Characterization of a calmodulin-binding protein that is deficient in trifluoperazine-resistant variants of the macrophage-like cell line J774.

A calmodulin-binding protein is present in extracts of the macrophage-like mouse cell line J774 and in extracts of thioglycollate-stimulated mouse peritoneal macrophages; it is deficient in variants of J774 resistant to trifluoperazine and in resident peritoneal macrophages. The calmodulin-binding protein [CaMBP (J7)0.5] was purified from J774 and resolved from endogenous cyclic nucleotide phosphodiesterase and protein kinase activities. The protein has an apparent native Mr of 125,000-150,000 and binds calmodulin in a calcium-dependent manner with a Kd of 20 nM. It inhibits the ability of calmodulin to activate phosphodiesterase. Its sedimentation constant in glycerol gradients containing calmodulin was dependent upon the relative concentrations of calmodulin and the calmodulin-binding protein.     

Pseudophakic bullous keratopathy

We reviewed the records of all patients with pseudophakic bullous keratopathy (271 eyes, 251 patients) seen during a six-month period to determine predisposing factors, associated problems, current management, and visual outcome. Pseudophakic bullous keratopathy was associated most frequently with anterior chamber intraocular lenses in general (155 of 271), and with Leiske style lenses in particular (100 of 271). It was associated with a visual acuity of 20/200 or less in 206 eyes and a visual acuity of counting fingers or less in 129 of the eyes at the initial examination. Penetrating keratoplasties had been performed in 189 of the eyes. After penetrating keratoplasty, 108 of 189 of the eyes had a visual acuity of 20/200 or less (mean follow-up, 15 months). Visual acuity improved with longer follow-up, and among patients with a minimum follow-up of two years, 23 of 36 eyes had a visual acuity of 20/100 or better. Most grafts were clear (145 of 189). Pseudophakic bullous keratopathy was associated with marked visual loss, which was permanent despite clear grafts in 29 of 92 eyes followed-up for one year or longer.     

A quantitative luminescence assay for measuring cell uptake of aqueous-based microcapsules in vitro

We recently developed a system of microencapsulation consisting of aqueous-based polymers (e.g. alginate) and aqueous amines (e.g. spermine). We found that microencapsulation enhanced virus-specific protective immune responses. In addition, we found that microencapsulation may enhance virus-specific immune responses by selecting for antigen-presenting cells (APC) that are more efficient at processing and presenting viral antigens than those involved after natural infection. To determine the intracellular trafficking patterns and fate of microcapsules within APC, we developed a luminescence assay that permits the determination of specific quantities of proteins introduced into cells by microcapsules. We found that the time-dependent uptake of horseradish peroxidase (HRP)-labeled microcapsules was accurately detected in lysates of peritoneal exudate cells using luminol. The amplitude of HRP-catalyzed chemiluminescence in cell lysates correlated with the capture efficiency and retention kinetics of HRP in three different microcapsule preparations. HRP was most efficiently captured and retained by linking biotinylated HRP to microcapsulses chemically modified at the amine moiety with egg avidin. This preparation yielded more accurate and sensitive quantitation of HRP contained within cells than preparations capturing HRP or HRP-conjugated goat antibody into the microcapsular matrix by ionic interactions.     

Methamphetamine and core temperature in the rat: Ambient temperature, dose, and the effect of a D2 receptor blocker

Rationale

Methamphetamine (METH) induces hyperthermia in warm and hypothermia in cool environments. Our first goal was to further study the role of ambient temperature in METH’s effect on core temperature in rats. Previously, these effects were primarily demonstrated in high doses; we extended this investigation to the low-dose range (1 mg/kg METH). Our second goal was to identify the role of the D2 receptor in METH’s effects in cool ambient temperatures.

Method

Rats received METH (saline, 1, 5, and 10 mg/kg), raclopride (saline, 0.3, 0.6, and 1.2 mg/kg), or a combination (all doses of raclopride combined with 10 mg/kg METH). Treatments occurred in ambient temperatures of 18, 24, or 30 °C.

Results and conclusions

Consistent with prior research, 5 and 10 mg/kg METH caused hyperthermia or hypothermia in a dose- and ambient temperature-dependent manner (60 min after METH). In contrast, 1 mg/kg produced similar levels of hyperthermia at all ambient temperatures. These findings suggest that a threshold METH dose exists; below this dose, METH still changes core temperature, but CNS control over temperature regulation is left intact. In our experiments regarding D2 blockade, raclopride decreased METH-induced core temperature at 30 and 24 °C (60 min after METH), consistent with previous findings. We extended these findings by demonstrating that in a cool ambient temperature (18 °C), raclopride pretreatment also lowered the core temperature response to METH. Although the D2 receptor is known to mediate hypothermia, the combination of METH and D2 blockade suggests a complex mediation of the core temperature response, perhaps involving neurotransmitter interactions.     

Experimental myocardial ischemia and infarction Production of diffuse myocardial lesions in unanesthetized calves

To produce a chronic, ischemic heart failure preparation of controllable severity, plastic microspheres (6 to 14 μ in diameter) were injected into the left main coronary artery of unanesthetized calves. Two dose levels were used (4 and 5 mg/kg, Groups I and II, respectively). Hemodynamic and electrocardiographic changes were not observed during the microsphere infusion. Arterial-coronary sinus lactate differences shifted from positive control values (mean +5.3 mg/ 100 ml) to negative during and throughout the injection (mean −6.4 mg/100 ml). Significant hemodynamic abnormalities at rest (Group II only) were first noted on postinfusion day 1, and unaccompanied by subsequent significant physiologic recovery. These included an increase in left ventricular end-diastolic pressure (+18 to 21 mm Hg), reduction in stroke volume (−32 percent), cardiac output (−25 percent), dP/dt (−22 percent), mean ejection rate (−18 percent) and left ventricular ejection time, and a prolongation of preejection period. Cardiac response to angiotensin-induced stress was impaired in both test groups. Mean heart weight and left ventricular wall thickness increased by 65 and 45 percent, respectively, in both groups. Histopathologic examination demonstrated microinfarcts widely distributed throughout the myocardium supplied by the left coronary artery at various stages of healing, sometimes in the same heart, with typical time-dependent morphologic characteristics. The myocardial fibers were hypertrophied. In addition, atypical areas were found on glycogen selective staining.

The results suggest that the extent of microcirculatory ischemia and infarction determines the degree of myocardial impairment at rest and during stress irrespective of compensatory hypertrophy.     

Effect of punctal plugs in patients with low refractive errors considering refractive surgery

PURPOSE: To study the effect of punctal plug placement in patients undergoing refractive surgery for correction of poor vision. METHODS: A retrospective study was performed of 12 patients (21 eyes) who presented for initial refractive surgery or retreatment after LASIK or photorefractive keratectomy to improve visual acuity. Uncorrected visual acuity (UCVA) was documented in each eye. If tear normalization test was positive (indicating dry eye), silicone punctal plugs were placed in the lower lids of both eyes. Subjective patient UCVA and Snellen UCVA were assessed at 1 month. RESULTS: At 1 month, two eyes improved by three Snellen lines, nine eyes improved by two lines, seven eyes improved by one line, and three eyes remained unchanged. Uncorrected visual acuity was considered statistically significant (P < .0001) using the paired t test. No eye demonstrated decrease in visual acuity. One patient experienced punctal plug extrusion in both eyes. No other adverse outcomes were reported. One patient pursued additional refractive surgery after plug placement. No patient requested plug removal. CONCLUSIONS: Patients with low refractive errors note improvement in visual acuity after punctal plug placement.