Interaction of measles virus vectors with Auger electron emitting radioisotopes

A recombinant measles virus (MV) expressing the sodium iodide symporter (NIS) is being considered for therapy of advanced multiple myeloma. Auger electrons selectively damage cells in which the isotope decays. We hypothesized that the Auger electron emitting isotope 125I can be used to control viral proliferation. MV was engineered to express both carcinoembryonic antigen and NIS (MV-NICE). Cells were infected with MV-NICE and exposed to 125I with appropriate controls. MV-NICE replication in vitro is inhibited by the selective uptake of 125I by cells expressing NIS. Auger electron damage is partly mediated by free radicals and abrogated by glutathione. In myeloma xenografts, control of MV-NICE with 125I was not possible under the conditions of the experiment. MV-NICE does not replicate faster in the presence of radiation. Auger electron emitting isotopes effectively stop propagation of MV vectors expressing NIS in vitro. Additional work is necessary to translate these observations in vivo.     

Synthesis and evaluation of 18F-hexafluorophosphate as a novel PET probe for imaging of sodium/iodide symporter in a murine C6-glioma tumor model

Noninvasive imaging of iodide uptake via the sodium/iodide symporter (NIS) has received great interest for evaluation of thyroid cancer and reporter imaging of NIS-expressing viral therapies. In this study, we investigate ¹⁸F-labeled hexafluorophosphate (HFP or PF₆^?) as a high-affinity iodide analog for NIS imaging. ¹⁸F-HFP was synthesized by radiofluorination of phosphorus pentafluoride·N-methylpyrrolidine complex and evaluated in human NIS (hNIS)-expressing C6 glioma cells and a C6 glioma xenograft mouse model. ¹⁸F-HFP was obtained in radiochemical yield of 10?±?5%, radiochemical purity of >96% and specific radioactivity of 604?±?18?MBq/µmol. Specific uptake of ¹⁸F-HFP and high affinity of ¹⁹F-HFP were observed in hNIS+ C6-glioma cells. PET imaging showed robust uptake of ¹⁸F-HFP in NIS-expressing tissues (thyroid, stomach, and hNIS+ C6 glioma xenografts), and the uptake of ¹⁸F-HFP was blocked by NaClO₄ pretreatment. Specific accumulation in hNIS-expressing xenograft (hNIS+) was observed relative to isogenic control tumor (hNIS?). Clearance of ¹⁸F-HFP was predominantly through renal excretion. The biodistribution showed consistent results with PET imaging. Minimal bone uptake was observed over 2?h period post-injection, indicating excellent in vivo stability of ¹⁸F-HFP. Although improvement in specific radioactivity is desirable, the results indicate that ¹⁸F-HFP is a promising candidate radiotracer for further evaluation for NIS imaging.     

Computational modeling of the N‐terminus of the human dopamine transporter and its interaction with PIP2‐containing membranes

The dopamine transporter (DAT) is a transmembrane protein belonging to the family of neurotransmitter:sodium symporters (NSS). Members of the NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their translocation back into the presynaptic nerve terminal. The DAT contains long intracellular N‐ and C‐terminal domains that are strongly implicated in the transporter function. The N‐terminus (N‐term), in particular, regulates the reverse transport (efflux) of the substrate through DAT. Currently, the molecular mechanisms of the efflux remain elusive in large part due to lack of structural information on the N‐terminal segment. Here we report a computational model of the N‐term of the human DAT (hDAT), obtained through an ab initio structure prediction, in combination with extensive atomistic molecular dynamics (MD) simulations in the context of a lipid membrane. Our analysis reveals that whereas the N‐term is a highly dynamic domain, it contains secondary structure elements that remain stable in the long MD trajectories of interactions with the bilayer (totaling >2.2 μs). Combining MD simulations with continuum mean‐field modeling we found that the N‐term engages with lipid membranes through electrostatic interactions with the charged lipids PIP₂ (phosphatidylinositol 4,5‐Biphosphate) or PS (phosphatidylserine) that are present in these bilayers. We identify specific motifs along the N‐term implicated in such interactions and show that differential modes of N‐term/membrane association result in differential positioning of the structured segments on the membrane surface. These results will inform future structure‐based studies that will elucidate the mechanistic role of the N‐term in DAT function. Proteins 2015; 83:952–969. © 2015 Wiley Periodicals, Inc.     

High-throughput analytical gel filtration screening of integral membrane proteins for structural studies

Background

Structural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks; proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning.

Methods

Here we present a screening approach that prioritizes IMP targets based on three criteria: expression level, detergent solubilization yield and homogeneity as determined by high-throughput small-scale immobilized metal affinity chromatography (IMAC) and automated size-exclusion chromatography (SEC).

Results

To validate the strategy, we screened 48 prokaryotic IMPs in two different vectors and two Escherichia coli strains. A set of 11 proteins passed all preset quality control checkpoints and was subjected to crystallization trials. Four of these crystallized directly in initial sparse matrix screens, highlighting the robustness of the strategy.

Conclusions

We have developed a rapid and cost efficient screening strategy that can be used for all IMPs regardless of topology. The analytical steps have been designed to be a good mimic of preparative purification, which greatly facilitates scale-up.

General significance

The screening approach presented here is intended and expected to help drive forward structural biology of membrane proteins.     

Inhibition of thyroid-restricted genes by follicular thyroglobulin involves iodinated degree

Follicular thyroglobulin (TG) reflects the storage of both iodine and thyroid hormone. This is because it is a macromolecular precursor of thyroid hormone and organic iodinated compound in follicular lumen. Thus, it may have an important feedback role in thyroid function. In this study, monolayer cells were cultured and follicles were reconstituted with primary pig thyroid cells in vitro. Reconstituted follicles were treated with iodine and methimazole (MMI), a drug that blocks iodine organification and reduces the degree of TG iodination in follicular lumen. The high degree of iodinated TG in follicular lumen was observed to inhibit thyroid-restricted gene expression. To confirm this finding, monolayer thyroid cells were treated with a different degree of TG iodination at the same concentration. These iodinated TG were extracted from reconstituted follicles of different groups. In this manner, this study provides firsthand evidence suggesting that follicular TG inhibits the expressions of thyroid-restricted genes NIS, TPO, TG, and TSHr.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

The presence of psicofuranine in the fermentation medium caused the accumulation of a copious amount of 5′–XMP by Brevibacterium ammoniagenes. The accumulation of 5′–XMP in the medium was considered to be due to the inhibition of converting 5′–XMP to 5′–GMP by psicofuranine, which is known as a specific inhibitor of XMP aminase.

It was previously reported that in 5′–IMP fermentation with Br. ammoniagenes pantothenate and thiamine, in addition to biotin which was required for the growth of the microorganism, were exclusively required. This requirement for both vitamins was also observed in 5′–XMP production induced by the antibiotic.

The addition of manganese in excess to the fermentation medium promoted the bacterial growth greatly and inhibited IMP production, whereas XMP production induced by piscofuranine was not affected by the addition of excess manganese.

The accumulation of XMP induced by the antibiotic was completely suppressed by the presence of purine derivatives such as guanine, and xanthine derivatives, and partially by hypoxanthine.

5′–XMP was identified by chemical and enzymatic analyses and by UV absorption spectrum.     

Molecular mechanisms of urea transport in plants

Urea is a soil nitrogen form available to plant roots and a secondary nitrogen metabolite liberated in plant cells. Based on growth complementation of yeast mutants and “in-silico analysis”, two plant families have been identified and partially characterized that mediate membrane transport of urea in heterologous expression systems. AtDUR3 is a single Arabidopsis gene belonging to the sodium solute symporter family that cotransports urea with protons at high affinity, while members of the tonoplast intrinsic protein (TIP) subfamily of aquaporins transport urea in a channel-like manner. The following review summarizes current knowledge on the membrane localization, energetization and regulation of these two types of urea transporters and discusses their possible physiological roles in planta.     

钠／碘同向转运体在人不同组织学分型胃癌中的表达

目的研究钠／碘同向转运体（sodium-iodide symporter,NIS）在人不同组织学分型胃癌组织中的表达,分析NIS表达与胃癌组织学分型的关系。方法选取于中国医科大学附属第一医院肿瘤外科行手术治疗的180例患者的胃癌组织为研究对象。依据胃癌的组织学分型分为6组,其中乳头状腺癌30例,管状腺癌30例,低分化腺癌30例,黏液腺癌30例,印戒细胞癌30例,未分化癌30例。选取上述180例患者的癌旁组织及30例慢性胃炎患者的胃组织作为对照。采取免疫组织化学SP法对上述组织的石蜡切片进行染色。结果在人胃癌组织、人胃癌旁组织及人慢性胃炎组织中,NIS阳性细胞率分别为10．6％（19／180）、28．3％（51／180）和90％（27／30）。人胃癌组织与人胃癌旁组织相比（P〈0．01）、人胃癌组织与人慢性胃炎组织相比（P〈0．01）、人胃癌旁组织与人慢性胃炎组织相比（P〈0．01）,NIS阳性细胞率均具有显著统计学差异。在人乳头状腺癌、管状腺癌、低分化腺癌、黏液腺癌、印戒细胞癌及未分化癌中,NIS阳性细胞率分别为30％（9／30）、10％（3／30）、13．3％（4／30）、6．7％（2／30）、3．3％（1／30）和0（0／30）。人乳头状腺癌与人黏液腺癌相比（P〈0．05）、人乳头状腺癌与人印戒细胞癌相比（PGO．01）以及人乳头状腺癌与人未分化癌相比（P〈0．01）,NIS阳性细胞率均具有统计学差异。在人慢性胃炎组织及人胃癌旁组织中,NIS仅表达于胃黏膜细胞的细胞膜上,而在人胃癌组织中,NIS在癌细胞膜有表达,在细胞质也有较明显的表达。结论在人胃癌组织、癌旁组织、慢性胃炎组织中,NIS阳性细胞率依次增高。在本研究人各种组织学分型的胃癌中,随着胃癌恶性程度的增高,NIS表达呈现出减少的趋势。人胃癌组织NIS在癌细胞的细胞膜和细胞质均有表达,而?