Structural and functional comparison of hexahistidine tagged and untagged forms of small multidrug resistance protein, EmrE.

EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli. EmrE confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by proton motive force. The purification yield of most membrane proteins are challenging because of difficulties in over expressing, isolating and solubilizing them and the addition of an affinity tag often improves purification. The purpose of this study is to compare the structure and function of hexahistidinyl (His6) tagged (T-EmrE) and untagged (UT-EmrE) versions of EmrE. In vivo QCC resistance assays determined that T-EmrE demonstrated reduced resistance as compared to UT-EmrE. We isolated EmrE using the two different purification methods, an organic solvent extraction method used to isolate UT-EmrE and nickel affinity chromatography of T-EmrE. All proteins were solubilized in the same buffered n-dodecyl-ß-d-maltopyranoside (DDM) detergent and their conformations were examined in the presence/absence of different QCCs. In vitro analysis of protein multimerization using SDS-Tricine PAGE and dynamic light scattering analysis revealed that both proteins predominated as monomers, but the formation of dimers was more constant and uniform in T-EmrE compared to UT-EmrE. The aromatic residue conformations of both proteins indicate that T-EmrE form is more aqueous exposed than UT-EmrE, but UT-EmrE appeared to have a more dynamic environment surrounding its aromatic residues. Using fluorescence to obtain QCC ligand-binding curves indicated that the two forms had differences in dissociation constants (Kd ) and maximum specific one-site binding (Bmax ) values for particular QCCs. In vitro analyses of both proteins demonstrated subtle but significant differences in multimerization and QCC binding. In vivo analysis indicates differences caused by the addition of the tag, we also observed differences in vitro that could be a result of the tag and/or the different purification methods.

Conjunctival metastasis as the presenting sign for stage IV lung cancer.

PURPOSE: Lung cancer is the leading cause of cancer-related death in North America. It is often diagnosed at an advanced stage, lending to a poor prognosis. Symptoms of lung cancer often do not present until more advanced stages. Common sites of lung cancer metastasis are the bones, liver, and brain. The etiology of eye masses ranges from the relatively benign to those with tremendous risk of morbidity, and the differentiation is often difficult clinically. This case highlights the importance of more detailed workup, including biopsy, to determine the exact nature of the lesion. CASE REPORT: A 50-year-old white man was referred for evaluation of a "bump" on his right upper eyelid. He had noticed it for 1 month and noted enlargement during the past 2 weeks. He also reported that he had been smoking about one pack per day since 1969. External examination was remarkable for a 1.5-cm nodule pushing up from under the right upper lid. When the lid was everted, there was a 0.9-cm red and black vascularized sessile lesion on the palpebral conjunctiva. The patient was referred to an oculoplastics specialist to rule out a malignant or metastatic conjunctival neoplasm. The oculoplastics service performed an excisional biopsy, and the pathologic examination showed a poorly differentiated and highly aggressive non-small-cell lung cancer (NSCLC). After systemic evaluation, he was diagnosed as having stage IV NSCLC, with metastases to the right eyelid, brain, liver, and right lung. He underwent multiple radiotherapy sessions. He died 5 months after our initial examination. CONCLUSIONS: Stage IV NSCLC is incurable, and its treatment is often palliative. Conjunctival metastasis of stage IV NSCLC is rare, and it is clinically difficult to differentiate eyelid tumors as benign or concerning by examination alone. This case highlights the importance of a thorough history, referral, proper imaging, and biopsy to diagnose a metastatic neoplasm in a patient at high risk for cancer.

Destruction of the orbit and globe by recurrence of basal cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) is the most common skin malignancy and represents 90% of eyelid malignancies. Of those that occur on the eyelids, most involve the lower lid. Risk factors for BCC include environmental and genetic factors. There are several clinical presentations, the 2 main forms of which are 1) nodular and 2) morpheaform (or sclerosing). Several treatment options exist, including surgical excision, cryotherapy, radiotherapy, laser surgery, chemotherapy, and photodynamic therapy. The average rate of recurrence is 5%, depending on the type of BCC, the size, the location, and therapeutic approach. CASE REPORT: The patient described in this case report had basal cell carcinoma of the upper right lid 4 to 5 years prior to examination. At that time the patient was treated with surgical excision and radiotherapy. Subsequently, he had not received any eye care since the initial surgery to remove the malignancy. His entering complaint was drainage and an odor emanating from his right eye. The upper lid was retracted and had a lesion suspicious for BCC. The globe was keratinized, inflamed, and proptotic. He was referred to the oculoplastics service to confirm the recurrence of BCC. They found that the BCC had infiltrated the right globe and the retro-orbital region and probably invaded the adjacent bony margins. He had extensive surgery to remove the tumor and subsequent skin grafting. CONCLUSION: BCC, when treated early, has excellent surgical outcomes. However, there is no procedure that has a zero recurrence rate. BCC, although rarely metastatic, can be invasive. This case underscores the importance of proper follow-up protocol for all surgical patients as well as patient education that reinforces the importance of follow-up care and self-monitoring on the part of the patient.