Histochemical Observations on the Distribution of Adenosine Triphosphatase in Phloem and Motor Organs in Higher Plants

In a previous communication (Kuo 1964), it was shown by histochemical means that the phloem of cucurbit, as compared with the ground parenchyma, is particularly rich in adenosine triphosphatase (ATPase). A concurrent research by Yen et al. (1965) has succeeded in actually extracting from the vascular bundle of tobacco a crude protein fraction which not only possesses high ATPase activity, but also exhibits viscosity changes on addition of ATP or AMP. Such properties have been known to be characteristic of the contractile proteins constituting the muscle in animals. Although little is known about the actual role played in food transport by the living tissues in phloem, evidence has been accumulating that the active participation of the protoplasm in the phloem is necessary to facilitate sap movement. All these results have tempted us to think that higher plants, just like animals, also derive the energy required for performing mechanical work directly from ATP through the mediation of the contractile protein which is the ATPase itself. Accordingly, the present investigation is engaged in further histochemical observations to locate more precisely ATPase in the sieve element and to map out the distribution of the enzyme in various motor organs of higher plants, with the purpose in mind to see whether the map of enzyme distribution in the tissue has anything to do with its peculiar way of movement. 1. Presence of ATPase in the slime body of the functioning sieve element. In the functioning sieve element, high ATPase activity is displayed at the slime body and the connecting strands traversing the pores of the sieve plate (Plate Ⅰ, figs. 2, 3 & 4). However, as the pores of the sieve plate are partially blocked by the callose, the enzyme reaction becomes lessened. If the plate is completely covered by the callose pad, none could be detected. Since the slime body has been claimed by many workers to be pro- teinaceous in nature (cf. Esau 1950), there should be no surprise that it also possesses enzyme activity and contractile action. This seems to indicate that the slime body and the connecting strands are not mere hindrance to the sap flow but may actively participate in propelling the translocation stream. The fact that the intense ATPase reaction is given in the companion cell (Plate I, figs. 1 & 3) is in conformity with the common notion that the cell is actively involved in the normal functioning of phloem. 2. Enzyme distribution in various motor organs of different sensitivity. The ATPase content in the various motor organs (pulvinus, stamen, tendril, etc.) of different sensitivity has been compared histochemically, ranging from. 1) the highly sensitive organs; e.g. the pulvinus of Mimosa pudica (Plate Ⅱ, fig. 5) and of Oxalis piota (Plate Ⅱ, fig. 6), the stamen of Berberis Watsonae (Plate Ⅲ, fig. 10), the young tendril of Cucurbita maxima Duchesne and Vitis vinifera (Plate Ⅲ, figs. 11 & 12); 2) the moderately sensitive organs; e.g. the pulvinus of Sesbania cannabina (Plate Ⅱ, fig. 8); 3) the feebly sensitive or insensitive organs, e.g. the pulvinus of Robinia pseudoacacia (Plate Ⅱ, fig. 7), the stamen of Brassica campestris var. oleifera (Plate Ⅲ, fig. 9). From the results, it can be seen that the higher the sensitivity of the organ, the greater its enzyme activity. In addition to the morphological assymetry which is typical of plant motor organs, there also exists a physiological gradient, as evidenced by the uneven distribution of ATPase, usually being more intense on the more irritable side. The claim made recently by Aimi (1963) and others that the sudden collapse of the pulvinus of Mimosa on stimulation is not a direct result of the loss of turgidity as is generally assumed, but an immediate manifestation of the active vacuolar contraction in the motor cells is supported by the high ATPase content in the motor cells in our investigation.     

The Effect of Dietary Prebiotics and Probiotics on Body Weight,Large Intestine Indices,and Fecal Bile Acid Profile in Wild Type and IL10?/? Mice

Previous studies have suggested roles of probiotics and prebiotics on body weight management and intestinal function. Here, the effects of a dietary prebiotic, inulin (50 mg/g diet), and probiotic, Bfidobacterium animalis subsp. lactis (Bb12) (final dose verified at 10⁵ colony forming unit (cfu)/g diet, comparable to human consumption), were determined separately and in combination in mice using cellulose-based AIN-93G diets under conditions allowed for the growth of commensal bacteria. Continuous consumption of Bb12 and/or inulin did not affect food intake or body, liver, and spleen weights of young and adult mice. Fecal bile acid profiles were determined by nanoESI-MS/MS tandem mass spectrometry. In the presence of inulin, more bacterial deconjugation of taurine from primary bile acids was observed along with an increased cecal weight. Consumption of inulin in the absence or presence of Bb12 also increased the villus cell height in the proximal colon along with a trend of higher bile acid sulfation by intestinal cells. Feeding Bb12 alone at the physiological dose did not affect bile acid deconjugation and had little effect on other intestinal indices. Although interleukin (IL)10-null mice are susceptible to enterocolitis, they maintained the same body weight as the wild type mice under our specific pathogen-free housing condition and showed no signs of inflammation. Nevertheless, they had smaller cecum suggesting a mildly compromised intestinal development even before the disease manifestation. Our results are consistent with the notion that dietary factors such as prebiotics play important roles in the growth of intestinal microbiota and may impact on the intestinal health. In addition, fecal bile acid profiling could potentially be a non-invasive tool in monitoring the intestinal environment.     

Synthesis, and cytotoxic and antiplatelet activities of oxime- and methyloxime-containing flavone, isoflavone, and xanthone derivatives

A series of oxime- and methyloxime-containing flavone, isoflavone, and xanthone derivatives (1-12) were synthesized (Scheme) and evaluated for their cytotoxic (Table 1) and antiplatelet activities (Table 2). The in vitro anticancer assay indicated that the cytotoxicity of structurally related compounds decreases in the order isoflavones (7a-7c) > flavones (8a-8c) > xanthones (9a-9c), electron-releasing substituents (R) on the Ph ring being favorable (mean GI50 values of 2.84, 12.3, and 20.9 microM for 7c, 8c, and 9c, resp.). The inhibition of platelet aggregation induced by arachidonic acid (AA) similarly decreased from the isoflavone 1 (IC50 = 2.97 microM) to the flavone 2 (7.70 microM) to the xanthone 3 (inactive). Thereby, compound 1 seems to be a promising lead, since it was not only the most-potent aggregation inhibitor (IC50 = 2.97 microM), but was also found to be noncytotoxic at a concentration of 100 microM.     

Application of bovine pituitary extract and polyglycolide/poly(lactide-co-glycolide) scaffold to the cultivation of bovine knee chondrocytes

In vitro cultivation of primary bovine knee chondrocytes (BKCs), using bovine pituitary extract (BPE) and porous scaffolds composed of polyglycolide (PGA) and 85/15 poly(lactide-co-glycolide) (PLGA), was investigated. Here, BPE was prepared from fresh bovine pituitaries, and cylindrical PGA/PLGA scaffolds with various chemical compositions were fabricated by solvent merging/particulate leaching method. Experimental results showed that in microcarrier systems, the rate of BKC growth on PGA surfaces is faster than that on PLGA surfaces, and the decrease in the medium pH value of BKCs-adsorbed PGA particles is faster than that of BKCs-adsorbed PLGA particles. After 28-day construct cultivation, the BKC amount and the content of glycosaminoglycans and collagen per construct increased with BPE protein concentration. For a constant BPE protein concentration, a higher PGA percentage in scaffold leads to a better biological environment for the growth of BKCs and the synthesis of extracellar matrices.     

A general procedure for the purification of human beta-secretase expressed in Escherichia coli

Expression and purification of human beta-secretase (BACE1) in bacteria have been plagued with issues concerning solubility, inhomogeneous N-terminus, and lack of enzymic activity. Several forms of the mature human BACE1 have been expressed in Escherichia coli with different N-terminal extensions and without the C-terminus transmembrane domain. Although each of the proteins expresses in inclusion bodies, a generalized protocol has been developed to solubilize, refold, and purify these BACE1 variants. The resultant proteins are homogeneous and monodispersed in solution. Each possesses a unique N-terminus. Activity assays using the peptide substrate 7-methoxycoumarin-4-yl-SEVNLDAEFK-2,4-dinitrophenyl-RR, corresponding to the beta-secretase cleavage sequence in the amyloid precursor protein with the Swedish mutations of N(670)L(671) substituting for the residues K(670)M(671), reveal a kcat and KM of 9.3 min(-1) and 55 microM, respectively.     

Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence

Tetrodotoxin-resistant (TTX-R) Na(+) channels are much less susceptible to external TTX but more susceptible to external Cd(2+) block than tetrodotoxin-sensitive (TTX-S) Na(+) channels. Both TTX and Cd(2+) seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La(3+), Zn(2+), Ni(2+), Co(2+), and Mn(2+) also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd(2+), the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na(+) flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na(+) currents than those in outward Na(+) current, signaling exit of the blocker along with the Na(+) flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na(+) current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na(+) flow-dependent unblocking of the bound La(3+) from the open Na(+) channel, whereas channel inactivation cannot happen with any discernible speed in the La(3+)-blocked channel. Thus, the selectivity filter of Na(+) channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.     

Block of inactivation-deficient Na+ channels by local anesthetics in stably transfected mammalian cells: evidence for drug binding along the activation pathway

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na(+) channels with higher affinities. However, an alternative view suggests that activation of Na(+) channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na(+) channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC(50)) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 +/- 3.3 microM (n = 5) and 81.7 +/- 10.6 microM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC(50) values for resting-channel block at -140 mV were >12-fold higher than those for open-channel block. With 300 microM benzocaine, rapid time-dependent block (tau approximately 0.8 ms) of inactivation-deficient Na(+) currents occurred at +30 mV, but such a rapid time-dependent block was not evident at -30 mV. The peak current at -30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na(+) channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.