Gramicidin-induced hexagonal HII phase formation in negatively charged phospholipids and the effect of N- and C-terminal modification of gramicidin on its interaction with zwitterionic phospholipids

The effect of gramicidin on macroscopic structure of the negatively charged membrane phospholipids cardiolipin, dioleoylphosphatidylglycerol and dioleoylphosphatidylserine in aqueous dispersions was investigated and compared with the effect of gramicidin on dioleoylphosphatidylcholine. It was shown by small-angle X-ray diffraction, 31P nuclear magnetic resonance and freeze-fracture electron microscopy that in all these lipid systems gramicidin is able to induce the formation of a hexagonal HII phase. 31P-NMR measurements indicated that the extent of HII phase formation in the various lipids ranged from about 40% to 60% upon gramicidin incorporation in a molar ratio of peptide to lipid of 1 : 10. Next, the following charged analogues of gramicidin were prepared: desformylgramicidin, N-succinylgramicidin and O-succinylgramicidin. The synthesis was verified with 13C-NMR and the effect of these analogues on lipid structure was investigated. It was shown that, as with gramicidin itself, the analogues induce HII phase formation in dioleoylphosphatidylcholine, lower and broaden the bilayer-to-HII phase transition in dielaidoylphosphatidylethanolamine and form lamellar structures upon codispersion with palmitoyllysophosphatidylcholine. Differential scanning calorimetry measurements indicated that, again like gramicidin, in phosphatidylethanolamine the energy content of the gel-to-liquid-crystalline phase transition is not affected by incorporation of the analogues, whereas in phosphatidylcholine a reduction of the transition enthalpy is found. These observations were explained in terms of a similar tendency to self-associate for gramicidin and its charged analogues. The results are discussed in the light of the various factors which have been suggested to be of importance for the modulation of lipid structure by gramicidin.     

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。     

Antigen-specific helper activity in serum of mice primed with sheep red cells. I. Definition of the test system and comparison with other systems

An adoptive transfer system is described to measure serum helper activity in the primary antibody response to sheep red blood cells (SRBC). Mice injected with a high dose of cyclophosphamide and reconstituted with rabbit anti-thymocyte serum-treated spleen cells were used as recipients. Serum obtained 9 hr after ip injection of normal mice with 2 × 10⁸ SRBC (S(SRBC)) injected i.v. in the recipients caused a significant enhancement of the antibody response to 2 × 10⁷ SRBC. The serum helper activity was not generated in thymectomized animals and could be absorbed from S(SRBC) by normal and formalinized SRBC. The SRBC-specific serum helper activity (SSHA) is heat labile (30 min 56 °C) and shows allogeneic restriction. Another test system described in literature for measuring T-cell help in vivo was less suited to measure SSHA in the response to 2 × 10⁷ SRBC. A system using normal mice injected with 10⁵ SRBC for determining specific immune response-enhancing factor (SIREF), demonstrated SIREF activity in S(SRBC). It did, however, not measure SSHA, as absorption of S(SRBC) with formalinized SRBC did not abolish the activity in that system.     

Isolation and structure elucidation of a novel adipokinetic hormone (Lom-AKH-III) from the glandular lobes of the corpus cardiacum of the migratory locust, Locusta migratoria

A new adipokinetic hormone (named Lom-AKH-III) was isolated from the glandular lobes of the corpora cardiaca of Locusta migratoria. At the N-terminus it is blocked by a 5-oxoproline (pyroglutamic acid) residue (less than Glu). After enzymatic deblocking, the amino acid sequence of the N-terminus was partly established by automatic Edman degradation to be [less than Glu]-Leu-Asn-Phe-Thr-Pro-. Fast-atom-bombardment spectrometry (FAB-MS) revealed that the new hormone is an octapeptide, which is amidated at the C-terminus, and has a relative molecular mass of 1072. Based on the FAB-MS data the complete sequence is less than Glu-Leu-Asn-Phe-Thr-Pro-Trp-Trp-NH2, which was confirmed by chemical synthesis. All characteristics from HPLC, FAB-MS and biological activity of the natural hormone and the synthetic peptide appeared to be identical. Although the structure of this new hormone resembles that of Lom-AKH-I (less than Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2), its amino acid sequence points to a completely different route for its biosynthesis, involving a third prohormone. High-[K+]-containing media can cause release of all three adipokinetic hormones in vitro. Interestingly, the new hormone is absent in another locust species. Schistocerca gregaria. Based on in vitro biosynthesis experiments the turnover for this hormone is very high, suggesting an important physiological function. Locusta migratoria is the first insect species in which three different adipokinetic hormones have been demonstrated.     

Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)

Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the T7 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the T7 RNAP by the T7 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.     

Sequence and hydropathy profile analysis of two classes of secondary transporters

A structural class in the MemGen classification of membrane proteins is a set of evolutionary related proteins sharing a similar global fold. A structural class contains both closely related pairs of proteins for which homology is clear from sequence comparison and very distantly related pairs, for which it is not possible to establish homology based on sequence similarity alone. In the latter case the evolutionary link is based on hydropathy profile analysis. Here, we use these evolutionary related sets of proteins to analyze the relationship between E-values in BLAST searches, sequence similarities in multiple sequence alignments and structural similarities in hydropathy profile analyses. Two structural classes of secondary transporters termed ST[3], which includes the Ion Transporter (IT) superfamily and ST[4], which includes the DAACS family (TC# 2.A.23) were extracted from the NCBI protein database. ST[3] contains 2051 unique sequences distributed over 32 families and 59 subfamilies. ST[4] is a smaller class containing 399 unique sequences distributed over 2 families and 7 subfamilies. One subfamily in ST[4] contains a new class of binding protein dependent secondary transporters. Comparison of the averaged hydropathy profiles of the subfamilies in ST[3] and ST[4] revealed that the two classes represent different folds. Divergence of the sequences in ST[4] is much smaller than observed in ST[3], suggesting different constraints on the proteins during evolution. Analysis of the correlation between the evolutionary relationship of pairs of proteins in a class and the BLAST E-value revealed that: (i) the BLAST algorithm is unable to pick up the majority of the links between proteins in structural class ST[3], (ii) ‘low complexity filtering’ and ‘composition based statistics’ improve the specificity, but strongly reduce the sensitivity of BLAST searches for distantly related proteins, indicating that these filters are too stringent for the proteins analyzed, and (iii) the E-value cut-off, which may be used to evaluate evolutionary significance of a hit in a BLAST search is very different for the two structural classes of membrane proteins.     

Fully Automated Enhanced Tumor Compartmentalization: Man vs. Machine Reloaded

ObjectiveComparison of a fully-automated segmentation method that uses compartmental volume information to a semi-automatic user-guided and FDA-approved segmentation technique.MethodsNineteen patients with a recently diagnosed and histologically confirmed glioblastoma (GBM) were included and MR images were acquired with a 1.5 T MR scanner. Manual segmentation for volumetric analyses was performed using the open source software 3D Slicer version 4.2.2.3 (www.slicer.org). Semi-automatic segmentation was done by four independent neurosurgeons and neuroradiologists using the computer-assisted segmentation tool SmartBrush® (referred to as SB), a semi-automatic user-guided and FDA-approved tumor-outlining program that uses contour expansion. Fully automatic segmentations were performed with the Brain Tumor Image Analysis (BraTumIA, referred to as BT) software. We compared manual (ground truth, referred to as GT), computer-assisted (SB) and fully-automated (BT) segmentations with regard to: (1) products of two maximum diameters for 2D measurements, (2) the Dice coefficient, (3) the positive predictive value, (4) the sensitivity and (5) the volume error.ResultsSegmentations by the four expert raters resulted in a mean Dice coefficient between 0.72 and 0.77 using SB. BT achieved a mean Dice coefficient of 0.68. Significant differences were found for intermodal (BT vs. SB) and for intramodal (four SB expert raters) performances. The BT and SB segmentations of the contrast-enhancing volumes achieved a high correlation with the GT. Pearson correlation was 0.8 for BT; however, there were a few discrepancies between raters (BT and SB 1 only). Additional non-enhancing tumor tissue extending the SB volumes was found with BT in 16/19 cases. The clinically motivated sum of products of diameters measure (SPD) revealed neither significant intermodal nor intramodal variations. The analysis time for the four expert raters was faster (1 minute and 47 seconds to 3 minutes and 39 seconds) than with BT (5 minutes).ConclusionBT and SB provide comparable segmentation results in a clinical setting. SB provided similar SPD measures to BT and GT, but differed in the volume analysis in one of the four clinical raters. A major strength of BT may its independence from human interactions, it can thus be employed to handle large datasets and to associate tumor volumes with clinical and/or molecular datasets ("-omics") as well as for clinical analyses of brain tumor compartment volumes as baseline outcome parameters. Due to its multi-compartment segmentation it may provide information about GBM subcompartment compositions that may be subjected to clinical studies to investigate the delineation of the target volumes for adjuvant therapies in the future.