Interaction of haptoglobin with hemoglobin octamers based on the mutation <i>α</i>Asn78Cys or <i>β</i>Gly83Cys

Octameric hemoglobins have been developed by the introduction of surface cysteines in either the alpha or beta chain. Originally designed as a blood substitute, we report here the structure and ligand binding function;in addition the interaction with haptoglobin was studied. The recombinant Hbs (rHbs) with mutations alpha Asn78Cys or beta Gly83Cys spontaneously form octamers under conditions where the cysteines are oxidized. Oxygen binding curves and CO kinetic studies indicate a correct allosteric transition of the tetramers within the octamer. Crystallographic studies of the two rHbs show two disulfide bonds per octamer. Reducing agents may provoke dissociation to tetramers, but the octamers are stable when mixed with fresh human plasma, indicating that the reduction by plasma is slower than the oxidation by the dissolved oxygen, consistent with an enhanced stability. The octameric rHbs were also mixed with a solution of haptoglobin (Hp), which binds the dimers of Hb: there was little interaction for incubation times of 15 min;however, on longer timescales a complex was formed. Dynamic light scattering was used to follow the interaction of Hp with the alpha Asn78Cys octamer during 24 hours;a transition from a simple complex of 15 nm to a final size of 60 nm was observed. The results indicate a specific orientation of the αβ dimers may be of importance for the binding to haptoglobin.

小干扰RNA沉默OCT4基因表达对胰腺癌细胞株PANC1增殖与凋亡的影响

目的运用RNA干扰(RNAinteference,RNAi)技术阻断胰腺癌细胞株PANC1中OCT4基因表达,并研究该基因沉默后对细胞增殖与凋亡的影响。方法利用阳离子脂质体LipofectamineTM2000将化学合成的人Octamer binding factor4(OCT4)的小干扰RNA转染入胰腺癌细胞株PANC1中。RT-PCR法测定胰腺癌细胞内OCT4mRNA的表达。WesternBlot测定OCT4、PARP。CCK8法测定细胞生长曲线观察细胞增殖的抑制情况。流式细胞仪测定细胞凋亡率的变化。结果化学合成的人OCT4siRNA能有效地抑制PANC1细胞中OCT4的表达(P<0.05)。OCT4siRNA组中细胞增殖减慢,凋亡率较对照组明显增加(P<0.05)。干扰OCT4能够激活PARP的表达。结论体外实验初步证明OCT4基因在胰腺癌细胞株增殖分化与凋亡方面扮演重要角色。通过沉默其表达可抑制胰腺癌细胞株PANC1增殖并诱导其凋亡。

Expression of Transcription Factor Oct4 in Bladder Cancer Cell Line T24 and Its Effects on the Biological Characteristics of the Cells

The expression of octamer binding factor 4 （Oct4） gene in bladder cancer cell line T24 and its effects on the biological characteristics of the cells were investigated. RT-PCR and Western blot were employed to detect the expression of Oct4 in T24 cells. The changes of biological characteristics in T24 cells were analyzed before and after gene-silencing by Boyden chamber and MTT. The results showed that the expression of Oct4 gene was detectable in T24 cells by RT-PCR and Western blot. The expression of Oct4 gene and protein was down-regulated by siRNA, and average number of transwell cells in interference group, negative control group and blank control group was 101.40±54.56, 104.20± 10.03 and 111.00±11.90, respectively. There was significant difference in the proliferation abilit,） of the cells from 48 h, 72 h to 96 h after the interference by siRNA between interference group and negative group or blank control group （P〈0.05）. It was suggested that Oct4 gene was related with proliferation ability of T24 cells, but not with invasive capability.

Detection of Serine Octamer by Desorption Electrospray Ionization Mass Spectrometry in Resultant Mixture of Aspartic Acid Exposed to Sunshine Under Natural Conditions

Serine, one of the nonessential amino acids, is of principal interest because of its capability to form magic-number ionic clusters, which provide a remarkable preference for homochirality. With L-aspartic acid as the precursor, this study provides experimental evidence for serine formation in weak acidified aqueous solutions in the presence of iron, with exposure to sunlight, which simulates the natural conditions of the prebiotic aqueous environment. The resultant mixture is directly analyzed via desorption electrospray ionization mass spectrometry（ DESI-MS）, without any sample preseparation. The serine monomer is successfully detected as protonated molecules, giving a peak at m/z 106, which is confirmed by the MS/MS fragments. Protonated serine octamer（ m/z 841 ） is also observed with significant abundance in the MS spectra and is confirmed by the MS/MS data, which shows the formation of the serine octamer by a synthesized serine in the resultant mixture. It is also found that the serine octamer yielded equivalent abundance in the DESI mass spectra in a wide pH range（pH = 1-5 ）, and that existence of ferrous salt and sunshine are essential for the conversion of aspartic acid to serine in the acidic water solution.

An octamer of enolase from Streptococcus suis

Enolase is a conserved cytoplasmic metalloenzyme existing universally in both eukaryotic and prokaryotic cells.The enzyme can also locate on the cell surface and bind to plasminogen,via which contributing to the mucosal surface localization of the bacterial pathogens and assisting the invasion into the host cells.The functions of the eukaryotic enzymes on the cell surface expression(including T cells,B cells,neutrophils,monocytoes,neuronal cells and epithelial cells)are not known.Streptococcus suis serotype 2(S.suis 2,SS2)is an important zoonotic pathogen which has recently caused two large-scale outbreaks in southern China with severe streptococcal toxic shock syndrome(STSS)never seen before in human sufferers.We recently identified the SS2 enolase as an important protective antigen which could protect mice from fatal S.suis 2 infection.In this study,a 2.4-angstrom structure of the SS2 enolase is solved,revealing an octameric arrangement in the crystal.We further demonstrated that the enzyme exists exclusively as an octamer in solution via a sedimentation assay.These results indicate that the octamer is the biological unit of SS2 enolase at least in vitro and most likely in vivo as well.This is,to our knowledge,the first comprehensive characterization of the SS2 enolase octamer both structurally and biophysically,and the second octamer enolase structure in addition to that of Streptococcus pneumoniae.We also investigated the plasminogen binding property of the SS2 enzyme.

Cylindrical vector beam-excited frequency-tunable second harmonic generation in a plasmonic octamer

We report a method to tune the second harmonic generation(SHG) frequency of a metallic octamer by employing cylindrical vector beams as the excitation. Our method exploits the ability to spatially match the polarization state of excitations with the fundamental target plasmonic modes, enabling flexible control of the SHG resonant frequency.It is found that SHG of the octamer is enhanced over a broad band(400 nm) by changing the excitation from the linearly polarized Gaussian beam to radially and azimuthally polarized beams. More strikingly, when subjected to an azimuthally polarized beam, the SHG intensity of the octamer becomes 30 times stronger than that for the linearly polarized beam even in the presence of Fano resonance.

Investigation of L/D-threonine substituted L-serine octamer ions by mass spectrometry and infrared photodissociation spectroscopy

Threonine-substituted serine octamer ions were generated by electrospray ionization（ESI） and investigated by mass spectrometry and infrared photodissociation（IRPD） spectroscopy. IRPD spectra of[L-Ser_7＋ L/D-Thr_1]H~＋and [L-Ser_6＋ L/D-Thr_2]H~＋were obtained in the range of 2700–3600 cm^（-1）. Chiral differentiation was achieved by comparing their IRPD spectra. The main difference located in the range of 3300–3500 cm^（-1）. And the results indicate the substitution of L-Ser by D-Thr could weaken the intermolecular H-bonds and loosen the original structures of serine octamers.

NodD binds to target DNA in isologous octamer

NodD, the major regulatory protein of nodulation, was partially purified from Rhizobium leguminosarum 8401(pIJ1518), and its binding sequences within nodF promoter of R.l. bv. viciae were determined by DNase I footprinting. A series of techniques based on gel retardation were used to analyze the NodD_target DNA interaction, showing that NodD binds to target DNA in isologous octamer.