Phosphorylation by CDK1 regulates XMAP215 function in vitro.

XMAP215, a microtubule-associated protein isolated from Xenopus eggs, promotes microtubule assembly dynamics in an end-specific manner: addition of XMAP215 to purified porcine tubulin increases both elongation and shortening rates at microtubule plus ends, with minimal effects at minus ends. Previous results indicated that XMAP215 is phosphorylated during M phase, suggesting that its activity may be regulated by cell cycle phosphorylation. To test this hypothesis, we used video-enhanced DIC microscopy to examine the effects of XMAP215 phosphorylated by CDK1 on the assembly of purified tubulin. XMAP215 incubated with ATP at 30 degrees C for 10- 20 min in the absence of CDK1 exhibited a 4.1-fold increase in plus end elongation rate compared to purified tubulin. Elongation was promoted to a lesser degree (2.4-fold) by phosphorylated XMAP215. In contrast, XMAP215 phosphorylation did not alter the approximately 3-fold increase in shortening rate. XMAP215 binding to taxol microtubules was also not changed by phosphorylation. To further investigate mechanisms responsible for the faster microtubule shortening rate observed with XMAP215, we made microtubules with segments assembled prior to XMAP215 addition (proximal segments) and segments assembled in the presence of XMAP215 (distal segments). In 9 of 10 microtubules, the distal segment shortened faster (distal = 60.7 microm/min; proximal = 37.5 microm/min), suggesting that MTs assembled in the presence of XMAP215 have an altered lattice that results in subsequent faster shortening.

Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increases tubulin GTPase activity, we propose that nocodazole acts by generating TuD subunits that then alter dynamic instability.

XMAP from Xenopus eggs promotes rapid plus end assembly of microtubules and rapid microtubule polymer turnover.

We have used video-enhanced DIC microscopy to examine the effects of XMAP, a Mr 215,000 microtubule-associated protein from Xenopus eggs (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), on the dynamic instability of microtubules nucleated from axoneme fragments in vitro. Our results indicate that XMAP substantially alters the parameters of microtubule assembly at plus ends. Specifically, addition of 0.2 microM XMAP resulted in (a) 7-10-fold increase in elongation velocity, (b) approximately threefold increase in shortening velocity, and (c) near elimination of rescue (the switch from rapid shortening to elongation). Thus, addition of XMAP resulted in the assembly of longer, but more dynamic, microtubules from the plus ends of axonemes which upon catastrophe disassembled back to the axoneme nucleation site. In agreement with previous observations (Gard, D.L., and M. W. Kirschner. 1987. J. Cell Biol. 105:2203-2215), the effects of XMAP on the minus end were much less dramatic, with only a 1.5-3-fold increase in elongation velocity. These results indicate that XMAP, unlike brain MAPs, promotes both polymer assembly and turnover, and suggests that the interaction of XMAP with tubulin and the function of XMAP in vivo may differ from previously characterized MAPs.

Iontophoresis for eyelid anesthesia.

Local anesthesia is appropriate for eyelid surgery, but patients fear the pain of injections. We evaluated iontophoresis of lidocaine for eyelid skin anesthesia to pinprick pain sensation in normal subjects, and prior to regional infiltration of the anesthetic agent in patients undergoing eyelid surgery. After iontophoresis of lidocaine hydrochloride 4% solution to one randomly chosen eyelid, pinprick skin sensation was tested in a double-masked manner. Subjective pain scores of 10 subjects were significantly less (P less than .008) on the iontophoresis treated eyelids (0.25) than on the untreated eyelids (2.55). Nine patients undergoing bilateral upper eyelid surgery had iontophoresis applied to one randomly chosen eyelid prior to the usual anesthetic injections. In this double-masked evaluation, patients reported significantly less pain (P less than .02) on the treated eyelids (1.4) than on the untreated eyelids (4.7). We conclude that iontophoresis is effective for achieving short-term, superficial anesthesia of eyelid skin.

Factitious "calcification" in the lacrimal gland fossa.

Computed tomographic (CT) scans are valuable in the evaluation of lacrimal gland fossa masses. Calcification in this region is considered a sign of malignancy. Silicone implants are widely used in orbital surgery and may give the appearance of calcification on CT scan. These must be distinguished from true calcification by the proper choice of the CT scan window.

Adult vernier thresholds do not increase with age; vernier bias does.

Vernier acuity and vernier bias were examined in persons aged 20 to 79 years using a method of adjustments. Vernier bias (mean error) showed a sharp increase between 35 and 45. Vernier acuity (standard deviation or precision of alignment) did not vary significantly with age. These different results indicate the importance of separate evaluation of acuity and bias. Vernier acuity is little affected by minor optical changes that occur with age. Therefore, normal vernier acuity in older persons suggests that the neural substrates which underlie fine-grain discrimination of object location are unaffected by aging over the range investigated.

The anophthalmic socket and the prosthetic eye. A clinical and bacteriologic study.

Culture samples were obtained from the anophthalmic sockets of 40 consecutive patients using a prosthetic eye. We found an increased prevalence of bacteria in the conjunctivae of anophthalmic sockets. No significant differences in bacterial flora were observed between symptomatic and asymptomatic patients, suggesting that symptoms of irritation are not usually related to abnormal bacterial flora. Patients who frequently manipulated their prosthesis had a significantly higher proportion of gram-negative bacteria, suggesting that manipulation of the prosthesis should be avoided.

Aponeurotic ptosis repair under local anesthesia. Prediction of results from operative lid height.

The unpredictability of acquired ptosis repair is a difficult problem. Aponeurotic ptosis repair may be performed under local anesthesia, and past reports have suggested that operative lid position may be used to predict the final result. The authors prospectively studied 20 aponeurotic repairs under local anesthesia for patients with acquired ptosis and normal levator function. Photographs were taken during surgery, 1 week after surgery, and 3 months after surgery. Statistical analysis of vertical lid fissure measurements demonstrated a linear relationship between operative lid position and the 3-month result. When operative lid height was significantly greater than 10 mm, then a slight postoperative rise was observed, but when operative lid height was significantly less than 10 mm, then a slight postoperative fall was observed. Lid position at 1 week proved to be an excellent predictor of the 3-month result, establishing a reasonable basis for intervention in cases of overcorrection or undercorrection.

Histopathology of nasolacrimal duct obstruction compatible with localized sarcoidosis.

We report a case of nasolacrimal duct obstruction with histopathology compatible with localized sarcoidosis, in a patient with no evidence of systemic disease. Excisional biopsy of the nasolacrimal duct revealed multiple noncaseating granulomas. Previously reported cases of sarcoidosis involving the lacrimal drainage system all had systemic disease, and these case are reviewed. Our case illustrates the potential value of nasolacrimal duct biopsy during dacryocystorhinostomy.