Effects of molybdenum on ascorbate-glutathione cycle metabolism in Chinese cabbage (Brassica campestris L. ssp. pekinensis)

A hydroponic trial was conducted to investigate effects of molybdenum (Mo) on ascorbate-glutathione cycle (AsA-GSH cycle) metabolism in Chinese cabbage (Brassica campestris L. ssp. pekinensis). Mo was applied at four rates: 0, 0.01, 0.15 and 1.5 mg l⁻¹. The concentrations of ascorbate, dehydroascorbate, reduced- and oxidized- glutathione, and activities of five key enzymes in the AsA-GSH cycle were studied. The results showed that appropriate Mo application increased the fresh weight of Chinese cabbage, but excess application of Mo (1.5 mg l⁻¹ Mo) decreased the fresh weight. Total ascorbate and reduced ascorbate concentrations in the Chinese cabbage increased with Mo application rates. Although no significant differences existed in DHA concentration between the different Mo regimes, but it has an increase trend with the 0.01 mg l^-1 Mo treatment, and then decreased with the Mo level increasing. No significant difference in GSH concentration was found between the different Mo treatments. Compared with the control, the GSSG concentration decreased significantly in the 0.01 mg l⁻¹ Mo treatment. The activities of APX, MDHAR, DHAR and GR increased due to Mo application. But the activity of AAO decreased with increasing Mo application rates. It is hypothesized that Mo may promote the redox process and regeneration of ascorbic acid, and affect the ability of anti-oxidation in the Chinese cabbage. Responsible Editor: Jian Feng Ma.     

Involvement of nitric oxide in cerebroside-induced defense responses and taxol production in <Emphasis Type="Italic">Taxus yunnanensis</Emphasis> suspension cells

This work was to characterize the generation of nitric oxide (NO) in Taxus yunnanensis cells induced by a fungal-derived cerebroside and the signal role of NO in the elicitation of plant defense responses and taxol production. (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine at 10 μg/ml induced a rapid and dose-dependent NO production in the Taxus cell culture, reaching a maximum within 5 h of the treatment. The NO donor sodium nitroprusside (SNP) potentiated cerebroside-induced H₂O₂ production and cell death. Inhibition of nitric oxide synthase activity by phenylene-1,3-bis(ethane-2-isothiourea) dihydrobromide or scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide partially blocked the cerebroside-induced H₂O₂ production and cell death. Moreover, NO enhanced cerebroside-induced activation of phenylalanine ammonium-lyase and accumulation of taxol in cell cultures. These results are suggestive of a role for NO as a new signal component for activating the cerebroside-induced defense responses and secondary metabolism activities of plant cells. Taxol is a trademark of Bristol-Myers Squibb, Madison, NJ.     

A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine

Native smooth muscle L-type Ca(v)1.2 calcium channels have been shown to support a fraction of Ca(2+) currents with a window current that is close to resting potential. The smooth muscle L-type Ca(2+) channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca(v)1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Ca(v)1.2 channels. Here, we report a smooth muscle L-type Ca(v)1.2 calcium channel splice variant, Ca(v)1.2SM (1/8/9(*)/32/Delta33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca(v)1.2b clone (1/8/9(*)/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Ca(v)1.2CM clone (1a/8a/Delta9(*)/32/33) that is different from the established Ca(v)1.2a (1a/8a/Delta9(*)/31/33). Importantly, Ca(v)1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca(v)1.2b and cardiac Ca(v)1.2CM channels when currents were recorded in either 5 mM Ba(2+) or 1.8 mM Ca(2+) external solutions. This is the first time that a smooth muscle Ca(v)1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.     

The cytosolic protein talin induces an intermediate affinity integrin alphaLbeta2

The integrin alphaLbeta2 mediates leukocyte adhesion and migration that are required for a functional immune system. It is known that inside-out signaling triggers alphaLbeta2 conformational changes, which affect its ligand-binding affinity. At least three alphaLbeta2 affinity states (low, intermediate, and high) were described. The cytosolic protein talin connects alphaLbeta2 to the actin filament. The talin head domain is also known to activate alphaLbeta2 ligand binding. However, it remains to be determined whether talin promotes an intermediate or high affinity alphaLbeta2. In this study using transfectants and T cells, we showed that talin induced an intermediate affinity alphaLbeta2 that adhered constitutively to its ligand intercellular adhesion molecule (ICAM)-1 but not ICAM-3. Adhesion to ICAM-3 was induced when an additional exogenous activating agent was included. Similar profiles were observed with soluble ICAMs. In addition, the intermediate affinity alphaLbeta2 induced by talin allowed adhesion and migration of T cells on immobilized ICAMs.     

Distance-dependent processing of adeno-associated virus type 5 RNA is controlled by 5' exon definition

Adeno-associated virus type 5 (AAV5) is unique among human AAV serotypes in that it uses a polyadenylation site [(pA)p] within the single small intron in the center of the genome. We previously reported that inhibition of polyadenylation at (pA)p, necessary for read-through of P41-generated capsid gene pre-mRNAs which are subsequently spliced, requires binding of U1 snRNP to the upstream donor. Inhibition was reduced as the distance between the cap site and the donor was increased (increasing the size of the 5' exon). Here, we have demonstrated that U1-70K is a key component of U1 snRNP that mediates inhibition of polyadenylation at (pA)p. Furthermore, introduction of a U-rich stretch, predicted to target TIA-1 and thus increase the affinity of U1 snRNP binding to the intervening donor site, significantly augmented inhibition of (pA)p, while depletion of TIA-1 by siRNA increased (pA)p read-through. Finally, artificially tethering the cap binding complex (CBC) components CBP80 and CBP20 upstream of the intron donor increased inhibition of polyadenylation at (pA)p. Our results suggest that interaction with the CBC strengthens U1 snRNP binding to the downstream intron donor in a manner inversely proportional to the size of the 5' exon, thus governing the competition between intron splicing and polyadenylation at (pA)p. This competition must be optimized to program both the levels of polyadenylation of P7- and P19-generated RNA at (pA)p required to produce proper levels of the essential Rep proteins and the splicing of P41-generated RNAs to produce the proper ratio of capsid proteins during AAV5 infection.