The novel method for isolating chitinolytic bacteria and its application in screening for hyperchitinase producing mutant of Alcaligenes xylosoxydans.

AIMS: To develop a novel, rapid and effective screening method for chitinase producing bacteria. METHODS AND RESULTS: A simple and rapid technique for screening of potential chitinolytic bacteria has been developed using the chitin binding dye calcofluor white M2R in chitin agar. Microorganisms possessing high chitinolytic potential gave a clear zone under ultraviolet light after 24-48 h of incubation. This method was successfully applied for isolating the hyperchitinase mutant of Alcaligenes xylosoxydans. The mutant Alc. xylosoxydans EMS 33 was found to produce 3.4 times more chitinase than the wild type. CONCLUSIONS: In this study, the screening method for chitinase producing bacteria has been developed and it was applied to screen chitinase-overproducing mutant of Alc. xylosoxydans. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel screening method for chitinase producer is more sensitive, rapid, user-friendly and reliable, which can also be used for screening of recombinants having chitinase gene.

Biological control of Fusarium wilt of pigeonpea Cajanus cajan (L.) Millsp with chitinolytic Alcaligenes xylosoxydans.

Alcaligenes xylosoxydans protected pigeonpea from Fusarium wilt in a pot experiment and field trials. When seeds of pigeonpea (C. cajan) were treated with A. xylosoxydans and sown in soil infested with Fusarium, the incidence of wilt was reduced by 43.5% and resulted in 58% higher grain yield. The antifungal activity of A. xylosoxydans was based on chitinase production and was comparable in efficacy to commercial antifungal agents such as benlate, monitor WP, thiram and bavistin.

Characterization of carbamoyl phosphate synthetase of Streptomyces spp.

Carbamoyl phosphate synthetase (CPS) activity in Streptomyces lividans was repressed (70%) by addition of arginine and uracil in the growth medium. Enzyme activity was also inhibited by UMP and activated by ornithine and IMP. Pattern of inhibition and activation was similar irrespective of whether the cells were grown in medium supplemented with arginine or with uracil. A mutant of S. coelicolor with dual auxotrophy for arginine and uracil possessed only about 20% of CPS activity compared to the wild-type strain. An activity staining protocol has been developed for CPS enzyme. Using this method a single CPS band has been observed in the crude extracts of Escherichia coli as well as in S. lividans. Taken together, our results supported the conclusion that Streptomyces species might possess a single CPS enzyme unlike other gram-positive bacteria, which show the presence of two pathway-specific isozymes (Bacillus) or none (Lactobacillus and Leuconostoc).

A novel mechanism of action of tetracyclines: effects on nitric oxide synthases.

Tetracyclines have recently been shown to have "chondroprotective" effects in inflammatory arthritides in animal models. Since nitric oxide (NO) is spontaneously released from human cartilage affected by osteoarthritis (OA) or rheumatoid arthritis in quantities sufficient to cause cartilage damage, we evaluated the effect of tetracyclines on the expression and function of human OA-affected nitric oxide synthase (OA-NOS) and rodent inducible NOS (iNOS). Among the tetracycline group of compounds, doxycycline > minocycline blocked and reversed both spontaneous and interleukin 1 beta-induced OA-NOS activity in ex vivo conditions. Similarly, minocycline > or = doxycycline inhibited both lipopolysaccharide- and interferon-gamma-stimulated iNOS in RAW 264.7 cells in vitro, as assessed by nitrite accumulation. Although both these enzyme isoforms could be inhibited by doxycycline and minocycline, their susceptibility to each of these drugs was distinct. Unlike acetylating agents or competitive inhibitors of L-arginine that directly inhibit the specific activity of NOS, doxycycline or minocycline has no significant effect on the specific activity of iNOS in cell-free extracts. The mechanism of action of these drugs on murine iNOS expression was found to be, at least in part, at the level of RNA expression and translation of the enzyme, which would account for the decreased iNOS protein and activity of the enzyme. Tetracyclines had no significant effect on the levels of mRNA for beta-actin and glyceraldehyde-3-phosphate dehydrogenase nor on levels of protein of beta-actin and cyclooxygenase 2 expression. These studies indicate that a novel mechanism of action of tetracyclines is to inhibit the expression of NOS. Since the overproduction of NO has been implicated in the pathogenesis of arthritis, as well as other inflammatory diseases, these observations suggest that tetracyclines should be evaluated as potential therapeutic modulators of NO for various pathological conditions.

Solid-state CP/MASS 13C-NMR spectroscopy: a sensitive method to monitor enzymatic hydrolysis of chitin.

The time-course hydrolysis of colloidal chitin by the chitinase complex isolated from Myrothecium verrucaria was monitored using solution and solid-state 13C-NMR spectroscopy. The solution NMR studies showed the presence of N-acetylglucosamine (GlcNAc) as the sole product of hydrolysis. Solid-state 13C CP/MASS studies, on the other hand, indicated the presence of high molecular weight oligomers as well as GlcNAc. The linewidth of the C1 carbon of the oligomers obtained after hydrolysis is found to be less than that of the unhydrolyzed sample. The linewidths calculated from the spin-spin relaxation times (T2) of colloidal chitin and its products of hydrolysis were in the restricted range of 40-50 Hz, compared with the observed linewidths of 143-123 Hz. Peak area measurement on monomer to polymer/oligomer indicated an initial slow formation of the monomer, GlcNAc. From the NMR data, the involvement of endo-enzymes in the initial phase of hydrolysis is suggested.

Genetic transformation of Chainia and heat attenuation of its restriction system.

A PEG-mediated transformation system for Chainia (NCL 82-5-1) was developed using a broad host range Streptomyces vector, pIJ702. Protoplasts prepared from Chainia (NCL 82-5-1) were regenerated with 5% efficiency. Transformation of the protoplasts with pIJ702 gave 10-20 transformants/micrograms DNA. The low efficiency of transformation is attributed to a restriction system in Chainia; this could be inhibited by treating the protoplasts at 42 degrees C for 10 min just before transformation. The yield of transformants increased 100-fold when pIJ702 was modified by passage in Chainia. Because the plasmid replicon was functional in Chainia and the modified plasmid was stably maintained, the transformation system should be useful for self-cloning in Chainia NCL 82-5-1 of the many commercially important enzymes this strain is known to produce.

Tricuspid incompetence resulting from retrograde aortic dissection.

This paper presents a 51 year old black female with known hypertension and an acute illness characterized by aortic regurgitation, cerebrovascular insufficiency, renal insufficiency, aortic valvular insufficiency, mediastinal widening and other features characteristic of acute Type I aortic dissection. An unusual feature in this individual is dissection extending into the membranous septum of the heart and into the aorto-atrial space with large hematoma, which partially disrupted the conduction system as well as dislodging the tricuspid septal leaflet in such fashion that major tricuspid regurgitation was present and interfered with termination of cardiopulmonary bypass. This patient presents a very unusual complication of which we wish to inform the readers.

Experience with free grafts in urethral reconstruction.

A total of 33 patients underwent hypospadias or epispadias repair by patch or tubular grafts of skin or bladder epithelium from July 1980 to January 1985. Indications included previous circumcision, proximal primary hypospadias, severe chordee alone, inadequate local skin during extended urethroplasty at 2-stage hypospadias repair, multiple previous reconstructions and failed epispadias repair. Genital skin grafts were used in 25 patients, extragenital skin grafts in 4 and bladder epithelial grafts in 4. Complications occurred in 13 patients (39.4 per cent): 8 required reoperation for a fistula, stricture or diverticulum, and 5 underwent internal urethrotomy or urethral dilation. Of the 8 patients 3 needed more than 1 revision. These revisions were short and generally uncomplicated. Final results uniformly were good functionally and cosmetically. Based on our experience and that of others we believe that despite a high incidence of minor complications a free graft serves as an excellent substitute urethra for treatment of a variety of urethral problems in children.

Urinary tract deterioration associated with the artificial urinary sphincter.

In most recent series of artificial sphincter implantations there has been a reduction in mechanical and surgical complications, with continence rates approaching 90 per cent. Despite initially excellent results in 47 children, with increasing durations of followup we noted a transient hydronephrosis related to incomplete bladder emptying in 5 and a persistent physiological alteration of detrusor dynamics consisting of a rigid, noncompliant bladder in 7. Preoperatively, all patients had had indepth radiological studies and most had a urodynamic evaluation, and were considered to be excellent candidates. The etiology of these alterations is not understood at this time. Bladder and even upper tract deterioration can occur without the appearance of urinary leakage. Therefore, children with an artificial sphincter must be monitored indefinitely with semiannual assessment of the upper tracts and periodic urodynamic evaluation.

Flavobacterium meningosepticum as an opportunist.

Flavobacterium meningosepticum was isolated from the cerebrospinal fluid of an adult immunodeficient female. In spite of prompt therapy the patient succumbed to the infection. The opportunistic role of the organism is discussed.