Short, hydrophobic, alanine-based proteins based on the basic region/leucine zipper protein motif: overcoming inclusion body formation and protein aggregation during overexpression, purification, and renaturation.

GCN4 is a yeast transcriptional regulatory protein; its DNA-binding domain is a basic region/leucine zipper (bZIP) structure that comprises a dimer of alpha-helices capable of high-affinity, sequence-specific recognition of the DNA major groove. We are exploiting what nature has evolved by manipulating the bZIP motif as a molecular recognition scaffold; thus we reduced the elegantly minimal bZIP to an even more simplified structure by substitution with alanine residues-hence, a generic, Ala-based, helical scaffold. These Ala-based mutants are unusual proteins for expression as they are short ( approximately 100 amino acids) and hydrophobic (Ala-mutated basic regions, leucine-zipper dimerization domains). Hydrophobicity posed a major problem throughout the expression, isolation, and purification stages; inclusion body formation and protein aggregation were significant hurdles throughout protein production. We describe measures that solved these problems, including use of high concentrations of denaturant in all steps of protein isolation and purification and use of temperature-dependent renaturing techniques to obtain folded, functional protein. Despite these difficulties, we ultimately retrieved 5-10 mg/L of broth of active, correctly folded protein after the complete purification procedure. Homogeneity of the proteins was established by chromatography, electrophoresis, and mass spectrometry. Furthermore, characterization by circular dichroism and DNase footprinting analysis demonstrates that these alanine-based mutants retain the structure and function of the native GCN4 DNA-binding domain. Remarkably, the most heavily mutated protein, containing 24 alanines of 27 total amino acids in the DNA-binding basic region, still binds the AP-1 site, the target of native GCN4.

Decreased libido--a side effect of carbonic anhydrase inhibitor.

Thirty-nine cases of decreased libido in glaucoma patients on carbonic anhydrase inhibitors are reported. This symptom completely reversed or markedly improved after discontinuation of the drug in all cases. Twelve of these patients restarted their carbonic anhydrase inhibitor medication which resulted in a recurrence of decreased libido symptoms. There were 3 cases of impotency which reversed after discontinuation of the drug. Most likely, these symptoms are a result of the malaise and depression occurring in some patients on carbonic anhydrase inhibitor therapy.

Cryosurgery for ocular and periocular lesions.

This preliminary report suggests that cryosurgery has a definite place in the management of certain external periocular and ocular problems. Cryosurgery for basal- or squamous-cell carcinoma of the lid is easily performed, gives excellent cosmetic results, and has a low recurrence rate. It is not recommended for lesions involving the fornices, nor for sclerotic or morphea-type basal-cell carcinomas. Tumor recurrences following radiation, surgery, or cryosurgery can still be retreated with cryosurgery. To date, there is no evidence that cryosurgery at temperatures above --40 degrees C causes damage to the lacrimal outflow system. Cryosurgery is of value in the management of trichiasis, reactive lymphoid hyperplasia, spider hemangioma, molluscum contagiosum, and conjunctival dysplasia. Cryotherapy for management of intraepithelial epithelioma and squamous-cell carcinoma of the conjunctiva and cornea is still under investigation. Cryosurgery will, in our opinion, become the treatment of choice for basal- and squamous-cell carcinomas of the eyelids. The 96% cure rate with one treatment for these lesions reported here is artificially high since the follow-up period is too short. However, retreatment with cryosurgery is a simple 10- min outpatient procedure which is certainly not the case with recurrences after other forms of therapy.

The role of cryosurgery in external ocular and periocular disease.

This preliminary report suggests that cryosurgery has a definite place in the management of certain external periocular and ocular problems. Cryosurgery for basal or squamous cell carcinoma of the lid is easily performed, gives excellent cosmetic results, and has a low recurrence rate. It is not recommended for lesions involving the fornices, or sclerotic and morphea-type basal cell carcinomas. Tumor recurrences following radiation, surgery, or cryosurgery can still be retreated with cryosurgery. To date, there is no evidence that cryosurgery at temperatures above -40 C causes damage to the lacrimal outflow system. Cryosurgery is of value in the management of trichiasis, reactive lymphoid hyperplasia, spider hemangioma, molluscum contagiosum, and conjunctival dysplasia. Cryotherapy for management of intraepithelial epithelioma and squamous cell carcinoma of the conjunctiva and cornea is still under investigation. Cryosurgery will, in our opinion, become the treatment of choice for basal and squamous cell carcinomas of the eyelids. The 96% cure rate with one treatment for these lesions reported here is artificially high since the follow-up period is too short. However, retreatment with cryosurgery is a simple ten-minute outpatient procedure which is certainly not the case with recurrences after other forms of therapy.