Expression, purification and characterization of the interferon-inducible, antiviral and tumour-suppressor protein, human RNase L.

The interferon (IFN)-inducible, 2′,5′-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) plays key role in antiviral defense of mammalian cells. Induction by IFN and activation by double-stranded RNA lead to 2-5A cofactor synthesis, which activates RNase L by causing its dimerization. Active RNase L degrades single-stranded viral as well as cellular RNAs causing apoptosis of virus-infected cells. Earlier, we had reported that expression of recombinant human RNase L caused RNA-degradation and cell-growth inhibition in E. coli without the need for exogenous 2-5A. Expression of human RNase L in E. coli usually leads to problems of leaky expression, low yield and degradation of the recombinant protein, which demands number of chromatographic steps for its subsequent purification thereby, compromising its biochemical activity. Here, we report a convenient protocol for expression of full-length, soluble and biochemically active recombinant human RNase L as GST-RNase L fusion protein from E. coli utilizing a single-step affinity purification with an appreciable yield of the highly purified protein. Recombinant RNase L was characterized by SDS-PAGE, immunoblotting and MALDI-TOF analysis. A semi-quantitative agarose-gel-based ribonuclease assay was developed for measuring its 2-5A-dependent RNase L activity against cellular large rRNAs as substrates. The optimized expression conditions minimized degradation of the protein, making it a convenient method for purification of RNase L, which can be utilized to study effects of various agents on the RNase L activity and its protein– protein interactions.

Replacement of the C-terminal tetrapeptide (314PAPV317 to 314SSSM317) in interferon regulatory factor-2 alters its N-terminal DNA-binding activity.

Interferon regulatory factor-2 (IRF-2) is an important transcription factor involved in cell growth regulation, immune response and cancer. IRF-2 can function as a transcriptional repressor and activator depending on its DNA-binding activity and protein–protein interactions. We compared the amino acid sequences of IRF-2 and found a C-terminal tetrapeptide (314PAPV317) of mouse IRF-2 to be different (314SSSM317) from human IRF-2. Recombinant GST-IRF-2 with 314PAPV317 (wild type) and 314SSSM317 (mutant) expressed in Escherichia coli were assessed for DNA-binding activity with 32P-(GAAAGT) 4 by electrophoretic mobility shift assay (EMSA). Wild type- and mutant GST-IRF-2 showed similar expression patterns and immunoreactivities but different DNA-binding activities. Mutant (mt) IRF-2 formed higher-molecular-mass, more and stronger DNA–protein complexes in comparison to wild type (wt) IRF-2. Anti-IRF-2 antibody stabilized the DNA–protein complexes formed by both wt IRF-2 and mt IRF-2, resolving the differences. This suggests that PAPV and SSSM sequences at 314-317 in the C-terminal region of mouse and human IRF-2 contribute to conformation of IRF-2 and infl uence DNA-binding activity of the N-terminal region, indicating intramolecular interactions. Thus, evolution of IRF-2 from murine to human genome has resulted in subtle differences in C-terminal amino acid motifs, which may contribute to qualitative changes in IRF-2-dependent DNA-binding activity and gene expression.

Maxillary sinusitis caused by Lasiodiplodia theobromae.

Lasiodiplodia (monotypic) comprises a very small proportion of the fungal biota. It is a common plant pathogen in tropical and subtropical regions. Clinical reports on its association with onychomycosis, corneal ulcer and phaeohyphomycosis are available. However, Lasiodiplodia theobromae causing fungal sinusitis has not been reported. We present here a case of fungal sinusitis in a 30-year-old woman, who came to the ENT OPD (out patient department) with complaints of intermittent bleeding and nasal discharge from the left side for a week. The patient complained of headache, predominantly on the left side and heaviness on and off since two months. Diagnosis was based on radiological and mycological evidence; the patient underwent endoscopic surgery and was started on antifungal treatment.

Myasthenia gravis: towards a safe anaesthesia technique, five years clinical experience in Hamad medical corporation, Qatar and review of literature

Myasthenia gravis [MG] is a disease with many implications for the safe administration of anesthesia and involves considerable morbidity and mortality. Myasthenia gravis is caused by autoantibodies to postsynaptic nicotinic acetylcholine receptors at the neuromuscular junction, causing weakness of skeletal muscles. Patients with thymoma-associated MG produce autoantibodies to a variety of neuromuscular antigens, including antibodies to the skeletal muscles calcium release channel and antibodies to cytoplasmic filamentous proteins. Thymectomy is a common surgical procedure in patients with myasthenia gravis.. Our aim is to review and document retrospectively our initial experience of providing a safe general anaesthesia technique involving continuation of preoperative anticholinesterase doses, use of non-paralyzing technique, and use of ultra-short acting anesthetics and cautious reintroduction of anticholinesterase after surgery. Eight patients underwent thymectomy from January 2002 to March 2006 under general anaesthesia technique involving continuation of preoperative anticholinesterase doses, avoidance of muscle relaxants, and use of ultra-short acting anesthetics and cautious reintroduction of anticholinesterase after surgery. Demographic and clinical data, preoperative symptomatology, treatment history, intraoperative and post-operative data, and distribution of patients according to intubating grade were recorded and analysed. All our patients who underwent trans-sternal thymectomy using the above technique were extubated on operating table following administration of intravenous anticholinesterase and anticholinergic drugs. There was no postoperative morbidity or mortality. None of our patients needed ventilatory assistance in the postoperative period. Patients can be extubated on table safely after thymectomy if we avoid muscle relaxants and use ultrashort acting anesthetics intraoperatively. Surgical exposure and technique was adequate in all our patients. This technique was safe and effective in all of our 8 patients who underwent trans-sternal thymectomy