behaviour. We evaluated patients' views having attended the school, and after surgery we asked how it had changed their behaviour with a lifestyle questionnaire. The school was launched in May 2016 and was attended by 586/1017 (58%) of invited patients. Patients who did not attend: lived further away, median (IQR [range]) 8 (4-19 [0-123]) miles vs. 5 (3-14 [0-172]) miles, p < 0.001; and were more deprived, Index of Multiple Deprivation Rank decile median (IQR [range]), 6 (4-8 [1-10]) vs. 7 (4-9 [1-10]), p = 0.04. Of the 492/586 (84%) participants who completed an evaluation questionnaire, 462 (94%) would recommend the school to a friend having surgery and 296 (60%) planned lifestyle changes. After surgery, 232/586 (40%) completed a behavioural change questionnaire, 106 (46%) of whom reported changing at least one lifestyle factor, most commonly by increasing exercise. The pre-operative school was acceptable to patients.

Factors associated with first thrombosis in patients presenting with obstetric antiphospholipid syndrome (APS) in the APS Alliance for Clinical Trials and International Networking Clinical Database and Repository: a retrospective study.

OBJECTIVE: To evaluate the subsequent rate of thrombosis among women with obstetric antiphospholipid syndrome (Ob-APS) in a multicentre database of antiphospholipid antibody (aPL)-positive patients, and the clinical utility of the adjusted Global Antiphospholipid Syndrome Score (aGAPSS), a validated tool to assess the likelihood of developing new thrombosis, in this group of patients. DESIGN: Retrospective study. SETTING: The Antiphospholipid Syndrome Alliance for Clinical Trials and International Networking Clinical Database and Repository. POPULATION: Women with Ob-APS. METHODS: Comparison of clinical and laboratory characteristics and measurement of aGAPSS in women with Ob-APS, with or without thrombosis, after initial pregnancy morbidity (PM). MAIN OUTCOME MEASURES: Risk factors for thrombosis and aGAPSS. RESULTS: Of 550 patients, 126 had Ob-APS; 74/126 (59%) presented with thrombosis, and 47 (63%) of these women developed thrombosis after initial PM, in a mean time of 7.6 ± 8.2 years (4.9/100 patient years). Younger age at diagnosis of Ob-APS, additional cardiovascular risk factors, superficial vein thrombosis, heart valve disease, and multiple aPL positivity increased the risk of first thrombosis after PM. Women with thrombosis after PM had a higher aGAPSS compared with women with Ob-APS alone [median 11.5 (4-16) versus 9 (4-13); P = 0.0089]. CONCLUSION: Based on a retrospective analysis of our multicentre aPL database, 63% of women with Ob-APS developed thrombosis after initial obstetric morbidity; additional thrombosis risk factors, selected clinical manifestations, and high-risk aPL profile increased the risk. Women with subsequent thrombosis after Ob-APS had a higher aGAPSS at entry to the registry. We believe that aGAPSS is a valid tool to improve risk stratification in aPL-positive women. TWEETABLE ABSTRACT: More than 60% of women with obstetric antiphospholipid syndrome had thrombosis after initial pregnancy morbidity.

Hydroxychloroquine in the primary thrombosis prophylaxis of antiphospholipid antibody positive patients without systemic autoimmune disease.

Objective The objective of this study was to determine the efficacy of hydroxychloroquine (HCQ) in the primary thrombosis prevention of antiphospholipid antibody (aPL)-positive patients with no other systemic autoimmune diseases. Methods Under the auspices of Antiphospholipid Syndrome Alliance for Clinical Trials and International Networking, a multicenter, international, randomized controlled trial (RCT) was initiated, in which persistently aPL-positive but thrombosis-free patients without systemic autoimmune diseases were randomized to receive HCQ or no treatment in addition to their standard regimen. The primary objective was the efficacy of HCQ in preventing the first thrombosis. The secondary objectives were the thrombosis incidence rate, and the effects of HCQ on aPL profile and mortality rate. Patients were risk-stratified based on antiplatelet agent use. The goal was to follow patients every 6 months for 5 years. Results We recruited 20 persistently aPL-positive patients (female: 19, mean age: 46.6 ± 9.9 years, and baseline antiplatelet medication: 14); 9/20 were randomized to HCQ. During the mean follow-up of 1.7 years, no patients developed thrombosis or a serious adverse event. The study was terminated early due to the low recruitment rate, exacerbated by the prolonged manufacturing shortage and significant price increase of HCQ in the United States. Conclusion Given that a small number of patients with a relatively short follow-up were enrolled in our RCT, and no patients developed thrombosis, we cannot accurately assess the effectiveness of HCQ for primary thrombosis prevention in persistently aPL-positive patients with no other systemic autoimmune diseases. Our experience suggests that conducting an international RCT, especially without pharmaceutical support, is an extremely challenging undertaking.

Bilateral versus unilateral botulinum toxin injections for chronic anal fissure: a randomised trial.

BACKGROUND: Botulinum toxin injected into the internal anal sphincter is used in the treatment of chronic anal fissure but there is no standardised technique for its administration. This randomised single centre trial compares bilateral (either side of fissure) to unilateral injection. METHODS: Participants were randomised to receive bilateral (50 + 50 units) or unilateral (100 units) Dysport® injections into the internal anal sphincter in an outpatient setting. Injection-related pain assessed by visual analogue scale was the primary outcome measure. Secondary outcomes were healing rate, fissure pain, incontinence, and global health scores. RESULTS: Between October 2008 and April 2012, 100 patients with chronic anal fissure were randomised to receive bilateral or unilateral injections. Injection-related pain was comparable in both groups. There was no difference in healing rate. Initially, there was greater improvement in fissure pain in the bilateral group but at 1 year the unilateral group showed greater improvement. Cleveland Clinic Incontinence score was lower in the unilateral group in the early post-treatment period and global health assessment (EuroQol EQ-VAS) was higher in the unilateral group at 1 year. CONCLUSIONS: Injection-related pain was similar in bilateral and unilateral injection groups. Unilateral injection was as effective as bilateral injections in healing and improving fissure pain without any deterioration in continence.

Molecular and biochemical characterisation and recognition by the immune host of the enolase of the abomasal nematode parasite Teladorsagia circumcincta.

A 1299 bp full length cDNA encoding Teladorsagia circumcincta enolase (TeciENO) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. Helminth enolase sequences were used to construct a phylogenetic tree. The predicted protein consisted of 433 amino acids and was present as a single band of about 50 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TeciENO with homologues from other helminths showed 98% similarity with Haemonchus contortus enolase, 78-95% similarity to other nematode sequences and 72-75% similarity to cestode and trematode enolases. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. The optimum pH for TeciENO activity at 25 °C was pH 7, the Km for 2-phophoglycerate 0.09 ± 0.04 mM and the Vmax was 604 ± 6 nmol min-1 mg-1 protein (both mean ± SD, n = 2). TeciENO activity was inhibited by 11.5% by 1 mM citrate (p < 0.001). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TeciENO in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native enolase indicates similar antigenicity of the two proteins.

Molecular and biochemical characterisation and recognition by the immune host of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of the abomasal nematode parasite Teladorsagia circumcincta.

A 1023 bp full length cDNA encoding Teladorsagia circumcincta GAPDH (TeciGAPDH) was cloned, expressed in Escherichia coli and the recombinant protein purified and its kinetic properties determined. A phylogenetic tree was constructed using helminth GAPDH sequences. The predicted protein consisted of 341 amino acids and was present as a single band of about 38 kDa on SDS-PAGE. Multiple alignments of the protein sequence of TeciGAPDH with homologues from other helminths showed that the greatest similarity (93%) to the GAPDH of Haemonchus contortus and Dictyocaulus viviparus, 82-86% similarity to the other nematode sequences and 68-71% similarity to cestode and trematode enzymes. Substrate binding sites and conserved regions were identified and were completely conserved in other homologues. At 25 °C, the optimum pH for TeciGAPDH activity was pH 8, the Vmax was 1052 ± 23 nmol min-1 mg-1 protein and the apparent Km for the substrate glyceraldehyde-3-phosphate was 0.02 ± 0.01 mM (both mean ± SD, n = 2). Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TeciGAPDH in enzyme-linked immunosorbent assays. The recognition of the recombinant protein by antibodies generated by exposure of sheep to native GAPDH indicates similar antigenicity of the two proteins.

Immunomodulatory effects of adult Haemonchus contortus excretory/secretory products on human monocyte-derived dendritic cells.

The levels of expression of surface molecules and release of cytokines and chemokines of human monocyte-derived dendritic cells were determined after their exposure to adult H. contortus excretory/secretory (ES) products or a combination of ES products and bacterial lipopolysaccharide (LPS). Worm products provoked a weak response and only partial maturation of the dendritic cells, consistent with the hyporesponsiveness and more tolerogenic immune environment present in parasitized animals and humans. Co-stimulation with LPS demonstrated that H. contortus secretions, like those of other helminths, contain immunomodulators capable of reducing some aspects of the strong T(H)1/T(H)2 response evoked by bacterial LPS. There were significant reductions in the release of some cytokine/chemokines by LPS-stimulated mdDCs and a trend (although not significant at P < 0.05) for reduced expression levels of CD40, CD80 and HLA-DR. A prominent feature was the variability in responses of dendritic cells from the four donors, even on different days in repeat experiments, suggesting that generalized conclusions may be difficult to make, except in genetically related animals. Such observations may therefore be applicable only to restricted populations. In addition, previous exposure to parasites in a target population for immunomodulatory therapy may be an important factor in assessing the likelihood of adverse reactions or failures in the treatment to worm therapy.

Molecular and biochemical characterisation of abomasal nematode parasites Teladorsagia circumcincta and Haemonchus contortus phosphofructokinases and their recognition by the immune host.

Full length cDNAs encoding phosphofructokinase (PFK) were cloned from Teladorsagia circumcincta (TcPFK) and Haemonchus contortus (HcPFK). TcPFK (2361 bp) and HcPFK (2367 bp) cDNA encoded 787 and 789 amino acid proteins respectively. The predicted amino acid sequences showed 98% similarity with each other and 70% with a Caenorhabditis elegans PFK. Substrate binding sites were completely conserved in both proteins. Soluble N-terminal His-tagged PFK proteins were expressed in Escherichia coli strain BL21, purified and characterised. The recombinant TcPFK and HcPFK had very similar kinetic properties: the pH optima were pH 7.0, Km for fructose 6-phosphate was 0.50 ± 0.01 and 0.55 ± 0.01 mM respectively when higher (inhibiting concentration, 0.3 mM) ATP concentration was used and the curve was sigmoidal. The Vmax for TcPFK and HcPFK were 1110 ± 16 and 910 ± 10 nM min(-1 )mg(-1) protein respectively. Lower ATP concentration (non-inhibiting, 0.01 mM) did not change the Vmax for TcPFK and HcPFK (890 ± 10 and 860 ± 12 nM min(-1 )mg(-1) protein) but the substrate affinity doubled and Km for fructose 6-phosphate were 0.20 ± 0.05 and 0.25 ± 0.01 mM respectively. Recognition of TcPFK and HcPFK by mucosal and serum antibodies in nematode exposed animals demonstrates antigenicity and suggests involvement in the host response to nematode infection.

Molecular and biochemical characterisation of arginine kinases in Haemonchus contortus and Teladorsagia circumcincta.

Full length cDNA encoding arginine kinases (AK) were cloned from Teladorsagia circumcincta (TcAK) and Haemonchus contortus (HcAK). The TcAK and HcAK cDNA (1080 bp) encoded 360 amino acid proteins. The predicted amino acid sequence showed 99% similarity with each other and 94% with a Caenorhabditis elegans AK. Soluble N-terminal His-tagged AK proteins were expressed in Escherichia coli strain BL21, purified and characterised. All binding sites were completely conserved in both proteins. The recombinant TcAK and HcAK had very similar kinetic properties: K(m) arginine was 0.35 mM, K(m) ATP was 0.8-0.9 mM and the pH optima were pH 7.5. Arginine analogues strongly inhibited recombinant enzyme activities (up to 80%), whilst other amino acids decreased activities by a maximum of 20%. TcAK and HcAK are potential vaccine candidates because of the strong antigenicity of invertebrate phosphagens and kinases and presence in metabolically active parts of the worm.

Sarcosine metabolism in Haemonchus contortus and Teladorsagia circumcincta.

Sarcosine (N-methylglycine) is an intermediate in glycine degradation and can also be synthesised from glycine in mammals. Sarcosine metabolism in Haemonchus contortus and Teladorsagia circumcincta differed from that of mammals in that creatinase activity was present and sarcosine was demethylated only by sarcosine oxidase (SOX) and not by sarcosine dehydrogenase (SDH). The mean SOX activity was 30 nmolmin(-1)mg(-1) protein in homogenates of L3 and adult worms of both parasites and the apparent Km for sarcosine was 1.1 mM. Addition of 2 mM Cd(2+) inhibited activity by 30%. There was no SDH activity with either NAD(+) or NADP(+) as co-factor. Mean creatinase activity in L3 T. circumcincta and adult worms of both species was 31±6 nmolmin(-1)mg(-1) protein, but was undetectable in L3 H. contortus. Activity was inhibited by up to 70% by Cu(2+), Fe(2+), Fe(3+) and Zn(2+). Possessing creatinase would allow host creatine to be incorporated into amino acids by the parasites.

A meta-analysis of the effectiveness of the opioid receptor antagonist alvimopan in reducing hospital length of stay and time to GI recovery in patients enrolled in a standardized accelerated recovery program after abdominal surgery.

BACKGROUND: Despite accelerated recovery programs and the widespread uptake of laparoscopic surgery, postoperative ileus remains a significant factor affecting length of stay after abdominal surgery. Alvimopan, an opioid-receptor antagonist, may reduce the incidence of postoperative ileus and expedite hospital discharge. OBJECTIVE: The aim of this study was to perform a meta-analysis to determine the role of alvimopan in accelerating GI recovery and hospital discharge after laparoscopic and open abdominal surgery performed within an accelerated recovery program. DATA SOURCES AND STUDY SELECTION: Cochrane (1999-2010), Embase (1980-2010), MEDLINE (1980-2010), and International Pharmaceutical Abstracts (1970-2010) were searched for relevant double-blinded, randomized controlled trials. INTERVENTIONS: Twelve milligrams of alvimopan and placebo were given to patients enrolled in an accelerated recovery program after abdominal surgery. MAIN OUTCOME MEASURES: The primary outcomes measured were the length of stay as defined by the writing of the hospital discharge order and GI-3 and GI-2 GI tract recovery. RESULTS: : Three trials were included that reported on a pooled modified intention-to-treat population of 1388 patients; 685 (49%) patients received alvimopan. On meta-analysis, alvimopan reduced time to the hospital discharge order (HR 1.37 (1.21, 1.62), p < 0.0001), GI-3 recovery (HR 1.42 (1.25, 1.62), p < 0.001), and GI-2 recovery (HR 1.49 (1.32, 1.68), p < 0.0001). LIMITATIONS: The search criteria identified only a small number of trials of alvimopan after abdominal surgery with no randomized trials of alvimopan after laparoscopic surgery. In addition, the use of length of hospital stay as the primary outcome measure may be inappropriate, because it is open to many confounding factors. Finally, adverse events, in particular, adverse cardiovascular events, were not considered. CONCLUSIONS: Alvimopan 12 mg can further reduce time to GI recovery and hospital discharge in patients undergoing abdominal surgery within an accelerated recovery program. Investigation into the effect of alvimopan following laparoscopic surgery and additional cost-benefit analyses are required to further define the role of this intervention.

A prospective case-control study of extralevator abdominoperineal excision (ELAPE) of the rectum versus conventional laparoscopic and open abdominoperineal excision: comparative analysis of short-term outcomes and quality of life.

BACKGROUND: Conventional abdominoperineal excision (APE) of the rectum is associated with higher circumferential resection margin (CRM) involvement, increased local recurrence, and reduced survival compared to anterior resection. A more radical extralevator APE (ELAPE) technique may improve oncological outcome. However, this technique may confer additional morbidity, and little comparative data on short-term outcomes have been reported. This study compares short-term outcomes and quality of life (QOL) after open and laparoscopic ELAPE, laparoscopic APE (LAPE), and open APE (OAPE). METHODS: Data on all ELAPE and 10 consecutive LAPE and OAPE were extracted from a prospective database. Perioperative care and follow-up were standardized. QOL was assessed using EORTC questionnaires. RESULTS: Sixteen ELAPE (14 laparoscopic), 10 LAPE, and 10 OAPE were included. Demographics, tumour stage, and neoadjuvant therapy use were comparable. Operative time was higher with ELAPE than LAPE and OAPE (295, 207.5, and 157.5 min, respectively, p = 0.01). A porcine collagen perineal mesh was used in 9 patients undergoing ELAPE but in no LAPE or OAPE patients. No difference in 30-day complications, re-admission, or length of stay was noted. ELAPE and LAPE were associated with earlier removal of urinary catheter (p = 0.02), yet other enhanced recovery after surgery (ERAS) parameters were equivalent. All ELAPE resections were R0 with no positive CRM identified. One LAPE and 2 OAPE were R1 resections. Analysis revealed no deterioration in QOL with ELAPE, with equivalent global health status. CONCLUSIONS: The results of this study suggest that ELAPE is not associated with deterioration in short-term outcomes or QOL when compared with LAPE or OAPE.

Molecular and biochemical characterisation of a Teladorsagia circumcincta glutamate dehydrogenase.

A full length cDNA encoding glutamate dehydrogenase was cloned from Teladorsagia circumcincta (TcGDH). The TcGDH cDNA (1614 bp) encoded a 538 amino acid protein. The predicted amino acid sequence showed 96% and 93% similarity with Haemonchus contortus and Caenorhabditis elegans GDH, respectively. A soluble N-terminal 6xHis-tagged GDH protein was expressed in the recombinant Escherichia coli strain BL21 (DE3) pGroESL, purified and characterised. The recombinant TcGDH had similar kinetic properties to those of the enzyme in homogenates of T. circumcincta, including greater activity in the aminating than deaminating reaction. Addition of 1mM ADP and ATP increased activity about 3-fold in the deaminating reaction, but had no effect in the reverse direction. TcGDH was a dual co-factor enzyme that operated both with NAD(+) and NADP(+), GDH activity was greater in the deaminating reaction with NADP(+) as co-factor and more with NADH in the aminating reaction.

National UK audit of procedure for prolapsing haemorrhoids on behalf of the Association of Coloproctology of Great Britain and Ireland.

OBJECTIVE: To assess the safety and short term outcomes of the procedure for prolapsing haemorrhoids (PPH), a relatively new procedure for the treatment of symptomatic haemorrhoids. METHOD: In 2005, the Association of Coloproctology of Great Britain and Ireland set up an online electronic database to audit the indications and outcomes for patients undergoing a PPH procedure. RESULTS: During the audit period, 695 patients were entered onto the database by 61 surgeons (range 1-50 patients per surgeon). The main indications for surgery were bleeding (90.5%) and prolapse (83.9%). Three hundred and ninety-seven (57.1%) patients had grade III or IV haemorrhoids. PPH was performed under general anaesthetic in 602 (86.6%) cases and a consultant surgeon performed the procedure in 572 (82.3%) cases. The median length of stay was 1 day (range 0-6 days). Two hundred and eighty-nine (41.6%) procedures were performed as a day case. Immediate complications were recorded in 75 (10.8%) patients, the commonest being bleeding (21) and urinary retention (24). At 6-week follow-up, 626 (90.1%) patients were pain free. Five patients required hospital re-admission for secondary haemorrhage (3), peri-anal abscess (1) and pain (1). The commonest problems were minor bleeding (48), urgency (22), pain (14), continued prolapse (12) and pruritus (11). Four patients required an open haemorrhoidectomy for persistent symptomatic haemorrhoids. CONCLUSION: Procedure for PPH is a safe and effective procedure for symptomatic haemorrhoids with good short-term outcomes. Long-term follow-up is required perhaps through a compulsory national register.

Use of fluorescent lectin binding to distinguish eggs of gastrointestinal nematode parasites of sheep.

The binding of a panel of 19 lectins to carbohydrates on the eggs of economically important nematode parasites of sheep has been assessed as the basis of a rapid test to distinguish parasite eggs, at least at the genus level. A total of six lectins can be used to identify eggs of six nematode parasites: peanut agglutinin (PNA) for Haemonchus contortus; Lens culinaris agglutinin (LCA) for Teladorsagia sp; Aleuria aurantia agglutinin (AAL) for Trichostrongylus sp; Psophocarpus tetragonolobus­II (PTLII) for Nematodirus sp; Lotus tetragonolobus lectin (LTL) for Cooperia sp and wheat germ agglutinin (WGA) for Chabertia ovina. For WGA, LCA and LTL, weak binding was also observed to H. contortus and Teladorsagia sp, Trichostrongylus sp and C. ovina eggs, respectively. Nematode eggs in two faecal samples were identically identified by both lectin binding and PCR, except for PCR identification of the eggs of Nematodirus sp, as these did not lyse. Lectins bound best to H. contortus eggs extracted from fresh faecal samples or after storage at room temperature or 4 °C for up to 24 h, but eggs stored at -20 °C or -80 °C did not bind PNA.

Excretory/secretory products of adult Haemonchus contortus and Teladorsagia circumcincta which increase the permeability of Caco-2 cell monolayers are neutralised by antibodies from immune hosts.

The onset of abomasal pathophysiology due to parasitism coincides with the presence of adult worms in the lumen, implicating worm excretory/secretory (ES) products acting on the surface mucosa. Caco-2 cell monolayers were grown to confluence on Transwell plates and exposed on the apical side to ES products of adult Haemonchus contortus and Teladorsagia circumcincta. ES products of both species significantly (p<0.001) reduced transepithelial electrical resistance after 2h to 81.1±1.0% and 82.9±1.1% respectively. Immunocytochemical staining of the Caco-2 monolayers for zona occludens-1 and occludin confirmed that the tight junctions remained intact in control medium, but these proteins were internalised from disrupted junctions after exposure to ES products. The components of H. contortus ES products responsible for increased epithelial permeability were partially blocked by phage displaying single chain antibodies derived from sheep immune to field infection and enriched by panning with H. contortus ES products. Immune hosts may therefore be able to reduce the effects of worm chemicals on the gastric epithelium. Permeabilisation of the abomasal surface mucosa by worm chemicals would also explain how cells deep in the gastric glands could rapidly be affected by parasites emerging from the glands or within a day of transplantation of adult worms into naïve hosts, resulting in the pathophysiology typically caused by abomasal nematode parasitism.

Phenotypic characterization of a comprehensive set of bicyclomycin-resistant mutants.

A comprehensive set of bicyclomycin-resistant mutants of transcription termination protein Rho has been characterized in Escherichia coli by in vivo and in vitro assays. Several of the mutant Rho proteins have functional defects. Strains with either the L208R or the S266A mutation in the bacterial chromosome have a higher intracellular concentration of the Rho protein than strains containing a wild-type copy of the rho gene. Strains carrying the L187R, L208R or S266A mutations in the chromosome also have a mutant phenotype; a plasmid-located arabinose promoter is constitutively de-repressed in these strains. The L208R and S266A mutant strains also have a rate of growth defect. When the S266A mutation is located on a high-copy plasmid, the mutant grows more slowly than a wild-type strain. In contrast to the majority of the bicyclomycin-resistant mutants, these two mutants show clear phenotypic differences from wild-type cells. These differences are also seen in vitro. In vitro transcription termination by RhoL208R and RhoS266A is defective at the lambda tR1 terminator, but can be enhanced by NusG. These functionally defective Rho mutations have been located near the putative catalytic site on a model of Rho based on the F1-ATPase. This indicates that this region of the Rho molecule is crucial for Rho function. The crucial region overlaps the putative bicyclomycin-binding site, suggesting an explanation for the efficacy of bicyclomycin as an antibiotic.

The bicyclomycin sensitivities of 38 bicyclomycin-resistant mutants of transcription termination protein rho and the location of their mutations support a structural model of rho based on the F(1) ATPase.

A total of 38 bicyclomycin-resistant mutants of Escherichia coli transcription termination protein Rho have been isolated. The locations of their mutations identify the ATP-binding region as the functional domain inhibited by bicyclomycin. Strains containing the S266C, S266A and L208R Rho mutations are very resistant to bicyclomycin in vivo. In a similar way, the mutant Rho proteins containing these mutations are very resistant to bicyclomycin in vitro. These data suggest that Ser266 and Leu208 might make direct contact with the antibiotic. These two residues are close to each other in the tertiary structure of a model of Rho based on the alpha and beta subunits of the F(1) ATPase, supporting the validity of the model. The strain containing the G337S Rho mutation also has high bicyclomycin resistance, and the proximity of L208, S266 and G337 in the quaternary structure of the Rho model has enabled a candidate bicyclomycin-binding pocket to be delineated. As a whole, the bicyclomycin sensitivities of the mutants are consistent with the locations of their respective mutations in the model of Rho based on the F(1) ATPase, therefore supporting the emerging consensus model of Rho structure.

Alteration of the Amount of the Chloroplast Phosphate Translocator in Transgenic Tobacco Affects the Distribution of Assimilate between Starch and Sugar.

Tobacco plants (Nicotiana tabacum L.) transformed with sense and antisense constructs of a cDNA encoding the tobacco phosphate-triose phosphate-3-phosphoglycerate translocator (phosphate translocator) were shown to contain altered amounts of phosphate translocator mRNA and protein. Phosphate translocator activity in intact chloroplasts isolated from transformed plants showed a 15-fold variation, from 20% of the wild-type activity in antisense transformants to 300% of the wild-type activity in sense transformants. However, the maximal rates of photosynthesis and the rates of photosynthetic carbon assimilation in ambient CO2 showed no consistent differences between transformants. Starch content was decreased by 20% and total soluble sugars were increased by 20% in leaves of antisense transformants compared to sense transformants. The 40% decrease in the ratio of starch to total soluble sugars in antisense transformants relative to sense transformants indicates that distribution of assimilate between starch and sugar had been altered. However, the amount of sucrose in the leaves was unchanged. The changes in total soluble sugars were accounted for completely by changes in glucose and fructose, suggesting the existence of a homeostatic mechanism for maintaining sucrose concentrations in the leaves at the expense of glucose and fructose.

Manipulating photosynthesis.

The levels of individual photosynthetic proteins can be independently decreased by the Agrobacterium-mediated transformation of plants with antisense RNA constructs. Protocols for the introduction of such constructs into Agrobacterium, the Agrobacterium-mediated transformation of tobacco leaf disks, and the screening and analysis of the transgenic plants produced are described.