Directed discovery of bivalent peptide ligands to an SH3 domain

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.     

The C-terminal domain of dimeric serine hydroxymethyltransferase plays a key role in stabilization of the quaternary structure and cooperative unfolding of protein: domain swapping studies with enzymes having high sequence identity

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share approximately 77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal-5'-phosphate microenvironment similar to that of the wild-type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins. Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding. These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway.     

Zymosan-induced luminol-dependent chemiluminescence response of circulating and extravasated leukocytes in experimental sepsis

This study examines a concurrent profiling of circulating and extravasated polymorphonuclear leukocytes (PMNs) in a rat model of experimental sepsis. Fecal peritonitis was induced in Wistar male rats by intraperitoneal instillation of a fecal suspension in saline (1:1 w/v). Blood and peritoneal fluid were collected 8 h following fecal inoculation for the evaluation of inflammatory response of PMNs using zymosan-induced luminol-dependent chemiluminescence. Fifty microliters of pre-diluted blood or peritoneal fluid samples were mixed with 150 microl of reaction mixture (4 x 10(-4) M luminol+50 microg opsonized zymosan+0.1% gelatin in Hank's balanced salt solution) and the chemiluminescence signal was measured in a luminometer at 37 degrees C. Fecal peritonitis caused a significant leukocytopenia (3540+/-297 mm(-3) versus control value of 7525+/-711 mm(-3), p < 0.001) accompanied by massive infiltration of PMNs in the peritoneal cavity (34700+/-4006 versus 7325+/-425 mm(-3), p < 0.001). The phagocytic activity of circulating blood PMNs was down-regulated whereas a significant up-regulation was observed in the activity of PMNs from peritoneal fluid. In conclusion, this study clearly demonstrates sepsis-induced alterations in both blood and peritoneal fluid PMNs and their quantitative assessment may be helpful in disease evaluation and designing effective therapies.     

Synthesis and immunomodulatory properties of selected oxazolone derivatives

Eleven oxazolone derivatives were synthesized and characterized by (1)H NMR, EI, IR and UV spectroscopic and CHN analysis. Three compounds, 4-[(E)-(4-nitrophenyl)methylidene]-2-phenyl-1,3-oxazol-5(4H)-one (11), 4-[(E)-(4-methoxyphenyl)methylidene]-2-methyl-1,3-oxazol-5-one (12) and 4-[(E)-(4-nitrophenyl)methylidene]-2-methyl-1,3-oxazol-5(4H)-one (13) were screened for phagocyte chemiluminescence, neutrophil chemotaxis, T-cell proliferation, cytokine production from mononuclear cells and cytotoxicity. 4-[(E)-(4-Nitrophenyl)methylidene]-2-methyl-1,3-oxazol-5(4H)-one (13) was found to be the most potent immunomodulator in the series.     

培养基成分对杜仲愈伤组织生长及次生代谢产物含量的影响

以Bs＋0．5mg／L NAA＋0．5mg／L BA为基本培养基,研究了B5培养基中8种主要无机盐浓度对杜仲愈伤组织生长及绿原酸和总黄酮两种次生代谢产物含量的影响。结果表明：在1000～5000mg／L范围内增加培养基中KNO3的含量有利于愈伤组织生长,B5培养基中当KNO3的浓度达到2／3时,绿原酸和总黄酮含量及产量最高;(NH4)2SO4以4／3原浓度时对愈伤组织生长量、总黄酮含量及产量最高,对绿原酸的含量则是其为原浓度的1／3时最高;MgSO4以2／3浓度对生长量及1／3浓度对绿原酸、总黄酮积累最高;NaH2PO4、CaCl2和MnSO4以原浓度的愈伤组织生长和次生代谢产物合成最好;ZnSO4和FeSO4的原浓度愈伤组织的生长量最大,而1／3浓度的绿原酸和总黄酮含量最高。     

Noninvasive remote ischemic preconditioning for global protection of skeletal muscle against infarction

The aim of this study was to investigate the efficacy and mechanism of action of a noninvasive remote ischemic preconditioning (IPC) technique for the protection of multiple distant skeletal muscles against ischemic necrosis (infarction). It was observed in the pig that three cycles of 10-min occlusion and reperfusion in a hindlimb by tourniquet application reduced the infarction of latissimus dorsi (LD), gracilis (GC), and rectus abdominis (RA) muscle flaps by 55%, 60%, and 55%, respectively, compared with their corresponding control (n = 6, P < 0.01) when they were subsequently subjected to 4 h of ischemia and 48 h of reperfusion. This infarct-protective effect of remote IPC in LD muscle flaps was abolished by an intravenous bolus injection of the nonselective opioid receptor antagonist naloxone (3 mg/kg) 10 min before remote IPC and a continuous intravenous infusion (3 mg/kg) during remote IPC and by an intravenous bolus injection of the selective delta 1-opioid receptor antagonist 7-benzylidenealtrexone maleate (3 mg/kg). However, this infarct-protective effect of remote IPC was not affected by an intravenous bolus injection of the ganglionic blocker hexamethonium chloride (20 mg/kg) or the nonspecific adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (10 mg/kg) or by a local intra-arterial injection of the adenosine1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (3 mg/muscle flap) given 10 min before remote IPC. It was also observed that this remote IPC of skeletal muscle against infarction was associated with a slower rate of muscle ATP depletion during the 4 h of sustained ischemia and a reduced muscle neutrophilic myeloperoxidase activity after 1.5 h of reperfusion. These observations led us to speculate that noninvasive remote IPC by brief cycles of occlusion and reperfusion in a pig hindlimb is effective in global protection of skeletal muscle against infarction. This infarct-protective effect is most likely triggered by the activation of opioid receptors in the skeletal muscle, and remote IPC is associated with an energy-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.     

沙木蓼和沙枣对地下水位变化的生理生态响应Ⅰ.叶片养分、叶绿素、可溶性糖和淀粉的变化

在甘肃民勤沙生植物园内利用植物蒸腾耗水量观测场,研究了两种优势旱生植物沙木蓼（Atraphaxis frutescens)和沙棘（Elaeagnus angustifolia)叶片中的叶绿素、可溶性糖,淀粉和N、P、K、含量等对不同地下水深度（1－3．4m）的响应。结果表明：1）1.4m,2.4m和3．4m 3种不同地的地下水深度处理,产生了3种差异显著的土壤水分梯度;2）地下水深度的变化导致了这两种旱生植物叶绿素a、叶绿素b、叶绿素总量、叶绿素a与叶绿素b的比值等的显著变化（P＜0．01）;3）地下水深度的增加引起了两种植物叶片可溶性糖含量的升高和淀粉含量的降低;4）地下水深度的增加引起了两种植物叶片中N、P、K含量的降低;5）不同的地下水深度引起沙棘和沙木蓼叶绿素a、叶绿素b、叶绿素总量、叶绿素a与叶绿素b的比值、N、P、K含量,可溶性糖和淀粉增加或减少的程度不同。沙棘是非豆科固氮植物,两者的差异是否与固氮作用相关还有待于进一步研究。     

Ribozymes: A modern tool in medicine

Since the discovery of ribozymes and self-splicing introns, it has been estimated that this biological property of RNA combined with other recombinant DNA technologies would become a tool to combat viral diseases and control oncogenes. These goals seem like a distinct possibility now. However, there is still a lot to be learned about the mobility of RNA inside the cells and the cellular factors that can impede ribozyme action in order to capitalize fully on the targeted RNA inactivation property of ribozymes. The most effective approach to maximize ribozyme function in a complex intracellular environment is to understand as much as possible about the intracellular fate of the RNA that is being targeted. As new techniques in cell biology become available, such understanding will be less problematic. Fundamental studies of ribozyme structure and mechanism of catalysis are flourishing both at the academic and industrial level and it can be expected that many new developments will continue to take place in these areas in the near future. Here, we review the design, stability and therapeutic application of these technologies illustrating relevant gene targets and applications in molecular medicine. Relevant problems in implementation of the technology, group I and II introns and the differences in applications, ribozyme structure and the application of this technology to virus attack and oncogene downregulation are discussed. Also some of the latest RNA-based technologies such as siRNA, RNA/DNA duplexes and RNA decoys have been introduced.     

Effects of the trematode Microphallus turgidus on locomotion and prey capture in the grass shrimp Palaemonetes pugio

The grass shrimp, Palaemonetes pugio, is common in estuaries and marshes along the east coast of the USA and is frequently infected with metacercariae of the trematode, Microphallus turgidus. To test whether or not M. turgidus has an effect on intermediate host behaviour, the length of time spent swimming and walking over 1 min and 3 min intervals and prey (Artemia) capture rates of uninfected grass shrimp and those infected with 1-10, 11-20 or 21-30 metacercarial cysts were compared. Uninfected shrimps spent significantly more time swimming than infected shrimps during the first minute of observation. There were no differences between the control and infected groups in terms of swimming at 3 min, walking at 1 and 3 min, or in numbers of prey captured. These results indicate that M. turgidus may induce little or no change in grass shrimp locomotion nor in prey capture behaviour. This is in contrast to other parasites that modify intermediate host behaviour to enhance their transmission to definitive hosts. Furthermore, these data support earlier studies indicating that M. turgidus does not affect the growth and survival of P. pugio.     

A study of prey-predator relations for mammals

In this paper, we present a prey-predator nonlinear model for mammals, consisting of large- and small-size prey species with group defence, in a partially protected habitat. If the prey size is small, then it is more prone to the predator at higher densities. Conversely, large prey size at higher densities tend to develop group defence. Therefore, the predator will be attracted towards that area where prey are less in number. A new physical constant has been introduced into the radiation-type condition on that part of the boundary where interaction between prey and predator takes place. This constant allows us to efficiently model group defence capabilities of the herds and its numerical values have to be determined for different pairs of prey-predator species from field observations. A way of measuring the constants involved in the model is suggested. Numerical results are provided and thoroughly discussed for a habitat of circular shape. The obtained results show that in the region away from the protected area, the density of large-size prey species is higher than that of small-size prey species, a fact that is in accordance with observations.