Cell cycle regulation of myosin-V by calcium/calmodulin-dependent protein kinase II.

Organelle transport by myosin-V is down-regulated during mitosis, presumably by myosin-V phosphorylation. We used mass spectrometry phosphopeptide mapping to show that the tail of myosin-V was phosphorylated in mitotic Xenopus egg extract on a single serine residue localized in the carboxyl-terminal organelle-binding domain. Phosphorylation resulted in the release of the motor from the organelle. The phosphorylation site matched the consensus sequence of calcium/calmodulin-dependent protein kinase II (CaMKII), and inhibitors of CaMKII prevented myosin-V release. The modulation of cargo binding by phosphorylation is likely to represent a general mechanism regulating organelle transport by myosin-V.

A liquid-stable reagent for lactic acid levels. Application to the Hitachi 911 and Beckman CX7.

We evaluated the use of a new lactate oxidase-based reagent for the determination of serum and plasma lactic acid levels with the Hitachi 911 (Roche Diagnostics, Indianapolis, IN) and the Beckman CX7 (Beckman Instruments, Brea, CA). Evaluation studies demonstrated on-board stability of at least 3 months and a calibration stability of more than 5 months. Within- and between-day imprecision of this reagent was less than 2% for both applications. The reagent is free of the deleterious effects of triglyceride up to levels of 1,400 mg/dL (15.8 mmol/L), bilirubin to concentrations of 24.6 mg/dL (420 mumol/L), and hemoglobin, from lysed erythrocytes, to levels of more than 0.3 g/dL (3.0 g/L). When used on the Hitachi 911 for the determination of plasma lactate concentrations, the reagent correlates with the Dade aca III (Dade International, Deerfield, IL). When applied to the Beckman CX7 for the determination of serum lactate levels, the method correlates with the Beckman method.

Regulation of melanosome movement in the cell cycle by reversible association with myosin V.

Previously, we have shown that melanosomes of Xenopus laevis melanophores are transported along both microtubules and actin filaments in a coordinated manner, and that myosin V is bound to purified melanosomes (Rogers, S., and V.I. Gelfand. 1998. Curr. Biol. 8:161-164). In the present study, we have demonstrated that myosin V is the actin-based motor responsible for melanosome transport. To examine whether myosin V was regulated in a cell cycle-dependent manner, purified melanosomes were treated with interphase- or metaphase-arrested Xenopus egg extracts and assayed for in vitro motility along Nitella actin filaments. Motility of organelles treated with mitotic extract was found to decrease dramatically, as compared with untreated or interphase extract-treated melanosomes. This mitotic inhibition of motility correlated with the dissociation of myosin V from melanosomes, but the activity of soluble motor remained unaffected. Furthermore, we find that myosin V heavy chain is highly phosphorylated in metaphase extracts versus interphase extracts. We conclude that organelle transport by myosin V is controlled by a cell cycle-regulated association of this motor to organelles, and that this binding is likely regulated by phosphorylation of myosin V during mitosis.

Using a cutoff of <10 ppm for breath hydrogen testing: a review of five years' experience.

To assess the clinical use of the breath hydrogen test in a large community hospital using a <10 ppm cutoff, we reviewed 222 tests performed over a 5-year period to evaluate patients for disaccharidase deficiency or bacterial overgrowth of the small intestine. Of these, the vast majority (195) were for lactose malabsorption, although fructose (17), sucrose (8) and lactulose (2) were also occasionally administered. One hundred eleven tests (50 percent) were positive, with an increase of at least 10 ppm hydrogen above the fasting level and a maximum value most commonly observed (42.3 percent of the time) at 3 hours post-administration. Only 34 patients (15.3 percent) had symptoms noted during the test, as compared with 185 (83.3 percent) who had experienced persistent intestinal problems prior to the test. Recent conditions which may have caused intestinal distress, such as transient disaccharidase deficiency, infections, surgery or other disorders like Crohn's disease, ulcerative colitis or food poisoning, were recorded in only 14 cases. Patterns consistent with bacterial overgrowth of the small intestine were observed in only 3 cases. Of 111 positives, 9 cases had increases between 10 and 20 ppm hydrogen and 7 showed the increase in the 3-hour sample, possibly reflecting a delayed transit through the intestine. Final diagnoses in 6 of these where information was available were for conditions other than malabsorption. We conclude that using a rise of 10 ppm to interpret a positive test does not contribute significantly to an increased frequency of false positives, but that patients with increases between 10 and 20 ppm probably are not lactase deficient.

Comparison of the HemoCue beta-glucose photometer and reflotron for open heart surgery.

The HemoCue beta-glucose photometer (Angelholm, Sweden) was evaluated for use in monitoring blood glucose in both diabetic and nondiabetic patients undergoing open heart surgery. Because occasional discrepancies were noted in patients with low total proteins when the Reflotron (Boehringer Mannheim, Indianapolis, IN) was used for this purpose, the effects of protein and hematocrit on glucose results from both instruments were investigated and compared with plasma values from a Paramax 720 ZX (Baxter Healthcare, Irvine, CA). Linear-regression analysis of the HemoCue results (y) versus Paramax (x) yielded y = 0.956x + 0.35, r2 = 0.980, with Sy/x = 0.57 mmol/L (10.3 mg/dL). Results from the Reflotron (y) versus Paramax (x) yielded y = 1.075x - 0.10, r2 = 0.964, with Sy/x = 0.99 mmol/L (17.8 mg/dL). Bias plots of (HemoCue-Paramax) or (Reflotron-Paramax) versus glucose, hematocrit, or protein showed no effect of glucose on the results from either instrument and no effect of protein or hematocrit on the HemoCue findings. The Reflotron, however, showed a positive bias of up to 3.5 mmol/L (63 mg/dL) at protein concentrations between 30-40 g/L (3.0-4.0 g/dL) and a possible positive bias at low hematocrit levels.

A comparison of chromogen test strip (Chemstrip bG) and serum glucose values in newborns.

Although glucose oxidase-peroxidase chromogen test strips are frequently used to estimate serum glucose values in newborns, previous studies have not evaluated multiobserver variability of test strip readings and have included few infants with hypoglycemia. We compared values of 272 samples of serum glucose with values simultaneously obtained by chromogen test strips (Chemstrip bG) in newborns. The diagnostic sensitivity of a chromogen test strip less than 2.2 mmol/L for predicting a serum glucose level less than 1.9 mmol/L was 86% (95% confidence interval [CI], 75% to 94%), with 78% specificity (95% CI, 73% to 84%). The positive predictive value in our specimens, with a 21% prevalence of serum glucose levels less than 1.9 mmol/L, was 52% (95% CI, 41% to 62%), with a negative predictive value of 95% (95% CI, 91% to 100%). Fifty-eight of our serum glucose values were less than 1.9 mmol/L and the levels obtained by chromogen test strip were greater than or equal to 2.2 mmol/L in 8 of these cases. Review of these 8 cases showed that a delay in performing the laboratory glucose oxidase serum glucose could account for the discrepancy in 2 cases. Chromogen test strips are readily available and easy to use, but more sensitive, specific, accurate, and precise methods of serum glucose screening in newborns are needed.

Electrophoresis and the isomune-LD and LD-1 immuno methods compared for measurement of lactate dehydrogenase isoenzyme-1.

We evaluated three different methods for measuring lactate dehydrogenase (EC 1.1.1.27) isoenzyme LD-1 activity--agarose gel electrophoresis and two immunoassays, Isomune-LD (Roche) and LD-1 Immuno (Seragen)--in patients' samples for which measurement of creatine kinase-MB was ordered. Regression analyses of the comparisons gave the following results: LD-1 (%) from Isomune-LD (y) vs electrophoresis (x) (n = 51), y = 1.05x + 1.99, r = 0.92; LD-1 (%) from LD-1 Immuno (y) vs electrophoresis (n = 27), y = 1.05x + 3.94, r = 0.88; LD-1 (%) from LD-1 Immuno (y) vs Isomune-LD (x) (n = 41), y = 1.06x + 0.48, r = 0.95. Comparison by Student's paired t-test yielded significant differences between the mean values by electrophoresis and both Isomune-LD (P less than 0.005) and LD-1 Immuno (P less than 0.001), but no significant difference between the two immunoassays (P greater than 0.2). Analyzing these results by the overlap index, we conclude that electrophoresis shows the best clinical correlation followed, in order, by the Isomune-LD and the LD-1 Immuno methods. Both immunoassays are simpler and more rapid than electrophoresis, but in our hands the Isomune-LD method demonstrated greater precision and better correlation with electrophoretic values.

Alkaline phosphatase in the assessment of choledocholithiasis before surgery.

The pre-operative diagnosis of choledocholithiasis, or common bile-duct stones, is important in patients with cholelithiasis. Intraoperative cholangiography or choledochoscopy followed by exploration of the common bile duct could be limited to those patients with stones, if an adequate pre-operative diagnosis could be made. Many clinicians use pre-operative total alkaline phosphatase level alone or in combination with bilirubin level to determine the presence or absence of choledocholithiasis. Predictive value theory was used to analyze data reported by others to assess the value of alkaline phosphatase level alone or in combination with bilirubin level in identifying patients with choledocholithiasis. The authors conclude that alkaline phosphatase level is not useful either alone or in combination with bilirubin level in determining the presence of common bile duct stones in patients with cholelithiasis.

Comparative sensitivities of flame atomic absorption spectrophotometry (AAS) and molecular absorption spectrophotometry (MAS).

A sensitivity comparison was made between conventional atomic absorption spectrophotometry (AAS) and conventional molecular absorption spectrophotometry (MAS) for the serum trace metals, copper, iron, and zinc. The sensitivity aspect considered was absorbances was obtained for concentrations in the solutions analyzed using unit lightpaths of 1 cm and 10 cm for MAS and AAS, respectively. A distinction was made between procedural sensitivity and measurement sensitivity where the latter represents molar absorptivity. Some was given to the concepts of procedural sensitization by concentration of the analyte through the use of lyophilization, extraction or ashing. The misuse of data obtained with scale expanders is described as a commonly occurring phenomenon of both AAS and MAS and a source of confusion in the understanding of measurement sensitivity.