Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae

A novel chitinase was detected in extracellular culture fluids of the entomopathogenic fungus Metarhizium anisopliae (ATCC 20500) grown in liquid medium containing chitin as a sole carbon source. A chitinase was purified to near homogeneity from culture broth of M. anisopliae by DEAE-Sephacel, CM-Sepharose CL-6B ion-exchange chromatography, and gel filtration with Superose 12HR. The molecular mass of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 60 kDa and the optimum pH of the enzyme was 5.0. This molecular mass is different from values of 33, 43.5, and 45 kDa for endochitinases and 110 kDa for an exochitinase (N-acetylglucosaminidase) from M. anisopliae ME-1 published previously. In addition, N-terminal sequences of 60-kDa chitinase are different from those of 43.4- and 45-kDa endochitinases. The purified enzyme showed high chitinolytic activity against colloidal, crystalline chitin of crab shells as well as against p-nitrophenyl-beta-d-N-acetylglucosamide, p-nitrophenyl-beta-d-N, N'-diacetylchitobiose, and p-nitrophenyl-N, N'-N"-triacetylchitotriose, indicating that this enzyme has both endo- and exochitinase activity.     

Rectangular flap for horizontal upper vermilion tightness in secondary cleft lip deformity

Radical paring of the cleft edge during a primary cleft operation or repeated secondary surgeries can result in tightness of the upper lip. The degree of the resulting side-to-side tension can vary, from mild cases for which improvement is sought through realignment of the misplaced oral sphincter muscle in secondary revision, to severe cases for which the possibility of a lip switch flap must be considered. When the lip tightness accompanies more than three-quarters loss of the Cupid's bow, an Abbé flap is an alternative. However, the lip switch flap is far from ideal, in both artistic and functional perspectives, and should be avoided if at all possible in mild to moderate degrees of lip tightness. This study presents a method of correcting horizontal cleft upper lip tightness, especially of the vermilion. The method involves local transfer of an inferiorly based rectangular flap from the relatively redundant upper two-thirds to the lower one-third of the upper lip and vermilion. Primary indications for the technique include vermilion tightness with half to three-quarters loss of Cupid's bow. The method has the advantage of supplementing the horizontal lip dimension on the cleft side and restoring a natural Cupid's bow, thereby repositioning the shifted philtral column and adding fullness to the lower one-third of the upper lip. Incorporation of the upper lip scar in the rectangular flap removes ugly scars and spares the lower lip from surgical violation. The orbicularis sphincter function, as seen in facial animation, was well regained. Twenty unilateral and three bilateral cases with a maximal follow-up period of 4.5 years are presented.