Correlation of open cell-attached and excised patch clamp techniques

The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na⁺ channel, located on the apical surface of A₆ cells, to determine whether the nystatininduced open cell-attached patch could serve as an alternative configuration.Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and E _rev was zero in symmetrical Na⁺ solutions, indicating similar Na⁺ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3–4pS) and E _rev (75 mV), when the bath was perfused with a high K:low Na solution (E _Na=80 mV), were nearly equal in both patch configurations.Our results therefore indicate that the nystatininduced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.We thank Dr. Olaf S. Andersen for his suggestions in the development of the open cell-attached recording technique. This work was supported by a National Institutes of Health grant (DK-18061)     

Low night temperatures have a differential effect on the diurnal cycling of gene expression in cold–sensitive and tolerant tomatoes*

Abstract. Comparisons were made between the changes in mRNA levels induced by low night temperatures in the cold–sensitive tomato and two altitudinal ecotypes of the wild species L. hirsutum. Changes in mRNA levels were detected by resolving in vitro translation products of poly(A)⁺ RNA by 2-D PAGE. The treatment was applied by first growing plants in a thermoperiod of 25/18°C and then switching to 25/6°C. All tomatoes displayed a diurnal cycling in which a set of mRNAs accumulated at the end of the 18°C nights, whereas another accumulated at the end of the 25°C days. The accumulation of night specific mRNAs was inhibited by 6°C nights in the cold sensitive tomatoes while that of the tolerant one was only marginally affected. All tomatoes showed a similar reduction in the apparent turnover rate of the day specific mRNAs during the 6°C nights. Finally, low night temperatures induced the accumulation of six to eight mRNAs in all genotypes. This number increased by 15 in L. esculentum after the seventh night and are likely involved in stress response rather than acclimation/tolerance. The tomato is proposed as a genetic model to discriminate genes involved in acclimation/tolerance from those involved in stress response.     

Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians

Xanthomonas campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting.mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A⁺ RNA in rabbit reticulocyte lysate in the presence of ³⁵S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these had disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians.     

Increased Concentrations of 3-Hydroxykynurenine in Vitamin B6 Deficient Neonatal Rat Brain

Increased concentrations of the endogenous tryptophan metabolite 3-hydroxykynurenine (3-HK) were measured in the brains of vitamin B6 deficient neonatal rats. Mean concentrations of 3-HK in B6 deficient cerebellum, corpus striatum, frontal cortex, and pons/medulla ranged from 9.7 to 18.6 and 102 to 142 nmol/g of wet tissue at 14 and 18 days of age, respectively. 3-HK was not significantly increased in control neonatal or adult rat brain, vitamin B6 deficient rat brain at 7 days of age, or in brains from adult rats deprived of vitamin B6 for 58 days. The administration of daily intraperitoneal injections of vitamin B6 from the 14th to the 18th day of age decreased the concentration of 3-HK to control levels. 3-HK has been shown by other investigators to produce seizures when injected into the cerebral ventricles of adult rodents. Thus, our studies show the accumulation in brain of a putative endogenous convulsant as the result of a nutritional deficiency.     

Initiation of early breeding in a population of Microtus townsendii (Rodentia) with the secondary plant compound 6-MBOA

Summary Feeding the secondary plant compound 6-Methoxybenzoxazolinone (6-MBOA) during winter to a free living population of Microtus townsendii accelerated breeding in females. Both the recruitment of young and sexual maturation of these young were advanced by four weeks in comparison with a control population. Overwintered females supplied with 6-MBOA matured at a lower body weight than control females. But there was no such weightat-maturity difference between the grids for males. During April and May 72% of all juveniles captured came from the experimental population. Winter reproduction and early recruitment of young stimulated by 6-MBOA could have important population consequences for these voles.     

Phytochrome-mediated regulation of β-amylase mRNA level in mustard (Sinapis alba L.) cotyledons

Phytochrome, activated by continuous red light, increases the amount of total polyadenylated RNA during photomorphogenesis of mustard (Sinapis alba L.) cotyledons. In-vitro translation of total polyadenylated RNA in a reticulocyte translation system has shown that the activity of translatable -amylase mRNA is increased by phytochrome about threefold in the 3-d-old cotyledons, based on equal amounts of polyadenylated RNA, and about eightfold on a per-cotyledon basis. Cordycepin prevents the accumulation of translatable -amylase mRNA. It is concluded that the phytochrome-mediated control of -amylase synthesis is exerted on the level of mRNA synthesis. During seedling development in continuous red light, a phytochrome-dependent increase of -amylase mRNA can be observed at least 6 h before the onset of -amylase synthesis. If, after a period of enzyme synthesis, phytochrome action is interrupted by long-wavelength far-red light followed by darkness, -amylase mRNA as well as -amylase synthesis remain at a high level for 8–10 h and then decline sharply. It is concluded that -amylase mRNA, having an apparent lifetime of the order of 8–10 h, can be formed under the influence of phytochrome during early seedling development but it activates -amylase synthesis only after a lag-phase of about 8 h, when the cotyledons acquire competence to synthesize the enzyme. The consequences of these findings for the signal-transduction chain of phytochrome are discussed.Abbreviations EDTA Na₂-ethylenediaminotetraacetic acid - PAGE polyacrylamide gel electrophoresis - poly(A)⁺RNA polyadenylated mRNA - P_r, P_fr red- and far-red-absorbing forms of phytochrome - SDS sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Auxin-induced rapid changes in translatable mRNAs in tobacco cell suspension

When 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent tobacco cell suspensions, one normal and one transformed by Agrobacterium tumefaciens, were subcultured on hormone-lacking medium the stationary phase of the cell cycle was reached earlier than on medium containing 2,4-D. Addition of the auxin 2,4-D could restore cell division activity within 10–12 h for the most rapidly reacting cell line. The cell-division response was characterized as being auxin-specific and optimal with 2,4-D at 2.2 10^-6 M. Although the cell lines used showed different characteristics, both reacted with a rapid increase in at least three mRNA species within 1 or 2 h after 2,4-D application. Two, 2,4-D-induced protein spots, seen after in-vitro translation, had the same characteristics (MWs 35 kilodaltons (kDa) and 25 kDa with isoelectric points of 7.1 and 6.3, respectively) in both cell lines. Water-treated controls did not show alterations in the translatable mRNA populations. This indicates that the accumulation of the corresponding mRNAs is an early hormone-induced event. Since cell division is the only measurable reaction found after auxin application, cell systems as described here offer excellent possibilities for studying early auxin-induced changes at the molecular level preceding mitosis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - kDa kilodalton     

Inhibition and promotion by light of the accumulation of translatable mRNA of the light-harvesting chlorophyll a/b-binding protein of photosystem II

The amount of in-vitro translatable mRNA of the light-harvesting chlorophyll a/b-binding protein (LHCP) of photosystem II strongly increases in darkness (D) after a 5-min red-light pulse while continuous illumination of mustard seedlings with far-red (FR), red or white light leads only to a slight increase in the amount of translatable LHCP-mRNA. No increase can be observed after a long-wavelength FR (RG9-light) pulse. However, a FR pretreatment prior to the RG9-light pulse strongly increase LHCP-mRNA accumulation in subsequent D. This is not observed in the case of the mRNA for the small subunit of ribulose-1.5-bisphosphate carboxylase. The increase of LHCP-mRNA in D after a FR pretreatment can be inhibited by a reillumination of the seedlings with FR. The inhibition of LHCP-mRNA accumulation during continuous illumination with FR and the strong increase in D following a FR illumination was found to be independent of chlorophyll biosynthesis since no correlation between chlorophyll biosynthesis and translatable LHCP-mRNA levels could be detected. Even strong changes in the amount of intermediates of chlorophyll biosynthesis caused by application of levulinic acid or 5-aminolevulinic acid did not affect LHCP-mRNA levels. Therefore, we conclude that the appearance of LHCP-mRNA is inhibited during continuous illumination, even though illumination leads to a storage of a light singal which promotes accumulation of translatable LHCP-mRNA in D.Abbreviations c continuous - Chl chlorophyll - D darkness - FR far-red light (3.5 W·m^-2) - LHCP light-harvesting chlorophyll a/b-binding protein of photosystem II - NF Norfluration - PChl protochlorophyll(ide) - Pfr far-red absorbing form of phytochrome - Ptot total phytochrome - R red light (6.8 W·m^-2) - RG9-light long-wavelength FR (10 W·m^-2) - SSU small subunit of ribulose-1.5-bisphosphate carboxylase - WL white light - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system