An ultrasensitive stain for negative protein detection in SDS-PAGE via 4',5'-Dibromofluorescein.

A highly sensitive method for brief and economical staining of proteins in 1-D and 2-D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) by 4',5'-Dibromofluorescein (DBF) was developed in this study. Down to 0.025-0.05ng protein could be detected within 10min (only 2 steps) by DBF stain, which is approximately 10-fold more sensitive than those of Eosin Y (EY) and SYPRO Ruby stains, and 20-fold more sensitive than that of imidazole-zinc (IZ) negative stain. In addition, the LC-MS/MS results indicated that the newly developed staining method is compatible with the downstream protein identification. Moreover, the mechanism of DBF stain was investigated by molecular docking.

A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo.

Calcium imaging of individual neurons is widely used for monitoring their activity in vitro and in vivo. Synthetic fluorescent calcium indicator dyes are commonly used, but the resulting calcium signals sometimes suffer from a low signal-to-noise ratio (SNR). Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo. Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo. In neocortical neurons, calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetised mice with a high SNR and fast decay time. These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo.

Fluorescence imaging study of extracellular zinc at the hippocampal mossy fiber synapse.

Although synaptically released, vesicular Zn(2+) has been proposed to play a neuromodulatory or neuronal signaling role at the mossy fiber-CA3 synapse, Zn(2+) release remains controversial, especially when detected using fluorescent imaging. In the present study, we investigated synaptically released Zn(2+) at the mossy fiber (MF) synapse in rat hippocampal slices using three chemically distinct, fluorescent Zn(2+) indicators. The indicators employed for this study were cell membrane impermeable (or extracellular) Newport Green [K(DZn2+) approximatelly 1 microM] , Zinpyr-4 K(DZn2+) approximately 1 nM and FluoZin-3 K(DZn2+) approximately 15 nM, chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca(2+) K(DCa2+) approximately 200-300 microM which was present in the extracellular medium ([Ca(2+)](o)>2mM). Hippocampal slices loaded with either Newport Green or FluoZin-3 showed increases in fluorescence after electrical stimulation of the mossy fiber pathway. These results are consistent with previous studies suggesting the presence of synaptically released Zn(2+) in the extracellular space during neuronal activities; however, the rise in FluoZin-3 fluorescence observed was complicated by the data that the addition of exogenous Zn(2+) onto FluoZin-3 loaded slices gave little change in fluorescence. In the slices loaded with the high-affinity indicator Zinpyr-4, there was little change in fluorescence after mossy fiber activation by electrical stimulation. Further study revealed that the sensitivity of Zinpyr-4 was mitigated by saturation with Zn(2+) contamination from the slice. These data suggest that the sensitivity and selectivity of a probe may affect individual outcomes in a given experimental system.

Retrospective audit of the value of the pancreolauryl test in a district general hospital.

OBJECTIVES: To audit the specificity and value of the pancreolauryl test (PLT) for the diagnosis of pancreatic insufficiency. DESIGN: A retrospective case note review of 47 patients who had a PLT during three consecutive years. SETTING: A 650 bedded district general hospital. MAIN OUTCOME MEASURES: Patient demographics, symptoms, baseline blood, endoscopic and radiological investigations, and alcohol consumption were related to the results of the PLT to determine if the test could be better targeted. Response to therapy and final diagnoses were noted to determine the specificity of the test. RESULTS: Forty seven patients had a PLT during the three years studied. All successfully performed the test as outpatients. Seven inconclusive tests were repeated. Thirty eight patients (81%) had diarrhoea, of whom 10 (21%) described steatorrhoea; 23 (49%) had an abnormal PLT. All with an abnormal test had diarrhoea, more frequently describing watery stools than steatorrhoea. Those treated with pancreatic enzyme supplements had an excellent response, although two of these had a final diagnosis of coeliac disease. Nineteen patients with an abnormal PLT were thought to be suffering from pancreatic insufficiency. All patients with a normal PLT were clinically thought not to have pancreatic insufficiency. Age, sex, and alcohol intake correlated poorly with final diagnosis. CONCLUSIONS: The PLT can be used in a district general hospital setting as an outpatient test for pancreatic insufficiency and has a specificity of 83%-91%. Its use should be restricted to those presenting with diarrhoea, not necessarily steatorrhoea, and not restricted to those with a history of high alcohol consumption.

Effects of moderate hypothermia on sarcolemmal Na(+)/H(+) exchanger activity and its inhibition by cariporide in cardiac ventricular myocytes.

1. Specific inhibitors of the sarcolemmal Na(+)/H(+) exchanger (NHE) such as cariporide are being evaluated for cardioprotective therapy during cardiac surgery. We determined the effects of moderate hypothermia (25 degrees C), as occurs during cardiac surgery, on (1) sarcolemmal NHE activity and (2) the NHE-inhibitory potency of cariporide, in isolated adult rat ventricular myocytes. 2. As the index of NHE activity, trans-sarcolemmal acid efflux rate (J(H)) was determined by microepifluorescence in single cells (n = 8 to 11 per group), during recovery from intracellular acidosis in bicarbonate-free conditions. 3. Initially, myocytes were subjected to two consecutive acid pulses; these both occurred at 37 degrees C in the normothermic control group but the second pulse was at 25 degrees C in the moderate hypothermia group. J(H) values obtained after the first pulse were superimposed in both groups, indicating comparable cell populations. However, after the second pulse, J(H) values in the moderate hypothermia group were approximately 50% of those in the normothermic control group over the pH(i) range 6.80 - 7.10. 4. Similar results were obtained in cells subjected to a single acid pulse at 37 or 25 degrees C, with J(H) values in the latter group measuring approximately 60% of those in the former over the pH(i) range 6.80-7.10. 5. Cariporide (0.01, 0.03, 0.1, 0.3, 1.0 or 3.0 microM), present during recovery from a single acid pulse, reduced J(H) in a concentration-dependent manner, with IC(50) values of 150 and 130 nM at 37 and 25 degrees C, respectively. 6. We conclude that moderate hypothermia produces (1) a significant, but partial, inhibition of sarcolemmal NHE activity, and (2) no significant effect on the NHE-inhibitory potency of cariporide.

Quality control of fluorescein isothiocyanate-labeled reagents.

Fluorescein-labeled reagents are not amenable to the same precision of definition as are chemical substances. Nonetheless extensive experimental evidence shows that certain preparative requirements and selected measurable chemical parameters of the product are of considerable predictive value in determining whether a reagent will be suitable in use. Adherence to these criteria and their continuous reappraisal combined with performance assessment and information on stability are all essential to ensure that reagents are of a quality acceptable to laboratory workers and legislative bodies. The need for manufacturers to document each procedural step and record the results is an essential element of good manufacturing practice now imposed by regulatory bodies. All reputable manufacturers adhere to these requirements, which serve not only to maintain standards but also to form a basis for corrective action.

Fluress, fluorescein and benoxinate: recovery from bacterial contamination.

The ability to recover from bacterial contaminations with Staphylococcus auresus and Pseudomonas aeruginosa was determined for three optometric DPAs: Fluress, fluorescein and benoxinate. Results show that Fluress recovers from contamination more rapidly than benoxinate or fluorescein. This ability to recover from contamination and the relative ease of use of Fluress may make it the DPA choice for a number of optometric procedures including applanation tonometry.

The gingivitis fluorescein test in recruits.

The average individual status of gingival inflammation was evaluated in two groups of 39 and 47 subjects by means of the Gingivitis Fluorescein Test (GFT) and the Sulcus Bleeding Index. The subjects of the test group received a prophylaxis and hygiene instructions which were effective in reducing the gingivitis after 12 days. However, the reduction was not paralleled by a simultaneous decrease of fluorescein recovered in mouthwashings. The oral hygiene of the control group's subjects was not altered and examinations were performed after 7 days. In both groups the average SBI-score did not exceed 1.2 in all examinations. The fluorescein content of the mouthwashings was approximately 10 times reduced when compared with previous findings. No correlation was found between the amounts of fluorescein in mouthwashings and the severity or extent of the clinically assessed gingival inflammation.