The Sensitivity of the Human Kv1.3 (hKv1.3) Lymphocyte K+ Channel to Regulation by PKA and PKC is Partially Lost in HEK 293 Host Cells

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative PKA Ser⁴⁶⁸ phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K⁺ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35 mV (half activation, V _0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and −17.3 and −39.0 mV (V _0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at −30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni²⁺ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively. 8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced. Received: 18 June 1997/Revised: 7 October 1997     

Foraging tactics and home range of dark chub in a Japanese river

The foraging tactics of dark chub, Zacco temmincki were classified into sit-and-wait where fish remained stationary in running water, stop where fish remained stationary in still water, and cruising where fish moved widely near the water surface. Sit-and-wait foragers primarily fed upon invertebrates that were carried by the current or fell onto the water surface, whereas cruising foragers fed upon algae and invertebrates that fell onto the water surface. Stopping individuals only fed occasionally. The percentage of sit-and-wait foraging increased as current velocity increased, whereas the percentage of cruising foraging was negatively affected by both current velocity and fish density. Larger individuals and males employed sit-and-wait foraging more frequently than smaller individuals and females. Hom range was large for cruising foragers. Aggressive interactions were frequent among sit-and-wait foragers. Although behavioural patterns of dark chub markedly differed between running and still waters, the gut content and growth rate did not differ significantly between the two habitats.     

An Inactivating Inward-rectifying K Current Present in Prolactin Cells from the Pituitary of Lactating Rats

Primary cultures containing a high percentage of lactotrophs were obtained by dissociating the pituitary of rats following 14–18 days of lactation. Lactotrophs with a distinctive appearance were recorded after 1–35 days in vitro and identified by immunocytochemical staining for prolactin. Whole-cell voltage clamp measurements in isotonic KCl solution from a holding potential of −40 mV revealed the presence of inward-rectifying K currents with a time-dependent, Na⁺-independent inactivation at potentials negative to −60 mV. The time for complete inactivation was strikingly different between lactotrophs, varying between 1 sec and more than 5 sec at −120 mV, and was not related to time in culture. The reversal potential shifted 59 mV (25°C) for a tenfold change in external K⁺ concentration, demonstrating the selectivity of the channel for K⁺ over Na⁺. The inward-rectifying K current was blocked by 5 mm Ba²⁺ and partially blocked by 10 mm TEA. Chloramine-T (1 and 2 mm) produced a total block of the inward-rectifying K current in lactotrophs. Thyrotropin-releasing hormone (500 nm) significantly reduced the inward-rectifying K current in about half of the lactotrophs. This current is similar to the inward-rectifying K current previously characterized in clonal somatomammotrophic pituitary cells (GH₃B₆). The variability of the rate of inactivation of this current in lactotrophs and its responsiveness to TRH is discussed. Received: 28 September 1995/Revised: 11 December 1995     

Biosynthesis of proteoglycans by isolated rabbit glomeruli

Isolated rabbit glomeruli were incubated in vitro with 35SO4 in order to analyze the proteoglycans synthesized. Proteoglycans extracted with 4 M guanidine HCl from whole isolated glomeruli and from purified glomerular basement membrane (GBM) were analyzed by gel filtration chromatography. Two types of sulfated proteoglycans were found to be synthesized by rabbit glomeruli and these contained either heparan sulfate or chondroitin/dermatan sulfate glycosaminoglycan chains. These glycosaminoglycans were characterized by their sensitivity to selective degradation by nitrous acid or chondroitinase ABC, respectively. The major proteoglycan extracted from the whole glomeruli was a chondroitin/dermatan sulfate species (75%), while purified GBM contained mostly heparan sulfate (70%). The glycosaminoglycan chains were estimated to be about 12,000 molecular weight which is consistent with previous estimates for similar molecules extracted from the rat GBM.     

Proteoglycan changes during restoration of transparency in corneal scars

Corneal scars generated in rabbits by penetrating wounds are initially opaque but become transparent within a year. Previous studies have shown that the corneal stroma consists of proteoglycans and collagen fibrils spaced at regular intervals and that the interfibrillar spaces, the presumed location of proteoglycans, are abnormally large in opaque scars. In the present study, the size and glycosaminoglycan composition of the corneal stromal proteoglycans were determined in corneal scars during the restoration of transparency. The results showed that initially opaque scars which contained the large interfibrillar spaces also contained unusually large chondroitin sulfate proteoglycans with glycosaminoglycan side chains of normal size. These opaque scars also lacked the keratan sulfate proteoglycan but did contain hyaluronic acid. In the 1-year-old scars there was a restoration of normal interfibrillar spacing, and a return to corneal stromal proteoglycans of normal size and composition. These correlations suggest that the corneal stromal proteoglycans may play a fundamental role in regulating corneal collagen fibril spacing.     

Mathematical modelling of dynamics and control in metabolic networks. I. On Michaelis-Menten kinetics

As a starting point for modeling of metabolic networks this paper considers the simple Michaelis-Menten reaction mechanism. After the elimination of diffusional effects a mathematically intractable mass action kinetic model is obtained. The properties of this model are explored via scaling and linearization. The scaling is carried out such that kinetic properties, concentration parameters and external influences are clearly separated. We then try to obtain reasonable estimates for values of the dimensionless groups and examine the dynamic properties of the model over this part of the parameter space. Linear analysis is found to give excellent insight into reaction dynamics and it also gives a forum for understanding and justifying the two commonly used quasi-stationary and quasi-equilibrium analyses. The first finding is that there are two separate time scales inherent in the model existing over most of the parameter space, and in particular over the regions of importance here. Full modal analysis gives a new interpretation of quasi-stationary analysis, and its extension via singular perturbation theory, and a rationalization of the quasi-equilibrium approximation. The new interpretation of the quasi-steady state assumption is that the applicability is intimately related to dynamic interactions between the concentration variables rather than the traditional notion that a quasi-stationary state is reached, after a short transient period, where the rates of formation and decomposition of the enzyme intermediate are approximately equal. The modal analysis reveals that the generally used criterion for the applicability of quasi-stationary analysis that total enzyme concentration must be much less than total substrate concentration, et much less than St, is incomplete and that the criterion et much less than Km much less than St (Km is the well known Michaelis constant) is the appropriate one. The first inequality (et much less than Km) guarantees agreement over the longer time scale leading to quasi-stationary behavior or the applicability of the zeroth order outer singular perturbation solution but the second half of the criterion (Km much less than St) justifies zeroth order inner singular perturbation solution where the substrate concentration is assumed to be invariant. Furthermore linear analysis shows that when a fast mode representing the binding of substrate to the enzyme is fast it can be relaxed leading to the quasi-equilibrium assumption. The influence of the dimensionless groups is ascertained by integrating the equations numerically, and the predictions made by the linear analysis are found to be accurate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Temporal variations in some plant and soil P pools in two pasture soils of widely different P fertility status

Temporal variations in plant production, plant P and some soil P (and N) pools were followed over 21 months in two New Zealand pasture soils of widely different P fertility status. Plant growth rates, and herbage composition at the high-fertility site, were closely linked to soil water use, with growth rates falling when soil water deficits exceeded 60 mm. Herbage P concentrations reflected P fertility, and varied with season, being generally higher in winter and lower in summer. A similar temporal pattern was also observed for labile organic P (NaHCO₃-extractable P₀) in both soils. In the low-fertility soil in spring, net mineralization was especially strong, but from early winter net immobilization occurred. Surprisingly, Olsen P also changed temporally in the high-fertility soil. The microbial biomass remained fairly constant throughout the year, whereas the P content of the biomass varied seasonally. Although microbial biomass was not a useful index of soil fertility, highest microbial P₀ contents coincided with periods of maximum labile P₀ mineralization, when herbage production was also at a peak. Net N-mineralization in the low-fertility soil, in contrast to the high-fertility soil, was low but varied seasonally, under standardised incubation conditions. Soil P and N dynamics were apparently synchronised in the low-fertility soil through soil microbial processes, with mineral N being negatively correlated with microbial P₀ in samples collected two months later. The results of this investigation suggest that the demands of rapid and sustained pasture growth in spring and early summer can best be met by maximising the build-up of organic matter during the preceding autumn and winter. This practice could help to alleviate the common problem of feed shortage in North Island hill country pastures in late winter-early spring.     

土壤干旱条件下氮素营养对小麦水分状况和光合作用的影响

轻度土壤干旱下,小麦叶片仍能维持较好的水分状况,高氮营养对叶片光合作用有明显的促进作用。中度以上土壤干旱下,叶片水势和相对含水量明显降低,高氮叶片降低的幅度显著大于低氮,同时叶片净光合率(P_n)也趋于降低,高氮叶片降低的幅度较大。高氮叶片的叶肉光合活性明显大于低氮叶片,干旱下P_n降低与其气孔限制作用较大有关。高氮叶片的渗透调节大于低氮叶片,但渗透调节对气孔导度和P_n的维持有限。     

Accumulation of poly-β-hydroxybutyrate in a methane-enriched,halogenated hydrocarbon-degrading soil column: implications for microbial community structure and nutritional status

The prokarotic, endogenous storage polymer poly--hydroxybutyrate (PHB) accumulated in soil from a methane-enriched, halogenated hydrocarbon-degrading soil column. Based on phospholipid ester-linked fatty acid (PLFA) profiles, this mocrocosm has been previously reported to be significantly enhanced in type II methanotrophs. Two strains analysed of the type II methanotroph Methylobacterium organophilum were found to contain PHB, with PHB/PLFA ratios similar to those determined for the methane-enriched soil column, suggesting that methanotrophic bacteria enriched in the methane-amended column produced PHB. Control soil and sodium azide-inhibited material, in which methanotroph markers were below detection, did not contain PHB. Biochemical assays, based on the differences observed, can be used to monitor shifts in microbial biomass, community structure and nutritional status of systems used to model microbial biotransformation processes. Further manipulative experiments with pure methanotrophic bacteria will increase our understanding of the mechanism by which PHB is produced. This study illustrates, however, that biochemical procedures have the potential to monitor the stimulated populations of a native soil microbial community capable of degrading pollutants. Such data may ultimately provide information to assist in the selection and optimization of favorable conditions for the adaption of pollutant biotransformation processes to aquifers.