叶面喷施亚精胺对高温胁迫下番茄叶绿素合成代谢的影响

以设施番茄栽培品种‘金棚朝冠’幼苗为试验材料,研究叶面喷施0.25 mmol·L~(-1)亚精胺(Spd)对高温胁迫下(38℃/28℃,昼/夜)番茄幼苗生长及叶绿素合成过程中前体物质含量、关键酶活性和叶绿素含量的影响,探讨Spd缓解番茄高温胁迫伤害的生理机制。结果表明:(1)高温胁迫显著抑制了番茄幼苗的生长,叶面喷施Spd可有效缓解高温胁迫对番茄幼苗地上部生长的抑制作用,但对于地下部的生长无显著缓解作用。(2)高温胁迫下番茄叶片叶绿素合成前体物质δ-氨基酮戊酸(ALA)和胆色素原(PBG)的含量显著提高,而尿卟啉原Ⅲ(UroⅢ)、原卟啉Ⅸ(ProtoⅨ)、镁-原卟啉Ⅸ(Mg-protoⅨ)和原叶绿素酸(Pchl)的含量显著降低,证明叶绿素前体由PBG向UroⅢ的转化受阻,导致叶绿素a(Chla)、总叶绿素(Chlt)含量和Chla/Chlb显著降低。(3)叶面喷施Spd能增强高温胁迫下胆色素原脱氨酶(PBGD)活性,有效缓解了高温胁迫对PBG到UroⅢ转化的阻碍作用,促进PBG之后叶绿素合成前体物质的合成,Chla含量和Chla/Chlb显著增加。研究发现,高温胁迫显著抑制番茄幼苗的生长,叶面喷施Spd可通过显著增强PBGD活性来缓解由PBG向UroⅢ的受阻程度,促进高温胁迫下番茄叶片的叶绿素前体合成,提高叶绿素含量和番茄植株的生长量,减轻高温胁迫对番茄幼苗生长的伤害。     

Tartrate-resistant acid phosphatase 5B circulates in human serum in complex with alpha2-macroglobulin and calcium

Tartrate-resistant acid phosphatase (TRACP) is an enzyme with unknown biological function. In addition to its acid phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS) at neutral pH. Two forms of TRACP circulate in human serum, macrophage-derived TRACP 5a and osteoclast-derived TRACP 5b. Here we have studied the circulating forms of the osteoclast-derived TRACP 5b in rat and human serum. In human serum, TRACP 5b circulates in a large complex that contained α₂M and calcium. On the contrary, rat serum TRACP 5b circulates as a free molecule. Formation of the TRACP 5b complex in vitro decreased significantly the ROS generating activity of TRACP 5b without affecting its phosphatase activity. These results suggest that the complex formation may be necessary to eliminate the formation of the harmful ROS in the neutral pH of serum.     

Effect of phage therapy on the turnover and function of peripheral neutrophils

The aim of this investigation was to establish the impact of phage therapy on the turnover and function of circulating neutrophils in 37 patients with suppurative bacterial infections. We determined the levels of circulating neutrophils and their precursors before therapy, after 3 weeks of therapy, and at a distant time interval (3 months) following the beginning of therapy. In addition, we measured the ability of neutrophils to phagocytize Staphylococcus aureus in vitro. Eight healthy blood donors served as a control group. The results showed that, among the studied parameters, the significant changes involved neutrophil precursor count and the ability of neutrophils to phagocytize bacteria. The percentage of neutrophils in patients before therapy was lower than in healthy donors (mean 58.0, versus 61.4). This value dropped further in patients after 3 months of following the therapy (mean 55.6). The content of neutrophil precursors, on the other hand, was lower in healthy donors than in patients before therapy (mean 2.5, versus 3.8). After 3 weeks of the therapy and after 3 months, the levels of neutrophil precursors were significantly higher (mean 4.8 and 4.9, respectively) than in control donors. The phagocytic index was lower in patients before therapy than in control donors (mean 66.3, versus 70.1) and decreased further after 3 weeks of therapy (mean 59.0) and after 3 months (mean 59.6). The results of this investigation indicate that successful phage therapy accelerates the turnover of neutrophils, accompanied by a decrease in their ability to phagocytize bacteria.     

A TRPC1 Protein-dependent Pathway Regulates Osteoclast Formation and Function

Ca²⁺ signaling is essential for bone homeostasis and skeletal development. Here, we show that the transient receptor potential canonical 1 (TRPC1) channel and the inhibitor of MyoD family, I-mfa, function antagonistically in the regulation of osteoclastogenesis. I-mfa null mice have an osteopenic phenotype characterized by increased osteoclast numbers and surface, which are normalized in mice lacking both Trpc1 and I-mfa. In vitro differentiation of pre-osteoclasts derived from I-mfa-deficient mice leads to an increased number of mature osteoclasts and higher bone resorption per osteoclast. These parameters return to normal levels in osteoclasts derived from double mutant mice. Consistently, whole cell currents activated in response to the depletion of intracellular Ca²⁺ stores are larger in pre-osteoclasts derived from I-mfa knock-out mice compared with currents in wild type mice and normalized in cells derived from double mutant mice, suggesting a cell-autonomous effect of I-mfa on TRPC1 in these cells. A new splice variant of TRPC1 (TRPC1ϵ) was identified in early pre-osteoclasts. Heterologous expression of TRPC1ϵ in HEK293 cells revealed that it is unique among all known TRPC1 isoforms in its ability to amplify the activity of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, mediating store-operated currents. TRPC1ϵ physically interacts with Orai1, the pore-forming subunit of the CRAC channel, and I-mfa is recruited to the TRPC1ϵ-Orai1 complex through TRPC1ϵ suppressing CRAC channel activity. We propose that the positive and negative modulation of the CRAC channel by TRPC1ϵ and I-mfa, respectively, fine-tunes the dynamic range of the CRAC channel regulating osteoclastogenesis.     

前体物对石蒜悬浮细胞生长和生物碱积累的影响

为探究苯丙氨酸、酪氨酸和酪胺3种前体物对石蒜悬浮细胞系生长和生物碱积累的影响。通过向培养基添加不同浓度的3种前体物,以及同时添加苯丙氨酸和酪氨酸,考察其对细胞生长量及细胞中生物碱累积的影响。结果表明:苯丙氨酸对细胞的生长和生物碱的积累影响不明显;酪氨酸和酪胺作用显著:添加200μmol/L酪氨酸,细胞中生物碱的含量是对照组的2.56倍,其中力可拉敏和加兰他敏含量为3.77 mg/g和4.46 mg/g,分别是对照组的6.61倍和6.97倍;添加200μmol/L酪胺,细胞中生物碱含量是对照组的2.63倍,力可拉敏和加兰他敏含量为4.45 mg/g和5.14 mg/g分别是对照组的9.08倍和9.18倍;在200μmol/L酪氨酸的基础上添加苯丙氨酸没有明显的增效作用。表明添加酪氨酸和酪胺对细胞生长及生物碱生物合成具有显著的促进作用     

Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, -aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO₂. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA^Glu, ATP, Mg²⁺, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[³H]glutamate or 1-[¹⁴C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[¹⁴C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the subgroup of purple bacteria.Abbreviations ALA -aminolevulinic acid - DTT dithiothreitol - PALP pyridoxal phosphate - SDS sodium dodecyl sulfate - tricine N-tris-(hydroxymethyl)methylglycine     

RNase E cleavage in the 5' leader of a tRNA precursor

In this study, we have used various tRNA(Tyr)Su3 precursor (pSu3) derivatives that are processed less efficiently by RNase P to investigate if the 5' leader is a target for RNase E. We present data that suggest that RNase E cleaves the 5' leader of pSu3 both in vivo and in vitro. The site of cleavage in the 5' leader corresponds to the cleavage site for a previously identified endonuclease activity referred to as RNase P2/O. Thus, our findings suggest that RNase P2/O and RNase E activities are of the same origin. These data are in keeping with the suggestion that the structure of the 5' leader influences tRNA expression by affecting tRNA processing and indicate the involvement of RNase E in the regulation of cellular tRNA levels.     

The inhibitors of cyclin-dependent kinases and GSK-3β enhance osteoclastogenesis

Osteoclasts are multinucleated cells with bone resorption activity that is crucial for bone remodeling. RANK‐RANKL (receptor activator of nuclear factor κB ligand) signaling has been shown as a main signal pathway for osteoclast differentiation. However, the molecular mechanism and the factors regulating osteoclastogenesis remain to be fully understood. In this study, we performed a chemical genetic screen, and identified a Cdks/GSK-3β (cyclin-dependent kinases/glycogen synthase kinase 3β) inhibitor, kenpaullone, and two Cdks inhibitors, olomoucine and roscovitine, all of which significantly enhance osteoclastogenesis of RAW264.7 cells by upregulating NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) levels. We also determined that the all three compounds increase the number of osteoclast differentiated from murine bone marrow cells. Furthermore, the three inhibitors, especially kenpaullone, promoted maturation of cathepsin K, suggesting that the resorption activity of the resultant osteoclasts is also activated. Our findings indicate that inhibition of GSK-3β and/or Cdks enhance osteoclastogenesis by modulating the RANK–RANKL signaling pathway.     

Impaired Bone Homeostasis in Amyotrophic Lateral Sclerosis Mice with Muscle Atrophy

There is an intimate relationship between muscle and bone throughout life. However, how alterations in muscle functions in disease impact bone homeostasis is poorly understood. Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by progressive muscle atrophy. In this study we analyzed the effects of ALS on bone using the well established G93A transgenic mouse model, which harbors an ALS-causing mutation in the gene encoding superoxide dismutase 1. We found that 4-month-old G93A mice with severe muscle atrophy had dramatically reduced trabecular and cortical bone mass compared with their sex-matched wild type (WT) control littermates. Mechanically, we found that multiple osteoblast properties, such as the formation of osteoprogenitors, activation of Akt and Erk1/2 pathways, and osteoblast differentiation capacity, were severely impaired in primary cultures and bones from G93A relative to WT mice; this could contribute to reduced bone formation in the mutant mice. Conversely, osteoclast formation and bone resorption were strikingly enhanced in primary bone marrow cultures and bones of G93A mice compared with WT mice. Furthermore, sclerostin and RANKL expression in osteocytes embedded in the bone matrix were greatly up-regulated, and β-catenin was down-regulated in osteoblasts from G93A mice when compared with those of WT mice. Interestingly, calvarial bone that does not load and long bones from 2-month-old G93A mice without muscle atrophy displayed no detectable changes in parameters for osteoblast and osteoclast functions. Thus, for the first time to our knowledge, we have demonstrated that ALS causes abnormal bone remodeling and defined the underlying molecular and cellular mechanisms.