The C-terminus of neuronal Kv2.1 channels is required for channel localization and targeting but not for NMDA-receptor-mediated regulation of channel function.

The delayed rectifier voltage-gated potassium channel Kv2.1 underlies a majority of the somatic K(+) current in neurons and is particularly important for regulating intrinsic neuronal excitability. Various stimuli alter Kv2.1 channel gating as well as localization of the channel to cell-surface cluster domains. It has been postulated that specific domains within the C-terminus of Kv2.1 are critical for channel gating and sub-cellular localization; however, the distinct regions that govern these processes remain elusive. Here we show that the soluble C-terminal fragment of the closely related channel Kv2.2 displaces Kv2.1 from clusters in both rat hippocampal neurons and HEK293 cells, however neither steady-state activity nor N-methyl-d-aspartate (NMDA)-dependent modulation is altered in spite of this non-clustered localization. Further, we demonstrate that the C-terminus of Kv2.1 is not necessary for steady-state gating, sensitivity to intracellular phosphatase or NMDA-dependent modulation, though this region is required for localization of Kv2.1 to clusters. Thus, the molecular determinants of Kv2.1 localization and modulation are distinct regions of the channel that function independently.

Ca2+ release through ryanodine receptors regulates skeletal muscle L-type Ca2+ channel expression.

Skeletal muscle obtained from mice that lack the type 1 ryanodine receptor (RyR-1), termed dyspedic mice, exhibit a 2-fold reduction in the number of dihydropyridine binding sites (DHPRs) compared with skeletal muscle obtained from wild-type mice (Buck, E. D., Nguyen, H. T., Pessah, I. N., and Allen, P. D. (1997) J. Biol. Chem. 272, 7360-7367 and Fleig, A., Takeshima, H., and Penner, R. (1996) J. Physiol. (Lond.) 496, 339-345). To probe the role of RyR-1 in influencing L-type Ca(2+) channel (L-channel) expression, we have monitored functional L-channel expression in the sarcolemma using the whole-cell patch clamp technique in normal, dyspedic, and RyR-1-expressing dyspedic myotubes. Our results indicate that dyspedic myotubes exhibit a 45% reduction in maximum immobilization-resistant charge movement (Q(max)) and a 90% reduction in peak Ca(2+) current density. Calcium current density was significantly increased in dyspedic myotubes 3 days after injection of cDNA encoding either wild-type RyR-1 or E4032A, a mutant RyR-1 that is unable to restore robust voltage-activated release of Ca(2+) from the sarcoplasmic reticulum (SR) following expression in dyspedic myotubes (O'Brien, J. J., Allen, P. D., Beam, K., and Chen, S. R. W. (1999) Biophys. J. 76, A302 (abstr.)). The increase in L-current density 3 days after expression of either RyR-1 or E4032A occurred in the absence of a change in Q(max). However, Q(max) was increased 85% 6 days after injection of dyspedic myotubes with cDNA encoding the wild-type RyR-1 but not E4032A. Because normal and dyspedic myotubes exhibited a similar density of T-type Ca(2+) current (T-current), the presence of RyR-1 does not appear to cause a general overall increase in protein synthesis. Thus, long-term expression of L-channels in skeletal myotubes is promoted by Ca(2+) released through RyRs occurring either spontaneously or during excitation-contraction coupling.

Prolonged depolarization promotes fast gating kinetics of L-type Ca2+ channels in mouse skeletal myotubes.

The effects of prolonged conditioning depolarizations on the activation kinetics of skeletal L-type calcium currents (L-currents) were characterized in mouse myotubes using the whole-cell patch clamp technique. The sum of two exponentials was required to adequately fit L-current activation and enabled determination of both the amplitudes (A(fast) and A(slow)) and time constants (tau(fast) and tau(slow)) of each component comprising the macroscopic current. Prepulses sufficient to activate (200 ms) or inactivate (10 s) L-channels did not alter tau(fast), tau(slow), or the fractional contribution of either the fast (A(fast)/(A(fast) + A(slow)) or slow (A(slow)/(A(fast) + A(slow))) amplitudes of subsequently activated L-currents. Prolonged depolarizations (60 s to +40 mV) resulted in the conversion of skeletal L-current to a fast gating mode following brief repriming intervals (3-10 s at -80 mV). Longer repriming intervals (30-60 s at -80 mV) restored L-channels to a predominantly slow gating mode. Accelerated L-currents originated from L-type calcium channels since they were completely blocked by a dihydropyridine antagonist (3 microM nifedipine) and exhibited a voltage dependence of activation similar to that observed in the absence of conditioning prepulses. The degree of L-current acceleration produced following prolonged depolarization was voltage dependent. For test potentials between +10 and +50 mV, the fractional contribution of Afast to the total current decreased exponentially with the test voltage (e-fold approximately 38 mV). Thus, L-current acceleration was most significant at more negative test potentials (e.g. +10 mV). Prolonged depolarization also accelerated L-currents recorded from myotubes derived from RyR1-knockout (dyspedic) mice. These results indicate that L-channel acceleration occurs even in the absence of RyR1, and is therefore likely to represent an intrinsic property of skeletal L-channels. The results describe a novel experimental protocol used to demonstrate that slowly activating mammalian skeletal muscle L-channels are capable of undergoing rapid, voltage-dependent transitions during channel activation. The transitions underlying rapid L-channel activation may reflect rapid transitions of the voltage sensor used to trigger the release of calcium from the sarcoplasmic reticulum during excitation-contraction coupling.

Apoptosis in human Jurkat T cells after culture with live Taenia crassiceps cysticerci in vitro.

This study examines the effects of Taenia crassiceps cysticerci on the viability of a human T lymphocyte cell line (Jurkat). Both budding and non-budding T. crassiceps metacestodes were cultured over 24 and 48 h in the presence of Jurkat cells. Cell viability decreased with increasing numbers of cysticerci, particularly budding cysticerci. Single cell gel electrophoresis (comet) analysis, which grades DNA damage, showed a significant increase in apoptosis at 24 and 48 h. The morphology of treated cells was determined using acridine orange with the classical morphology of apoptotic bodies seen to increase with increasing cysticerci numbers over time. These results indicate that parasite-induced apoptosis occurs during murine cysticercosis. Such a mechanism may be important in survival of other metacestode infections of medical or veterinary importance.

The effect of recurrent practice at home on the acceptability of capillary blood glucose readings. Accuracy of self blood glucose testing.

OBJECTIVE: To judge how reliably patients perform capillary blood glucose testing over time with recurrent practice at home and to assess if a clinic glucose meter is an acceptable alternative to the clinical laboratory for monitoring patient performance. RESEARCH DESIGN AND METHODS: We compared capillary blood glucose readings obtained by patients with their own equipment and the venous blood glucose determinations by the clinical laboratory at three biweekly visits during the initial phase in 40 subjects attending the diabetes clinic at the Veterans' Affairs Medical Center in Phoenix, Arizona. We also compared patient-generated readings using their own equipment and the readings obtained by the clinic glucose meter and strips at five weekly visits during the second phase in 11 subjects who continued further participation. Error grid analysis was used for both comparisons. Capillary blood glucose readings obtained with clinic glucose meter and strips on one hand were correlated with venous blood glucose levels determined by the clinical laboratory. RESULTS: During the initial phase, 30 subjects consistently obtained clinically acceptable comparisons (zone A on the error grid, i.e., within 20% of the laboratory value) or improved over time, 9 subjects showed deterioration, and 1 subject failed to obtain zone A results on any of the visits. Three subjects who had consistently obtained zone A results during the initial phase maintained their performance, whereas eight subjects who had failed to achieve zone A values by the end of the initial phase gradually improved and ultimately achieved zone A values by the end of the study. A highly significant correlation was noted between clinic meter readings and laboratory values (r = 0.93, P < 0.00001). CONCLUSIONS: Clinically acceptable user proficiency in capillary blood glucose testing can be maintained in most subjects, with recurrent intensive education during follow-up clinic visits. Therefore, we recommend that these comparisons be performed and patient's technique be observed at each visit to monitor their performance. The clinic glucose meter is a suitable alternative to a clinical laboratory for user proficiency checks.

Acceptability of capillary blood glucose testing with Companion 2 after intensive individual patient training.

This study determined the accuracy and precision of the MediSense Companion 2 Blood Glucose Monitoring System in the hands of newly trained patients, after individual training. 101 subjects attending a diabetes clinic at a Veterans Affairs Medical Center participated in the study. Subjects were individually trained in the use of the Companion 2 system. They performed 10 practice control solution tests prior to two capillary blood glucose tests. Venous blood was collected within 15 minutes for comparative laboratory analysis. The comparisons were performed by both the linear regression and the total Grid analysis. The correlation between the first capillary blood glucose test results and the laboratory values was highly significant (r = 0.94, p = 0.0001). Total Error Analysis also revealed that the Systematic Error (SE), Random Error (RE) and Total Error (TE) were all well within the acceptable norms at two glucose levels of 5.5 mmol/(100 mg/dL) and 11.1 mmol/L (200 mg/dL); (SE < 15%, RE < 10% and TE < 15%). Therefore, this study demonstrates that Companion 2 provides precise and accurate values during capillary blood glucose testing by newly trained subjects following intensive individual training.

Frozen human serum reference material for standardization of sodium and potassium measurements in serum or plasma by ion-selective electrode analyzers.

Three interlaboratory round-robin studies (RR1, RR2, and RR3) were conducted to identify a serum-based reference material that would aid in the standardization of direct ion-selective electrode (ISE) measurements of sodium and potassium. Ultrafiltered frozen serum reference materials requiring no reconstitution reduced between-laboratory variability (the largest source of imprecision) more than did other reference materials. ISE values for RR3 were normalized by the use of two points at the extremes of the clinical range for sodium (i.e., 120 and 160 mmol/L), with values assigned by the flame atomic emission spectrometry (FAES) Reference Method. This FAES normalization of ISE raw values remarkably improved all sources of variability and unified the results from seven different direct ISE analyzers to the FAES Reference Method value. Subsequently, a three-tiered, fresh-frozen human serum reference material was produced to the specifications developed in RR1-RR3, was assigned certified values for sodium and potassium by Definitive Methods at the National Institute of Standards and Technology (NIST), and was made available in 1990 to the clinical laboratory community as a Standard Reference Material (SRM); it is now identified as SRM 956. Albeit retrospectively, we show how applying an FAES normalization step identical to that used in RR4/5 to the ISE data for SRM 956 after the NIST Definitive Method values were known, consistently moved the ISE results for RR3 closer to the true value for Na+ and K+.

The role of categorization in visual search for orientation.

Visual search for 1 target orientation is fast and virtually independent of set size if all of the distractors are of a single, different orientation. However, in the presence of distractors of several orientations, search can become inefficient and strongly dependent on set size (Exp. 1). Search can be inefficient even if only 2 distractor orientations are used and even if those orientations are quite remote from the target orientation (e.g. 20 degrees or even 40 degrees away, Exp. 2). Search for 1 orientation among heterogeneous distractor orientations becomes more efficient if the target orientation is the only item possessing a categorical attribute such as steep, shallow (Exp. 3), tilted left or tilted right (Exp. 4), or simply tilted (Exps. 5 and 6). Orientation categories appear to be 1 of several strategies used in visual search for orientation. These serve as a compromise between the limits on parallel visual processing and the demands of a complex visual world.

Different rates of metabolism of two chloroacetanilide herbicides in pioneer 3320 corn.

The in vivo rates of uptake and detoxification of alachlor and metolachlor were determined using Pioneer corn 3320 seedlings. Equal amounts of the radiolabeled herbicides were applied to etiolated coleoptiles and, at various intervals after treatment, the unabsorbed radioactivity was removed and quantified. Analysis of 80% methanol extracts by reverse phase liquid chromatography showed no significant differences in the rate of uptake of metolachlor and alachlor. However, the rate of glutathione conjugation of alachlor in vivo was two- to threefold greater than the rate for metolachlor at 2 and 4 hours after herbicide application. Since the initial step in detoxification is conjugation of the chloroacetanilide to glutathione, the activities of the enzymes responsible for conjugation, the glutathione-S-transferases (GST) were also analyzed in vitro, using crude extracts and the purified GST enzymes. The specific activities of the extracts were consistent with the results in vivo. Using alachlor as a substrate, the specific activity for glutathione conjugation was almost threefold higher than that for metolachlor. Kinetic analysis of purified GST III indicates that the enzyme has a higher affinity for alachlor (K(m)app = 1.69 millimolar) than for metolachlor (K(m)app = 8.9 millimolar).

Adaptation of the resting states of accommodation.

Without an adequate stimulus to focus, the eyes accommodate for a "resting state" that averages about 1.6 diopter (D) (62 cm). The resting state can be made more "myopic" by adaptation to a near accommodative or vergence stimulus. In the present experiment, 21 subjects were tested for adaptation to both types of stimuli. Resting state was measured before and after adaptation in the dark (dark focus) and in the presence of an illuminated empty field. The authors found that preadaptation resting states were more myopic in the light field than in the dark test conditions; accommodative and vergence stimuli produce myopic shifts in dark and light field resting states; and a subset of the subjects show much larger aftereffects in the light field conditions. These subjects also show the largest difference between preadaptation dark- and light-field measures. Differences between dark- and light-field measures of resting state in these and other experiments may require a re-examination of the hypothesis that there is a single resting state for each subject.

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants.

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid-derived plant transformation vectors. The progeny of the self-fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross-protection may be a general method for preventing viral disease in plants.

Expression of mouse dihydrofolate reductase gene confers methotrexate resistance in transgenic petunia plants.

Transgenic petunia plants containing an altered (Leu22----Arg22) mouse dihydrofolate reductase gene fused to the cauliflower mosiac virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming petunia leaf disks with an Agrobacterium tumefaciens strain carrying the chimeric gene. Transformants were directly selected for and rooted on medium containing 1 microM methotrexate (MTX). The chimeric gene was present in the regenerated plants at one to three copies and produced the expected 950-nucleotide-long transcript based on Southern and Northern hybridization analyses, respectively. Leaf pieces from the regenerated transgenic plants were able to form callus when cultured on medium containing 1 microM MTX and were able to incorporate 32P into high-molecular-weight DNA in the presence of greater than 100 microM MTX, thus demonstrating that the chimeric mouse dhfr gene was fully functional and useful as a selectable marker in plant transformation experiments. To date, this is the first report of successful expression of a vertebrate gene in transformed plant cells.

Cloning and expression of a cDNA encoding a maize glutathione-S-transferase in E. coli.

The isolation and characterization of a family of maize glutathione-S-transferases (GST's) has been described previously. These enzymes are designated GSTs I, II and III based on size, substrate specificity and responsiveness to safeners. GST III has been shown to act on the herbicide alachlor as well as the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Clones were isolated from a maize cDNA library in lambda gt10. Three clones contained the entire coding region for GST III. The sequences of these clones were consistent with the known amino terminal GST III protein sequence. Moreover, expression of one of these clones in E. coli resulted in a GST activity as measured with both CDNB and alachlor, proving that at least one of the clones encodes an active GST III species. With the enzyme expressed in E. coli it will become possible to study enzyme structure-function relationships ex planta. While a number of different GST proteins are present in maize tissue the GST III gene is present in single or low copy in the genome.

Fatigue and structural change: two consequences of visual pattern adaptation.

In a tilt aftereffect (TAE) paradigm, 2 min of adaptation produced an aftereffect that decayed almost completely within 4 min. Four minutes of adaptation produced a TAE that lasted more than 2 wk. Two modes of adaptation contribute to the TAE and account for other aftereffects: short-term fatigue, produced very quickly and long-term structural change, requiring more extended adaptation.

Pre-stereoptic binocular vision in infants.

In a preferential looking experiment, identical patterns (vertical stripes) were presented to both eyes on one of two screens while orthogonal patterns (vertical stripes in one eye and horizontal stripes in the other) were presented on the other screen. Most infants younger than 3.5 months of age originally showed a preference for the dichoptic (interocularly orthogonal) pattern. At an average age of 3.5 months, however, they showed a sudden shift of preference from this pattern to the interocularly identical pattern. The full shift from a preference for one stimulus to the other (both statistically significant) occurred within a few weeks in most cases. The onset age of the shift in preference agreed with the onset age of fusion-rivalry discrimination found in a previous study (Birch et al., 1985). The original preference for the bincularly orthogonal patterns may be interpreted as a preference for a grid (interocularly emergent intersections) over a grating, judging from results of two control experiments. These data suggest that the pre-stereoptic system non-selectively combines information from the two eyes without regard to edge orientation because it loses eye-of-origin information at a relatively early stage of binocular visual processing. Thus, the pre-stereoptic system does not have the capability of interocular suppression. The theoretical and clinical significance of the new findings are discussed along with a neuronal model of cortical development of ocular segregation and binocular pathways.

Kaposi's sarcoma in lymph nodes: histological study of lesions from 16 cases in Malawi.

Biopsy of lymph nodes containing Kaposi's sarcoma from 16 patients showed that the tumour is identical in appearance with that of Kaposi's sarcoma of the skin, regardless of the age of the patient or the mode of presentation. Spread of tumour along sinusoids throughout the lymph node was seen only in the cases of two children with generalised lymphadenopathy, but discrete deposits were present both in lymph nodes regional to skin lesions and in lymph nodes from patients who had presented with primary lymphadenopathy. The reaction of the remainder of incompletely involved nodes was variable. No transition was seen between Kaposi's sarcoma and malignant lymphoma.

Kaposi's sarcoma: histopathological study of 159 cases from Malawi.

The histological features of 168 lesions of Kaposi's sarcoma from 159 patients in Malawi, where the disease is commoner than elsewhere, were characteristic. All showed well-developed areas of tumour and could be grouped fairly readily into those with a mixed pattern, a predominantly spindle cell pattern, and an anaplastic group, though intermediate patterns were seen. Hyaline bodies were present in nearly all tumours of skin. The cell of origin of Kaposi's sarcoma is uncertain and possibly has multipotential capabilities. Differential diagnosis may be difficult. Clinically the lesion may be confused with granuloma pyogenicum and may also be like it histologically. The presence of hyaline bodies and deposits of haemosiderin indicate Kaposi's sarcoma. The spindle cell predominant type may be confused with leiomyoma, leiomyosarcoma, or fibrosarcoma. The presence of hyaline bodies and the formation of vascular channels between spindle cells point to a diagnosis of Kaposi's sarcoma.