A One-Day Sensitive Method to Detect and Distinguish Between the Citrus Black Spot Pathogen Guignardia citricarpa and the Endophyte Guignardia mangiferae.

If South African citrus exporters wish to retain their competitive edge in the European market and access new markets such as the United States of America, it is of quarantine importance to distinguish between the citrus black spot pathogen, Guignardia citricarpa, and the harmless endophyte, G. mangiferae. The endophyte is not a sanitary or phytosanitary concern. This paper describes the design of species-specific primers that are able to detect and distinguish between these two Guignardia species. Application of the primer set CITRIC1 and CAMEL2 in conjunction with the ITS4 primer yielded polymerase chain reaction (PCR) amplicons of approximately 580 bp and 430 bp for G. citricarpa and G. mangiferae, respectively. Results obtained with these primers are in accordance with sequence data, and repeated tests verified accuracy and sensitivity. A BLAST search revealed no matches other than G. citricarpa and G. mangiferae, and no positive PCR results were obtained with Colletotrichum gloeosporioides, which is the most common contaminant in black spot lesions. We are, therefore, able to distinguish G. citricarpa and G. mangiferae unequivocally using a PCR-based method. This method was further improved to directly isolate DNA from fruit lesions by means of the DNeasy Plant Mini Kit (Qiagen). This eliminates the prior need for culturing the slow-growing organism, thereby shortening the time required to one day to test for and verify the presence or absence of the pathogenic G. citricarpa in export consignments.

Comparison of cross inoculation potential of South African avocado and mango isolates of Colletotrichum gloeosporioides.

Three hundred and eighteen Colletotrichum gloeosporioides isolates from stem-end rot and anthracnose infected avocados as well as from stem-end rot/anthracnose and soft brown rot on mango, were compared using fruit inoculations. Isolates could be categorised according to lesion size and both avocado and mango isolates produced larger lesions when inoculated on their own hosts. Cross-inoculation potential of these isolates was also compared on strawberries, peppers, guavas, papayas and citrus. All isolates produced lesions on all hosts except citrus. Factors such as area of origin and symptom type from which original isolations were made, could not be correlated with lesion development on these hosts.

Phorbol dibutyrate-induced megakaryocytic differentiation increases susceptibility of K562 cells to SA11 rotavirus infection.

The role of integrins previously implicated as rotavirus receptors in determining cellular susceptibility to SA11 rotavirus was studied, using phorbol dibutyrate (PDB) treatment of K562 cells to induce megakaryocytic differentiation. Expression of alpha2beta1 integrin was detected after 2 days in PDB, and peaked after PDB treatment for 4-7 days. SA11 titres were increased by 1.8- to 10.8-fold over untreated cells after PDB treatment for 2-7 days, and correlated with levels of alpha2beta1 integrin expression in PDB-treated K562 cells.

Use of computerized tomography and chest x-rays in evaluating efficacy of aerosolized recombinant human DNase in cystic fibrosis patients younger than age 5 years: a preliminary study.

The aim of this study was to evaluate the ability of high-resolution computerized tomography (HRCT) of the chest and chest x-rays (CXR) to determine efficacy of inhaled recombinant human DNase (rhDNase) in cystic fibrosis (CF) patients younger than 5 years of age. A randomized, double-blind, placebo-controlled pilot study of 12 patients with CF younger than 5 years of age, attending the University of Michigan Cystic Fibrosis Center (Ann Arbor, MI) was conducted. The changes in the HRCT and CXR score from baseline to day 100 of therapy were assessed using a previously validated scoring system. The mean changes of HRCT scores between the rhDNase and placebo groups were found to be significant at the 95% level, with mean change +/- SE mean of - 1.00 +/- 0.53 and 0.58 +/- 0.24 for rhDNase and placebo groups, respectively (P = 0.02). The difference in CXR score was not significant between the two groups. An analysis was performed to relate HRCT subscores to CXR score; only thickening of the intra-interlobular septae was significantly correlated with the total CXR score (r = - 0.7, P < 0.01). There was improvement in the parents' assessments of the patients' well-being, with improvement in physical activity, decreased cough, sleep quality, and appetite in those subjects receiving rhDNase. We conclude that the administration of rhDNase was associated with improvement in the HRCT scan in CF patients younger than 5 years of age. Findings indicate that HRCT of the chest is useful and sensitive in studying responses to therapy in patients with CF lung disease. To our knowledge, this is the first report of the use of HRCT to assess the effectiveness of a therapeutic modality in so young a CF patient population.

Caudal ropivacaine and ketamine for postoperative analgesia in children.

In a prospective, randomised, double-blind clinical study, we studied 32 ASA grade I and II boys aged 18 months to 12 years, scheduled for circumcision under general anaesthesia on an outpatient basis. They were randomly allocated to one of two groups: those in the ropivacaine group received caudal ropivacaine 0.2% 1 ml. kg-1 for postoperative analgesia and those in the ketamine/ropivacaine group received caudal ropivacaine 0.2% 1 ml. kg-1 plus caudal ketamine 0.25 mg.kg-1. Postoperative pain was assessed using a modified 10-cm visual analogue scale and analgesia was administered if the pain score exceeded a value of 3. The median duration of analgesia was significantly longer in the ketamine/ropivacaine group (12 h) than in the ropivacaine group (3 h, p < 0.0001), and subjects in the ropivacaine group required significantly more doses of postoperative analgesia than those in the ketamine/ropivacaine group (p < 0.0001). There were no differences between the groups in the incidence of postoperative nausea, vomiting, sedation, emergence delirium, nightmares, hallucinations, motor block and urinary retention.

Radial nerve injury after intravenous cannulation at the wrist--a case report.

Peripheral venous cannulation is one of the commonest procedures performed in hospitals. The dorso-lateral aspect of the wrist is one of the favourite sites. Radial nerve injury, though extremely rare, can be a serious complication and has been reported twice. One patient was left with a permanent work disability due to a painful neuroma. Another patient required surgical intervention to remove a neuroma six months after the initial venous cannulation resulting in almost complete recovery. We report the first case of injury to the radial nerve at the wrist as a complication of venous cannulation where complete recovery occurred spontaneously. In our case, immediate removal of the cannula may be responsible for the improved outcome.

The reinforced laryngeal mask in paediatric outpatient dental surgery.

One hundred and twenty ASA I and II grade children aged 2-9 years scheduled for outpatient dental extractions under general anaesthesia were studied. They were allocated randomly to one of three groups for airway management: group R had anaesthesia with a reinforced laryngeal mask airway, group L with a standard laryngeal mask airway and group N with a nasal mask. Anaesthesia was induced in all children using halothane in 50% nitrous oxide with oxygen and maintained on halothane in 67% nitrous oxide with oxygen. An Ayre's T-piece with Jackson-Rees modification was used. The incidence of airway obstruction was significantly lower and surgical access significantly better with the reinforced laryngeal mask airway when compared with the standard laryngeal mask airway. However, the reinforced laryngeal mask airway was significantly more difficult to insert when compared with the standard laryngeal mask airway. On comparing the reinforced laryngeal mask airway with the nasal mask, there were significantly fewer episodes of airway obstruction, better oxygen saturation, less increase in heart rate and fewer arrhythmias in the reinforced laryngeal mask airway group. Total time for the procedures was the same for all three groups. Thus, the reinforced laryngeal mask airway was found to be a favourable alternative to the standard laryngeal mask airway and nasal mask for paediatric outpatient dental extractions.

Tracheal schwannoma presenting as status asthmaticus in a sixteen-year-old boy: airway considerations and removal with the CO2 laser.

A 16-year-old with clinical features of atypical asthma is presented, with a description of the workup leading to the diagnosis of an intratracheal mass. The mass was visualized with a flexible fiberoptic bronchoscope, then surgically removed through a rigid bronchoscope using a CO2 laser. We believe this is the first report of resection using this technique. A discussion of tracheal neurilemmomas (schwannoma) is included. This case reinforces the age-old adage that "not all that wheezes is asthma."

Is it feasible to use magnesium sulphate as a hypotensive agent in oral and maxillofacial surgery?

We report the results of a feasibility study using intravenous magnesium sulphate for deliberate hypotension in 16 ASA 1 patients undergoing major oral and maxillofacial surgery. All the patients received a standard nitrous oxide, oxygen, isoflurane, opioid and muscle relaxant anaesthetic. Magnesium sulphate was infused at 40 g/h until the mean arterial pressure reached 55 +/- 5 mmHg, followed by a maintenance dose of 5 g/h until 30 minutes prior to the end of surgery. The mean arterial pressure was significantly (P < 0.01) reduced by the magnesium sulphate when compared to baseline values. Control of the mean arterial pressure was satisfactory. No patient had reflex tachycardia, cardiac arrhythmia or rebound hypertension. In 14 patients the surgeons thought that the blood loss was less than when using other hypotensive anaesthetic techniques. In 2 patients the surgeons thought the blood loss was excessive. In another 2 patients, the surgeons thought that there was excessive facial swelling on completion of surgery. Postoperative muscle weakness and sedation were not problems clinically. Fourteen patients were extubated immediately after surgery and another 2 patients an hour later in the recovery room. Intraoperative urine output was well maintained. On completion of surgery, the prothrombin time was significantly increased (P < 0.05), and the partial thromboplastin time significantly decreased (P < 0.05) in all the patients (when compared to preoperative values); the clinical significance of this is unclear. The use of intravenous magnesium sulphate for deliberate hypotension is feasible in ASA 1 patients using a standard nitrous oxide, oxygen, isoflurane, opioid and muscle relaxant technique. This study forms the basis for a larger controlled study where the issues of postoperative sedation and weakness and coagulopathy can be dealt with in greater detail.

Dual targets of a transcriptional activator that tracks on DNA.

The sliding clamp of the bacteriophage T4 DNA polymerase, gp45, is also the proximal effector for activation of transcription of T4 late genes. We have identified the phage T4-encoded sigma factor gp55 and the co-activator gp33 as targets of gp45 in promoter complexes, and have shown that a conserved carboxy-terminal amino acid sequence of gp55 and gp33 is required for interaction with gp45. The respective contribution of each target-gp45 interaction to activation of transcription has been assessed by measuring promoter opening rates. The opening rate supported by interaction with both targets is far greater than the arithmetical sum of the separate contributions of each target, implying a synergistic activation of transcription through at least two separate interactions of the trimeric gp45.

Secondary structure of T4 gene 33 protein. Fourier transform infrared and circular dichroic spectroscopic studies.

The secondary structure of bacteriophage T4 gene 33 protein (gp33) has been quantitatively examined by using Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy were used to quantitate the spectral information from the amide I bands. The relative areas of these component bands indicate 21% alpha-helix, 25% beta-sheet, 34% turn, 12% random coil and 8% other undefined structures in gp33. An analysis of the CD spectrum of gp33 at the same pH and temperature revealed 19% alpha-helix, 25% beta-sheet, 13% turn and 43% random coil structures. The possible reasons for the discrepancies in estimates of the contributions to the secondary structure from turns and random coils are discussed.

Dynamics of DNA-tracking by two sliding-clamp proteins.

Bacteriophage T4 gene 45 protein (gp45) and Escherichia coli beta are DNA-tracking sliding-clamp proteins that increase processivity by tethering their conjugate DNA polymerases to DNA. gp45 also activates T4 late transcription. DNA loading of gp45 and beta requires ATP or dATP hydrolysis; efficient loading at primer-template junctions is assisted by single-stranded DNA-binding proteins. The kinetics of gp45 loading and tracking have been examined by DNase I footprinting of linear DNA with one blunt end, one primer-template junction, and binding sites for proteins that block gp45 tracking. DNA loading of gp45 can also be interrupted by adding the non-hydrolyzable ATP analog ATP-gamma-S. At saturation, DNA is very closely packed with gp45 or beta. When gp45 loading is interrupted, or when a segment of the track is blocked off, the gp45 footprint dissipates within seconds, but the DNA-tracking state of beta is much more stable. The stability of the tracking state of gp45 is, however, increased by the macromolecular crowding agent polyethylene glycol. We suggest that labile gp45 catenation directly generates the coupling of late transcription to DNA replication during bacteriophage T4 multiplication.

Rules governing the efficiency and polarity of loading a tracking clamp protein onto DNA: determinants of enhancement in bacteriophage T4 late transcription.

The bacteriophage T4 DNA polymerase accessory proteins confer processivity and high speed on replicative DNA chain elongation: the gene 45 protein, gp45, tracks along DNA and serves as the sliding clamp of the viral DNA polymerase; the gene 44/62 protein complex, gp44/62, is an ATP-dependent loading enzyme that mounts gp45 on DNA. Gp45 also activates T4 late transcription. Transcriptional enhancement by gp45 requires a particular orientation that is imposed by gp44/62 at the DNA loading site. Loading and orienting gp45 on DNA, tracking along DNA and interaction with RNA polymerase have been analyzed by measuring transcriptional activation. The efficiency of loading gp45 at different DNA structures and the resulting transcriptional activation have been compared, and sources of interference with transcriptional activation have been examined. All observations are compatible with a mechanism in which the loading enzyme recognizes the polarity of single-stranded DNA and imposes a corresponding polarity of DNA entry on gp45. Primer-template junctions are the most efficient DNA loading sites for gp45 and can generate very rapid opening at promoters that are located at a distance of > 1 kbp. In contrast, gp45 does not track efficiently across single-stranded DNA.