Conformational analysis of steroid hormone molecules in the lipid environment--a solid-state NMR approach

Solid-state ¹H/¹³C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy has been applied to two steroid compounds: dehydroepiandrosterone (DHEA) and spironolactone (SPI), to analyze their conformations at the atomic level. In the absence of lipid, the high-resolution ¹³C CP/MAS NMR signals of DHEA and SPI in a powder form reveal multiple patterns, with splittings of 30-160 Hz, indicating the existence of multiple conformations. In the mimic lipid environment formed by mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) in a molar ratio 3:1, the resulting DHEA and SPI spectra revealed mostly singlet patterns, suggesting that these steroids undergo a conformational change leading to a specific conformation in the lipid environment. Evidence from chemical shift isotropy and anisotropy analysis indicates that DHEA might adopt conformations subtly different from that seen in solution and in the powder form. In conclusion, we demonstrate by solid-state NMR that the structures of DHEA and SPI may adopt slightly different conformations in different chemical environments.     

Expansion of T cells targeting multiple antigens of cytomegalovirus, Epstein-Barr virus and adenovirus to provide broad antiviral specificity after stem cell transplantation

Background aimsHematopoietic stem cell transplant (HSCT) is the treatment of choice for a proportion of patients with hematologic malignancies as well as for non-malignant diseases. However, viral infections, particularly Epstein–Barr virus (EBV), cytomegalovirus (CMV) and adenovirus (Ad), remain problematic after transplant despite the use of antiviral drugs. We have shown that cytotoxic T lymphocytes (CTL) generated against CMV-pp65, EBV and Ad antigens in a single culture are capable of controlling infections with all three viruses after HSCT. Although pp65-specific CTL have proved efficacious for the control of CMV infection, several reports highlight the importance of targeting additional CMV antigens.MethodsTo expand multivirus-specific T cells with activity against both CMV-pp65 and CMV-IE-1, peripheral blood mononuclear cells (PBMC) were transduced with the adenoviral vector (Ad5f35-IE-1-I-pp65). After 9–12 days the CTL were restimulated with autologous EBV-transformed B cells transduced with the same Ad vector.ResultsAfter 18 days in culture nine CTL lines expanded from less than 1.5 × 10⁷ PBMC to a mean of 6.1 × 10⁷ T cells that recognized CMV antigens pp65 [median 273 spot-forming cells (SFC), range 47–995] and IE-1 (median 154 SFC, range 11–505), the Ad antigens hexon (median 153 SFC, range 26–465) and penton (median 37 SFC, range 1–353), as well as EBV lymphoblastoid cell lines (median 55 SFC, range 9–301). Importantly, the T cells recognized at least two antigens per virus and lysed virus peptide-pulsed targets.ConclusionsCTL that target at least two antigens each of CMV, EBV and Ad should have clinical benefit with broad coverage of all three viruses and enhanced control of CMV infections compared with current protocols.     

Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

Background

SXT is an integrating conjugative element (ICE) originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively.

Results

SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn²⁺ or Mg²⁺ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo.

Conclusions

The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V. cholerae cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.     

Affinity and kinetic modulation of polyamide–DNA interactions by N‐modification of the heterocycles

Synthetic N‐methyl imidazole and N‐pyrrole containing polyamides (PAs) that can form “stacked” dimers can be programmed to target and bind to specific DNA sequences and control gene expression. To accomplish this goal, the development of PAs with lower molecular mass which allows for the molecules to rapidly penetrate cells and localize in the nucleus, along with increased water solubility, while maintaining DNA binding sequence specificity and high binding affinity is key. To meet these challenges, six novel f‐ImPy*Im PA derivatives that contain different orthogonally positioned moieties were designed to target 5′‐ACGCGT‐3′. The synthesis and biophysical characterization of six f‐ImPy*Im were determined by CD, ΔT_M, DNase I footprinting, SPR, and ITC studies, and were compared with those of their parent compound, f‐ImPyIm. The results gave evidence for the minor groove binding and selectivity of PAs 1 and 6 for the cognate sequence 5′‐ACGCGT‐3′, and with strong affinity, K_eq = 2.8 × 10⁸ M^?1 and K_eq = 6.2 × 10⁷ M^?1, respectively. The six novel PAs presented in this study demonstrated increased water solubility, while maintaining low molecular mass, sequence specificity, and binding affinity, addressing key issues in therapeutic development. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 497–507, 2013.     

A Turn-like Structure ��KKPE�� Segment Mediates the Specific Binding of Viral Protein A27 to Heparin and Heparan Sulfate on Cell Surfaces

Vaccinia viral envelope protein A27 (110 amino acids) specifically interacts with heparin (HP) or heparan sulfate (HS) proteoglycans for cell surface attachment. To examine the binding mechanism, a truncated soluble form of A27 (sA27-aa; residues 21–84 of A27) with Cys⁷¹ and Cys⁷² mutated to Ala was used as the parent molecule. sA27-aa consists of two structurally distinct domains, a flexible Arg/Lys-rich heparin-binding site (HBS) (residues 21–32; ²¹STKAAKKPEAKR³²) and a rigid coiled-coil domain (residues 43–84), both essential for the specific binding. As shown by surface plasmon resonance (SPR), the binding affinity of sA27-aa for HP (K_A = 1.25 × 10⁸ m⁻¹) was approximately 3 orders of magnitude stronger than that for nonspecific binding, such as to chondroitin sulfate (K_A = 1.65 × 10⁵ m⁻¹). Using site-directed mutagenesis of HBS and solution NMR, we identified a “KKPE” segment with a turn-like conformation that mediates specific HP binding. In addition, a double mutant T22K/A25K in which the KKPE segment remained intact showed an extremely high affinity for HP (K_A = 1.9 × 10¹¹ m⁻¹). Importantly, T22K/A25K retained the binding specificity for HP and HS but not chondroitin sulfate, as shown by in vitro SPR and in vivo cell adhesion and competitive binding assays. Molecular modeling of the HBS was performed by dynamics simulations and provides an explanation of the specific binding mechanism in good agreement with the site-directed mutagenesis and SPR results. We conclude that a turn-like structure introduced by the KKPE segment in vaccinia viral envelope protein A27 is responsible for its specific binding to HP and to HS on cell surfaces.     

Genetic variation in South Indian castes: evidence from Y-chromosome, mitochondrial, and autosomal polymorphisms

Background

Major population movements, social structure, and caste endogamy have influenced the genetic structure of Indian populations. An understanding of these influences is increasingly important as gene mapping and case-control studies are initiated in South Indian populations.

Results

We report new data on 155 individuals from four Tamil caste populations of South India and perform comparative analyses with caste populations from the neighboring state of Andhra Pradesh. Genetic differentiation among Tamil castes is low (R_ST = 0.96% for 45 autosomal short tandem repeat (STR) markers), reflecting a largely common origin. Nonetheless, caste- and continent-specific patterns are evident. For 32 lineage-defining Y-chromosome SNPs, Tamil castes show higher affinity to Europeans than to eastern Asians, and genetic distance estimates to the Europeans are ordered by caste rank. For 32 lineage-defining mitochondrial SNPs and hypervariable sequence (HVS) 1, Tamil castes have higher affinity to eastern Asians than to Europeans. For 45 autosomal STRs, upper and middle rank castes show higher affinity to Europeans than do lower rank castes from either Tamil Nadu or Andhra Pradesh. Local between-caste variation (Tamil Nadu R_ST = 0.96%, Andhra Pradesh R_ST = 0.77%) exceeds the estimate of variation between these geographically separated groups (R_ST = 0.12%). Low, but statistically significant, correlations between caste rank distance and genetic distance are demonstrated for Tamil castes using Y-chromosome, mtDNA, and autosomal data.

Conclusion

Genetic data from Y-chromosome, mtDNA, and autosomal STRs are in accord with historical accounts of northwest to southeast population movements in India. The influence of ancient and historical population movements and caste social structure can be detected and replicated in South Indian caste populations from two different geographic regions.     

Oscillatory flow in a cone-and-plate bioreactor

Motivated by biometric applications, we analyze oscillatory flow in a cone-and-plate geometry. The cone is rotated in a simple harmonic way on a stationary plate. Based on assuming that the angle between the cone and plate is small, we describe the flow analytically by a perturbation method in terms of two small parameters, the Womersley number and the Reynolds number, which account for the influences of the local acceleration and centripetal force, respectively. Working equations for the shear stresses induced both by laminar primary and secondary flows on the plate surface are presented.     

Hybrid origin of Japanese mice "Mus musculus molossinus": evidence from restriction analysis of mitochondrial DNA

The Japanese mouse, Mus musculus molossinus, has long been considered an independent subspecies of the house mouse. A survey of restriction- site haplotypes of mitochondrial DNA (mtDNA) showed that Japanese mice have two main maternal lineages. The most common haplotype is closely related to the mtDNA of the European subspecies M. m. musculus. The other common haplotype and two minor ones are closely related to each other and to the mtDNA of an Asiatic subspecies, M. m. castaneus. Two other rare variants are probably the result of recent contamination by European M. m. domesticus. The musculus type of mtDNA is found in the southern two-thirds of Japan, whereas the common castaneus type is found in the northern third and the minor variants are found sporadically throughout Japan. The castaneus mtDNA lineage had a few minor variants, whereas the musculus lineage was completely monomorphic. By contrast, the native population of M. m. castaneus and the Chinese and Korean musculus populations were highly polymorphic. These results suggest that M. m. molossinus is a hybrid between ancestral colonies, possibly very small, of M. m. musculus and M. m. castaneus, rather than an independent subspecies.      

Flagellar adhesion in chlamydomonas induces synthesis of two high molecular weight cell surface proteins

Because our previous studies (Snell, W.J., and W.S. Moore, 1980, J. Cell Biol. 84:203- 210) on the mating reaction of chlamydomonas reinhardtii showed that there was an adhesion-induced turnover of proteins whose synthesis is induced during aggregation. Analysis by SDS PAGE and autoradiography showed that proteins of 220,000 M(r) and 165, 000 M(r) (designated A(1) and A(2) respectively) consistently showed a high rate of synthesis only in flagella or flagellar membrane-enriched fractions prepared from aggregating gametes. Since the two proteins were soluble in the non-ionic detergent NP-40 and were removed from intact cells by a brief pronase treatment, it is likely that A(1) and A(2) are membrane proteins expose on the cell surface. A(1) and A(2) were each synthesized by gametes of both mating types (mt(-) and mt(+)) and synthesis of these two proteins could be detected in the normal mating reaction (wild type mt(-) and mt(+)), in mixtures of mt(-) and impotent mt(+) gametes (which could aggregate but not fuse), and in mixtures of gametes of a single mating type with isolated flagella of the opposite mating type. Cells aggregating in tunicamycin, an inhibitor of protein glycosylation, lost their adhesiveness during aggregation and did not synthesize the 220,000 M(r) protein but instead produced a protein (possibly an underglycosylated form of A(1)) of slightly lower mol wt. The 220,000 and 165,000 M(R) proteins appeared to be flagellar proteins and not cell wall proteins because A(1) and A(2) did not co-migrate with previously identified cell wall proteins, and synthesis of the two proteins could not be detected in flagella-less (bald-2) mutant cells. Analysis of the adhesive activity of sucrose gradient fraction of detergent (octyl glucoside)-solubilized flagellar membranes revealed that fractions containing A(1) and A(2) did not have detectable adhesive activity. The possibility remains that A(1) and A(2) are adhesion molecules whose activity could not be measured in the assay we used. Alternatively, the 220,000 and 165,000 M(r) proteins may be inactivated adhesion molecules or else they may be flagellar surface proteins involved only indirectly in the adhesion process.     

Lymphocyte locomotion and attachment on two-dimensional surfaces and in three-dimensional matrices

The adhesion and locomotion of mouse peripheral lymph node lymphocytes on 2-D protein- coated substrata and in 3-D matrices were compared. Lymphocytes did not adhere to, or migrate on, 2-D substrata suck as serum- or fibronectin-coated glass. They did attach to and migrate in hydrated 3-D collagen lattices. When the collagen was dehydrated to form a 2-D surface, lymphocyte attachment to it was reduced. We propose that lymphocytes, which are poorly adhesive, are able to attach to and migrate in 3-D matrices by a nonadhesive mechanism such as the extension and expansion of pseudopodia through gaps in the matrix, which could provide purchase for movement in the absence of discrete intermolecular adhesions. This was supported by studies using serum-coated micropore filters, since lymphocytes attached to and migrated into filters with pore sizes large enough (3 or 8 mum) to allow pseudopod penetration but did not attach to filters made of an identical material (cellulose esters) but of narrow pore size (0.22 or 0.45 mum). Cinematographic studies of lymphocyte locomotion in collagen gels were also consistent with the above hypothesis, since lymphocytes showed a more variable morphology than is typically seen on plane surfaces, with formation of many small pseudopodia expanded to give a marked constriction between the cell and the pseudopod. These extensions often remained fixed with respect to the environment as the lymphocyte moved away from or past them. This suggests that the pseudopodia were inserted into gaps in the gel matrix and acted as anchorage points for locomotion.