Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins

Gap junctions are collections of intercellular channels composed of structural proteins called connexins (Cx). We have examined the functional interactions of the three rodent connexins present in the lens, Cx43, Cx46, and Cx50, by expressing them in paired Xenopus oocytes. Homotypic channels containing Cx43, Cx46, or Cx50 all developed high conductance. heterotypic channels composed of Cx46 paired with either Cx43 or Cx50 were also well coupled, whereas Cx50 did not form functional channels with Cx43. We also examined the functional response of homotypic and heterotypic channels to transjunctional voltage and cytoplasmic acidification. We show that all lens connexins exhibited sensitivity to cytoplasmic acidification as well as to voltage, and that voltage-dependent closure of heterotypic channels for a given connexin was dramatically influenced by its partner connexins in the adjacent cell. Based on the observation that Cx43 can discriminate between Cx46 and Cx50, we investigated the molecular determinants that specify compatibility by constructing chimeric connexins from portions of Cx46 and Cx50 and testing them for their ability to form channels with Cx43. When the second extracellular (E2) domain in Cx46 was replaced with the E2 of Cx50, the resulting chimera could no longer form heterotypic channels with Cx43. A reciprocal chimera, where the E2 of Cx46 was inserted into Cx50, acquired the ability to functionally interact with Cx43. Together, these results demonstrate that formation of intercellular channels is a selective process dependent on the identity of the connexins expressed in adjacent cells, and that the second extracellular domain is a determinant of heterotypic compatibility between connexins.     

Regulation of the raffinose permease of Escherichia coli by the glucose-specific enzyme IIA of the phosphoenolpyruvate:sugar phosphotransferase system

In enteric bacteria, chromosomally encoded permeases specific for lactose, maltose, and melibiose are allosterically regulated by the glucose-specific enzyme IIA of the phosphotransferase system. We here demonstrate that the plasmid-encoded raffinose permease of enteric bacteria is similarly subject to this type of inhibition.     

Toxicity of an antitumor ribonuclease to Purkinje neurons

Purkinje cell toxicity is one of the characteristic features of the Gordon phenomenon, a syndrome manifested by ataxia, muscular rigidity, paralysis, and tremor that may lead to death (Gordon, 1933). Two members of the RNase superfamily found in humans, EDN (eosinophil-derived neurotoxin) and ECP (eosinophil cationic protein), cause the Gordon phenomenon when injected intraventricularly into guinea pigs or rabbits. We have found that another member of the RNase superfamily, an antitumor protein called onconase, isolated from Rana pipiens oocytes and early embryos, will also cause the Gordon phenomenon when injected into the cerebrospinal fluid of guinea pigs at a dose similar to that of EDN (LD50, 3-4 micrograms). Neurologic abnormalities of onconase-treated animals were indistinguishable from those of EDN-treated animals, and histology showed dramatic Purkinje cell loss in the brains of onconase-treated animals. The neurotoxic activity of onconase correlates with ribonuclease activity. Onconase modified by iodoacetic acid to eliminate 70% and 98% of the ribonuclease activity of the native enzyme displays a similar decrease in ability to cause the Gordon phenomenon. In contrast, the homologous bovine pancreatic RNase A injected intraventricularly at a dose 5000 times greater than the LD50 dose of EDN or onconase is not toxic and does not cause the Gordon phenomenon. A comparison of the RNase activities of EDN, onconase, and bovine pancreatic RNase A using three pancreatic RNA substrates demonstrates that onconase is orders of magnitude less active enzymatically than EDN and RNase A. Thus, another member of the RNase superfamily in addition to EDN and ECP can cause the Gordon phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)     

The number of clonogenic cells in a mouse jejunal crypt

A study of eight deaths involving the benzodiazepine flunitrazepam was performed to assess the contribution of this drug to the fatalities. Coronial deaths in Victoria in the 2-year period to mid-1991 were selected in which either flunitrazepam or flunitrazepam and ethanol were the principal toxicological findings. All bodies were subject to a full autopsy by forensic pathologists, and a full toxicological examination. No significant pathology was found at autopsy in any case. Very high concentrations of 7-aminoflunitrazepam, a metabolite of flunitrazepam, were present in all cases. In four cases no other significant drug was detected, whereas in the other four cases there were significant concentrations of ethanol (mean 1.6 g/L). In these two groups of cases the concentrations of 7-aminoflunitrazepam were 0.45 mg/L and 0.16 mg/L, respectively. Only moderate levels of flunitrazepam were detected, suggesting that 7-aminoflunitrazepam is produced postmortem and may be an important marker of flunitrazepam usage. The causes of death in these eight cases were probably either flunitrazepam toxicity or a combination of flunitrazepam and ethanol toxicity. Only one case appeared likely to have been a suicide. Our observations in these cases suggest that flunitrazepam may cause death in the absence of other drugs or significant disease. The presence of ethanol reduces the amount of flunitrazepam needed to cause death.     

Site and parameters of microstimulation: evidence for independent effects on the properties of saccades evoked from the primate superior colliculus

1. Microstimulation is used to investigate how activity in the superior colliculus (SC) contributes to determining the properties of primate saccadic eye movements. The site of collicular stimulation, the duration of the stimulation train, and the frequency of the stimulation train are each varied to examine the relative contributions of the locus, duration, and level of collicular activity to determining saccade amplitude, direction, duration, and velocity. 2. For any given site of stimulation, a relationship between movement amplitude and train duration can be demonstrated. Movement amplitude is a monotonically increasing, but saturating, function of increasing train duration. The size of the largest movement is dictated by the site of stimulation. Within the range over which amplitude can be modulated, movement offset is linked to the offset of the stimulation train. As a result, each decrement or increment in train duration produces a corresponding decrement or increment in movement duration. 3. The peak velocity of an evoked movement is influenced by the frequency of stimulation; a higher frequency of stimulation produces a movement of higher velocity. 4. The effects of train duration and frequency can be traded to produce movements that have comparable amplitudes but different dynamic characteristics; high-velocity movements of short duration and low-velocity movements of long duration can be produced by stimulating with high-frequency, short-duration, and low-frequency, long-duration trains, respectively. Across stimulation frequencies, the amplitude of an evoked movement is best related to the total number of pulses in the stimulation train. 5. Because it is possible to compensate for reduced velocity by increasing the duration of the stimulation train, the same site-specific maximum amplitude can be attained with different frequencies of stimulation. 6. Small, but significant, changes in movement direction occur as a result of varying train duration or train frequency. 7. The latency to movement onset (i.e., interval from stimulation onset to movement onset) depends upon the frequency of stimulation. A higher frequency of stimulation produces a movement of shorter latency. 8. These data demonstrate that both the site of stimulation and the parameters of stimulation contribute to determining the properties of a movement evoked from the primate SC. In doing so, they contradict the results of early microstimulation studies that suggest that the properties of eye movements evoked from the primate SC are determined solely by the site of stimulation. The findings conflict with the traditional view of collicular function that suggests that the collicular motor representation is purely anatomic. Rather, these data support a revised view whereby the locus, duration, and level of collicular activity contribute to determining the properties of a primate saccadic eye movement. According to this view, independent information relating to desired displacement and saccade velocity are extracted from the spatiotemporal profile of collicular activity.     

Antihypertensive agent moxonidine enhances muscle glucose transport in insulin-resistant rats

The sympatholytic antihypertensive agent moxonidine, a centrally acting selective I1-imidazoline receptor modulator (putative agonist), may be beneficial in hypertensive patients with insulin resistance. In the present study, the effects of chronic in vivo moxonidine treatment of obese Zucker rats--a model of severe glucose intolerance, hyperinsulinemia and insulin resistance, and dyslipidemia--on whole-body glucose tolerance, plasma lipids, and insulin-stimulated skeletal muscle glucose transport activity (2-deoxyglucose uptake) were investigated. Moxonidine was administered by gavage for 21 consecutive days at 2, 6, or 10 mg/kg body weight. Body weights in control and moxonidine-treated groups were matched, except at the highest dose, at which final body weight was 17% lower in the moxonidine-treated animals compared with controls. The moxonidine-treated (6 and 10 mg/kg) obese animals had significantly lower fasting plasma levels of insulin (17% and 19%, respectively) and free fatty acids (36% and 28%, respectively), whereas plasma glucose was not altered. During an oral glucose tolerance test, the glucose response (area under the curve) was 47% and 67% lower, respectively, in the two highest moxonidine-treated obese groups. Moreover, glucose transport activity in the isolated epitrochlearis muscle stimulated by a maximally effective insulin dose (13.3 nmol/L) was 39% and 70% greater in the 6 and 10 mg/kg moxonidine-treated groups, respectively (P<.05 for all effects). No significant alterations in muscle glucose transport were elicited by 2 mg/kg moxonidine. These findings indicate that in the severely insulin-resistant and dyslipidemic obese Zucker rat, chronic in vivo treatment with moxonidine can significantly improve, in a dose-dependent manner, whole-body glucose tolerance, possibly as a result of enhanced insulin-stimulated skeletal muscle glucose transport activity and reduced circulating free fatty acids.     

Nonsulfhydryl-reactive phenoxyacetic acids increase aqueous humor outflow facility

PURPOSE: The phenoxyacetic acid, ethacrynic acid (ECA), has potential use in glaucoma therapy because it acts to increase aqueous outflow in vivo and in vitro. In human trabecular meshwork (HTM) cell culture, ECA acts to change cell shape and attachment, effects that have been correlated with microtubule (MT) alterations and chemical sulfhydryl (SH) reactivity. To further explore these actions, we evaluated two non-SH reactive phenoxyacetic acids, inadcrinone and ticrynafen, and the MT-disrupting drug vinblastine. METHODS: Excised bovine and porcine eyes were perfused and outflow facility measured. Calf pulmonary artery endothelial and HTM cells were grown in culture and cytoskeletal effects evaluated after drug treatment. RESULTS: Indacrinone, ticrynafen, and vinblastine all caused an increase in outflow facility. In contrast with ECA, the outflow effects of indacrinone and ticrynafen were not blocked by excess cysteine. Although indacrinone and ticrynafen produced changes in cell shape in vitro, the beta-tubulin staining pattern of treated cells was not altered. Vinblastine caused cell shape change and the expected MT disruption. CONCLUSIONS: Phenoxyacetic acids can increase aqueous outflow facility and alter HTM cell shape and attachment in vitro by a non-SH, non-MT mechanism (which is probably shared also by ECA). These findings suggest the possibility of a broader class of glaucoma drugs that may be directed at the HTM. An understanding of the cellular target for these drugs has implications both for potential glaucoma therapy and for the cytoskeletal mechanisms involved in normal outflow function.     

Humans: the missing link in manufacturing simulation?

Computer based discrete event simulation (DES) is one of the most commonly used aids for the design of automotive manufacturing systems. However, DES tools represent machines in extensive detail, while only representing workers as simple resources. This presents a problem when modelling systems with a highly manual work content, such as an assembly line. This paper describes research at Cranfield University, in collaboration with the Ford Motor Company, founded on the assumption that human variation is the cause of a large percentage of the disparity between simulation predictions and real world performance. The research aims to improve the accuracy and reliability of simulation prediction by including models of human factors.     

A semiautomated electron backscatter diffraction technique for extracting reliable twin statistics

A framework has been developed for extracting reliable twin statistics from a deformed microstructure using crystallographic twin identification techniques with spatially correlated electron backscatter diffraction (EBSD) data. The key features of this analysis are the use of the mathematical definition of twin relationships, the inclination of the common K ₁ plane at a twin boundary, and the correct identification of the parent orientation in a parent/twin pair. Methods for identifying the parent in a parent/twin pair will be briefly discussed and compared. Twin area fractions are then categorized by operative twin systems, number of active twin variants in each system, and corrected twin widths. These statistics are reported here for α-zirconium samples deformed in quasi-static four-point bend beams and in a 100 m/s Taylor cylinder impact test. Analysis of the statistics also begins to reveal the roles that deformation rate and relative orientation of the boundary conditions to the material’s symmetry axes play in determining the twinning activity that accommodates the imposed boundary conditions. These improved twin statistics can help quantify the deformation processes in materials that deform by twinning as well as serve to provide better validation of proposed models of the deformation processes.