Expression of VP1 protein of serotype A and O of foot-and-mouth disease virus in transgenic sunnhemp plants and its immunogenicity for guinea pigs.

UNLABELLED: Recently, transgenic plants expressing immunogenic proteins of foot-and-mouth disease virus (FMDV) have been used as oral or parenteral vaccines against foot-and-mouth disease (FMD). They exhibit advantages like cost effectiveness, absence of processing, thermostability, and easy oral application. FMDV VP1 protein of single serotype has been mostly used as immunogen. Here we report the development of a bivalent vaccine with tandem-linked VP1 proteins of two serotypes, A and O, present in transgenic forage crop Crotalaria juncea. The expression of the bivalent protein in the transgenic plants was confirmed by Western blot analysis. Guinea pig reacted to orally or parenterally applied vaccine by humoral as well as cell-mediated immune responses including serum antibodies and stimulated lymphocytes, respectively. The vaccine protected the animals against a challenge with the virus of serotype A as well as O. This is the first report on the development of a bivalent FMD vaccine using a forage crop. KEYWORDS: foot-and-mouth disease; sunnhemp; Agrobacterium tumefaciens; FMDV-VP1 gene; serotype O and A; in planta transformation; transgenic plants; bivalent vaccine.

Cyclodextrin supramacromolecules: unexpected formation in aqueous phase under ambient conditions.

The unexpected formation of supramacromolecules of ß-cyclodextrin in aqueous solution under ambient conditions is reported in this article. The shape and size of the supramacromolecules could be controlled by varying the stirring time and the concentration. The supramolecular interactions of the supramacromolecules were confirmed by ¹H NMR spectroscopy. Additionally, visual evidence of the supramacromolecules was provided by transmission and scanning electron microscopy. The thermal behavior of the supramacromolecules was examined by thermogravimetric analysis. The molecular mass determination by gel permeation chromatography showed that the supramacromolecules had a high molar mass.

Haemolytic effects of hypo-osmotic salt solutions on human erythrocytes.

BACKGROUND: While it is well known that hypotonic solutions of sodium chloride induce hemolysis, the effects of other salt solutions on human erythrocytes have not been well documented. OBJECTIVE: The study is to compare the effects of other salt solutions on human red cells. METHODS: Iso-osmotic and hypo-osmotic solutions of various salts were prepared after taking into account their molecular weight and osmotic pressure. Five healthy volunteers between the age of 22-30 years were randomly selected and ten blood samples were collected from them. The study was conducted from January 2009 to February 2009. Blood was collected from subjects by venepuncture into heparinised tubes. 20 fl of blood was pipetted into 1 ml of each solution and incubated for one hour at 37°C in a water bath. The solutions were centrifuged and the colour of the supernatant was read in a spectrophotometer. Supernatant from blood added to distilled water was considered 100% hemolysed. RESULTS: Iso-osmotic salt solutions were free of hemolysis. Among chloride salts, sodium chloride showed the least hemolysis and potassium chloride and nickel chloride resulted into greater hemolysis. Among potassium salts, potassium bromate caused highest amount of hemolysis whereas potassium sulphate showed the least. CONCLUSION: The significant differences in hemolytic pattern in hypo-osmotic salts solutions suggest that the hypo-osmotic stress causes morphological changes in red cells that alter their permeability to various ions leading to hemolysis. This probably occurs through opening of volume sensitive channels.

4-aminopyridine-induced contracture in frog ventricle is due to calcium released from intracellular stores.

The aim of the study is to demonstrate the presence of intracellular calcium store in frog ventricle based on contractures induced by 4-aminopyridine in calcium-free media. Frog-ventricular strips were subjected to field stimulation at 0.2 Hz and the force of contraction was recorded after stabilization. The preparation was then kept quiescent for some time in solutions with different sodium concentrations, containing 0 or 1 mmol/L calcium. Caffeine, 4-aminopyridine (4-AP), or tetraethylammonium chloride was then added. Frog skeletal muscle preparations were used as positive controls for the caffeine experiments. Frog ventricular preparations did not develop contractures (sustained contractions) in the presence of caffeine (25 mmol/L), while frog skeletal muscle preparations developed caffeine-induced contractures. However, 4-AP (16 mmol/L) was able to induce contractures in quiescent frog ventricular preparations, even when they were superfused with calcium-free solution. 4-AP contractures in frog ventricle were seen in the presence of nifedipine also. Amplitude of 4-AP evoked contractures in frog ventricle were much larger in low sodium (30 mmol/L) and sodium-free (sodium substituted by lithium) solutions than in normal sodium solution, suggesting that the route of extrusion of the cytosolic calcium (released from intracellular stores by 4-AP) is the sodium calcium exchanger, which gets reversed in low sodium solutions. Tetraethylammonium chloride (TEA) was not able to induce contractures in frog ventricle suggesting that the contracture evoked by 4-AP is not due to its potassium channel blocking effect. In quiescent frog skeletal muscle preparations, caffeine as well as 4-AP induced contractures in calcium-free solutions. We therefore conclude that there is a caffeine-insensitive, 4-AP sensitive intracellular calcium store in the frog ventricle.

Oligodendroglial tumor chemotherapy using "decreased-dose-intensity" PCV: a Singapore experience.

The authors propose "decreased-dose-intensity" PCV (procarbazine, lomustine [CCNU], and vincristine) chemotherapy for Asian patients with oligodendroglial tumors. In this study, all seven patients with oligodendroglioma (OD) and eight with anaplastic oligodendroglioma (AO) had objective responses or stable disease. Median progression-free survival was greater than 29 months (OD) and 36.5 months or greater (AO); 86% of patients with OD and 63% with AO remain progression-free. Twenty-four Common Toxicity Criteria Grade 3/4 adverse events were noted.

Expression of Chlamydia trachomatis genes encoding products required for DNA synthesis and cell division during active versus persistent infection.

During persistent infection, the intracellular bacterial pathogen Chlamydia trachomatis is viable but severely attenuates the production of new, infectious elementary bodies (EBs). To investigate the reasons for this lack of new EB output, we analysed the expression of chlamydial genes encoding products required for DNA replication and cell division, using in vitro models of active versus persistent infection and synovial tissue samples from patients with chronic Chlamydia-associated arthritis. Hep-2 cells were infected with K serovar C. trachomatis and harvested at t = 0-48 h post-infection (p.i; active). Human monocytes were infected similarly and harvested at t = 1-7 days p.i. (persistent). RNA preparations from infected/uninfected cells and patient samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) targeting polA, dnaA, mutS and parB mRNA, related to chlamydial DNA replication/segregation; these were expressed in infected Hep-2 cells from 11 to 48 h p.i; ftsK and ftsW, related to cell division, were expressed similarly. Real-time PCR analyses demonstrated that significant accumulation of chlamydial chromosome began at about 12 h p.i. in infected Hep-2 cells. In infected human monocytes, polA, dnaA, mutS and parB mRNA were produced from days 1-7 p.i. and were weakly expressed in patient samples. Real-time PCR indicated the continuing accumulation of chlamydial chromosome during the 7 day monocyte infection, although the rate of such accumulation was lower than that occurring during active growth. However, transcripts from ftsK and ftsW were detected only at 1 day p.i. in infected monocytes but not thereafter, and they were absent in all patient samples. Thus, genes whose products are required for chlamydial DNA replication are expressed during persistence, but transcription of genes whose products are required for cytokinesis is severely downregulated. These data explain, at least in part, the observed attenuation of new EB production during chlamydial persistence.

Chlamydia pneumoniae expresses genes required for DNA replication but not cytokinesis during persistent infection of HEp-2 cells.

Chlamydia pneumoniae causes community-acquired pneumonia and is associated with several chronic diseases, including asthma and atherosclerosis. The intracellular growth rate of C. pneumoniae slows dramatically during chronic infection, and such persistence leads to attenuated production of new elementary bodies, appearance of morphologically aberrant reticulate bodies, and altered expression of several chlamydial genes. We used an in vitro system to further characterize persistent C. pneumoniae infection, employing both ultrastructural and transcriptional activity measurements. HEp-2 cells were infected with C. pneumoniae (TW-183) at a multiplicity of infection of 3:1, and at 2 h postinfection gamma interferon (IFN-gamma) was added to the medium at 0.15 or 0.50 ng/ml. Treated and untreated cultures were harvested at several times postinfection. RNA was isolated and reverse transcribed, and reverse transcription (RT)-PCR analyses targeting primary transcripts from chlamydial rRNA operons as well as dnaA, polA, mutS, minD, ftsK, and ftsW mRNA were done. Some cultures were fixed and stained for electron microscopic analysis, and a real-time PCR assay was used to assess relative chlamydial chromosome accumulation under each culture condition. The latter assays showed that bacterial chromosome copies accumulated severalfold during IFN-gamma treatment of infected HEp-2 cells, although less accumulation was observed in cells treated with the higher dose. Electron microscopy demonstrated that high-dose IFN-gamma treatment elicited aberrant forms of the bacterium. RT-PCR showed that chlamydial primary rRNA transcripts were present in all IFN-gamma-treated and untreated cell cultures, indicating bacterial metabolic activity. Transcripts from dnaA, polA, mutS, and minD, all of which encode products for bacterial chromosome replication and partition, were expressed in IFN-gamma-treated and untreated cells. In contrast, ftsK and ftsW, encoding products for bacterial cell division, were expressed in untreated cells, but expression was attenuated in cells treated with low-dose IFN-gamma and absent in cells given the high dose of cytokine. Thus, the development of persistence included production of transcripts for DNA replication-related, but not cell division-related, genes. These results provide new insight regarding molecular activities that accompany persistence of C. pneumoniae, as well as suggesting requirements for reactivation from persistent to productive growth.

Nonarticular proximal tibia fractures: treatment options and decision making.

Nonarticular proximal-third fractures account for 5% to 11% of tibial shaft injuries and occur as a result of a variety of mechanisms. Treatment is more challenging than for more distal fractures, and the rates of compartment syndrome and arterial injury are higher, especially for displaced fractures. Closed management often leads to varus malunion, especially when the fibula is intact. Closed treatment should therefore be reserved for nondisplaced or minimally displaced fractures with little soft-tissue injury. Plating of the proximal tibia has become a less popular alternative because of the high incidence of infection and fixation failure. However, judicious use of lateral plates as an adjunct to medial external fixation in comminuted fractures can be effective. External fixation remains the most versatile method. It is indicated for fractures with short proximal fragments and in cases of extensive soft-tissue injury that would preclude use of other surgical techniques. Temporary joint-spanning external fixation has a role in the initial management of certain fracture patterns, particularly when accompanied by severe soft-tissue injury. Although intramedullary nailing can lead to valgus malunion in a sizable percentage of patients with this injury, it can be useful for stabilizing fractures with proximal fragments longer than 5 to 6 cm. Placing the entry portal more proximal and lateral, locking in extension, and using specific techniques, such as blocking screws, can improve alignment after nailing. Use of an algorithm that takes into account the severity of soft-tissue injury, the length of the fracture fragment, and the degree of fracture stability allows effective decision making among current treatment techniques.

Calmodulin mediated activation of acetyl-CoA carboxylase during aflatoxin production by Aspergillus parasiticus.

The relevance of Ca2+-calmodulin-mediated processes in channelling acetate for aflatoxin formation was investigated by studying the influence of trifluoperazine (an anticalmodulin agent) on [14C]-acetate incorporation and activity of acetyl-CoA carboxylase in Aspergillus parasiticus NRRL 2999. Culturing the organism in presence of 0.14 mmol l-1 trifluoperazine resulted in 55% decrease of [14C]-acetate incorporation into aflatoxin B1, along with an 80% decrease in acetyl-CoA carboxylase activity at periods corresponding to maximal aflatoxin production. Concomitant decrement (35%) in the activity of glucose-6-phosphate dehydrogenase indicated decreased availability of reduction potential (NADPH) required for aflatoxin biosynthesis. The ability of calmodulin to activate and trifluoperazine to inhibit acetyl-CoA carboxylase activity in a dose-dependent manner was also noted under in vitro conditions. The combined results suggest calmodulin-mediated activation of acetyl-CoA carboxylase as an important event for aflatoxin production.

Clinical evaluation of the Alta hip bolt in peritrochanteric hip fractures.

We reviewed the clinical and radiographic results of 58 patients with peritrochanteric fractures treated with the Alta hip bolt (a sliding compression device that inserts a dome plunger in the femoral head instead of a hip screw). This group was compared with a group of 53 patients treated with conventional hip screws. Three patients (5.2%) treated with the Alta hip bolt and three patients (5.7%) treated with conventional hip screw had failure of fixation. Failure of fixation consistently occurred in patients with unstable fracture patterns or significant osteopenia. There were no cases of bolt cut-out in stable intertrochanteric fractures. We conclude that the Alta hip bolt performs as well as sliding hip screws in peritrochanteric fractures, but the additional learning curve and increased cost do not justify its routine use at this point in time.

Irreducible superior dislocation of the patella requiring open reduction.

The authors describe a case of superior dislocation of patella that was irreducible by closed methods. Extensive review of the literature revealed that this is the first case reported in which an open reduction was needed to reduce a superior dislocation of the patella to unlock the osteophytes, which were blocking the closed reduction.

Results of arthroscopic versus open Bankart suture repair.

The purpose of this study was to compare patients with anterior shoulder instability who were treated with an open Bankart procedure with those treated with an arthroscopic procedure. During a 3-year period, 43 patients (44 shoulders) were surgically treated. Thirty-four patients were available for followup. Eighteen shoulders had open Bankart procedure, and 16 shoulders were treated arthroscopically. Capsular laxity can be better assessed with the open procedure. A Bankart lesion was found in all the patients in both series. Average followup for Group 1 was 34 months, and for Group 2, it was 23 months. Group 1 had 83% good to excellent results with no recurrent dislocation or reoperation. Group 2 had 50% good to excellent results, and 50% fair to poor results with 3 recurrent dislocations and 4 recurrent subluxations that required second operation. The average loss of external rotation in Group 1 was not significantly greater than that in Group 2. Sixteen patients in Group 1 and 8 patients in Group 2 were able to return to their primary work or sport. Results of arthroscopic Bankart repair do not equal those of the open Bankart procedure for the rate of recurrence and postoperative range of motion. The followup reported is short, and more dislocations can be anticipated with longer followup.

Bacterial lipopolysaccharide induces diarrhoea in caecectomized mice.

Castor oil, lipopolysaccharide of Escherichia coli, and endotoxin of Salmonella typhimurium were used for inducing diarrhoea in sham operated or caecectomized mice. Copious diarrhoea was induced by lipopolysaccharide (LPS) in caecectomized mice. Characteristics of diarrhoea induced by castor oil were not different between the two groups. It is concluded that caecectomized mice may be a good model to study lipopolysaccharide-induced diarrhoea.