Disruption of Sertoli-germ cell adhesion function in the seminiferous epithelium of the rat testis can be limited to adherens junctions without affecting the blood-testis barrier integrity: an in vivo study using an androgen suppression model

During spermatogenesis, both adherens junctions (AJ) (such as ectoplasmic specialization (ES), a testis-specific AJ type at the Sertoli cell-spermatid interface (apical ES) or Sertoli-Sertoli cell interface (basal ES) in the apical compartment and BTB, respectively) and tight junctions (TJ) undergo extensive restructuring to permit germ cells to move across the blood-testis barrier (BTB) as well as the seminiferous epithelium from the basal compartment to the luminal edge to permit fully developed spermatids (spermatozoa) to be sloughed at spermiation. However, the integrity of the BTB cannot be compromised throughout spermatogenesis so that postmeiotic germ cell-specific antigens can be sequestered from the systemic circulation at all times. We thus hypothesize that AJ disruption in the seminiferous epithelium unlike other epithelia, can occur without compromising the BTB-barrier, even though these junctions, namely TJ and basal ES, co-exist side-by-side in the BTB. Using an intratesticular androgen suppression-induced germ cell loss model, we have shown that the disruption of AJs indeed was limited to the Sertoli-germ cell interface without perturbing the BTB. The testis apparently is using a unique physiological mechanism to induce the production of both TJ- and AJ-integral membrane proteins and their associated adaptors to maintain BTB integrity yet permitting a transient loss of cell adhesion function by dissociating N-cadherin from beta-catenin at the apical and basal ES. The enhanced production of TJ proteins, such as occludin and ZO-1, at the BTB site can supersede the transient loss of cadherin-catenin function at the basal ES. This thus allows germ cell depletion from the epithelium without compromising BTB integrity. It is plausible that the testis is using this novel mechanism to facilitate the movement of preleptotene and leptotene spermatocytes across the BTB at late stage VIII through early stage IX of the epithelial cycle in the rat while maintaining the BTB immunological barrier function.     

Sleeping Beauty-mediated down-regulation of huntingtin expression by RNA interference

Huntington disease (HD) is a devastating neurologic disorder that is characterized by abnormal expansion of a CAG nt repeat in the first exon of the huntingtin (htt) gene, producing a mutant protein with an elongated polyglutamine stretch. The presence of this mutant protein is correlated with the characteristic loss of striatal neurons and the clinical manifestation of HD. Currently there is no effective treatment for the associated cell death. The aim of this study was to evaluate an innovative strategy combining RNA interference (RNAi) and gene transfer via the nonviral Sleeping Beauty (SB) transposon system to down-regulate Htt expression. siRNA expression vectors were designed to target exons 1, 4, 6, and 62 of the human htt gene. Real-time RT-PCR and Western blot analysis were used to quantify Htt mRNA and protein levels, respectively, in human cell lines. The results indicated that selected siRNA constructs significantly decreased Htt mRNA and protein levels relative to controls. In addition, SB transposition of the siRNA constructs into the genome reduced long-term protein expression of Htt by approximately 90%. The combination of siRNA, the SB transposon, and an accurate transgenic mouse model may permit evaluation of this approach in preventing the pathogenesis associated with expression of mutant Htt.     

1,2,5-Thiadiazolidin-3-one 1,1-dioxide-based heterocyclic sulfides are potent inhibitors of human tryptase

We describe herein the design, synthesis, and in vitro biochemical evaluation of a series of potent, time-dependent inhibitors of the mast cell-derived serine protease tryptase. The inhibitors were readily obtained by attaching various heterocyclic thiols, as well as a basic primary specificity residue P₁, to the 1,2,5-thiadiazolidin-3-one 1,1-dioxide scaffold. The inhibitors were found to be devoid of any inhibitory activity toward a neutral (elastase) or cysteine (papain) protease, however they were also fairly efficient inhibitors of bovine trypsin. The differential inhibition observed with trypsin suggests that enzyme selectivity can be optimized by exploiting differences in the S′ subsites of the two enzymes. The results described herein demonstrate the versatility of the heterocyclic scaffold in fashioning mechanism-based inhibitors of neutral, basic, and acidic (chymo)trypsin-like serine proteases.     

Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents

Cytochrome p450 BM-3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles. For many of these notoriously water-insoluble compounds, p450 BM-3's K(m) values are in the millimolar range. Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency. Using p450 BM-3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents. By directed evolution, we improved the activity of p450 BM-3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10-fold in 2% (v/v) THF and 6-fold in 25% (v/v) DMSO. The engineered p450 BM-3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction.     

Quartz crystal microbalance-based measurements of shear-induced senescence in human embryonic kidney cells

Fluid shear and other mechanical forces play an important role in the normal biophysical, biochemical, and gene regulatory responses of vertebrate tissue that are reflected in the expression of normal cell differentiation, growth, and function. Despite some promising work reported on the application of the quartz crystal microbalance (QCM) to both prokaryote and eukaryote cells over the last decade, QCM has yet to be successfully applied to cells in culture under conditions of flow-induced shear. In this study, high sensitivity QCM in conjunction with fluid modelling was used to monitor the onset of senescence in immortalised human embryonic kidney cells under laminar shear stresses of between 0.04 and 335 dyne/cm(2). The feasibility of this approach as a means of quantification and characterisation of cell physiological response and adhesion are explored and discussed.     

Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkali-extractable arabinoxylans

The results of a comparative study of two thermostable (1-->4)-beta-xylan endoxylanases using a multi-technical approach indicate that a GH11 xylanase is more useful than a GH10 xylanase for the upgrading of wheat bran into soluble oligosaccharides. Both enzymes liberated complex mixtures of xylooligosaccharides. 13C NMR analysis provided evidence that xylanases cause the co-solubilisation of beta-glucan, which is a result of cell-wall disassembly. The simultaneous use of both xylanases did not result in a synergistic action on wheat bran arabinoxylans, but instead led to the production of a product mixture whose profile resembled that produced by the action of the GH10 xylanase alone. Upon treatment with either xylanase, the diferulic acid levels in residual bran were unaltered, whereas content in ferulic and p-coumaric acids were unequally decreased. With regard to the major differences between the enzymes, the products resulting from the action of the GH10 xylanase were smaller in size than those produced by the GH11 xylanase, indicating a higher proportion of cleavage sites for the GH10 xylanase. The comparison of the kinetic parameters of each xylanase using various alkali-extractable arabinoxylans indicated that the GH10 xylanase was most active on soluble arabinoxylans. In contrast, probably because GH11 xylanase can better penetrate the cell-wall network, this enzyme was more efficient than the GH10 xylanase in the hydrolysis of wheat bran. Indeed the former enzyme displayed a nearly 2-fold higher affinity and a 6.8-fold higher turnover rate in the presence of this important by-product of the milling industry.     

Chiral 2-pyridyl alcoholate copper complexes: crystal structures of [bis(2-pyridyl alcoholate)copper(II)] and the use of [(2-pyridyl alcoholate)copper(II)]and [(2-pyridyl alcoholate)copper(I)] in asymmetric cyclopropanation of styene and allylic oxidation of cyclohexene

Chiral N,O pyridine alcohols HL¹-HL⁶ were used to form complexes with copper(II) ions. Ligands HL¹ and HL² formed complexes with copper(II) ions when Cu(OAc)₂ and HL were refluxed in methanol/ethanol mixture. Ligand HL³ formed a complex with copper(II) when deprotonated with NaH and stirred in a Cu(II) acetate THF solution. Ligands HL⁴-HL⁶ did not form complexes with copper(II) under similar conditions. Two complexes, [Cu(L¹)₂] and [Cu(L²)₂], were isolated as single crystals and characterized by X-ray crystallography. These complexes showed low catalytic activities in asymmetric reactions. However, they became active when reacted with triflic acid. Copper complexes, [Cu(L)] or [Cu(L)]⁺, formed in situ by reacting ligands HL with copper(I) or (II) ions, respectively, were also found to be active copper catalysts for asymmetric cyclopropanation of styrene with ethyl diazoacetate and allylic oxidation of cyclohexene with t-butylperoxybenzoate. Enantioselectivities up to 56% and 38% were obtained in asymmetric cyclopropanation of styrene and asymmetric allylic oxidation of cyclohexene, respectively.     

Synthesis, characterization and crystal structures of Cu(II) and Pd(II) complexes of the pentadentate mixed donor macrocycle, [17]aneNO2S2

An aza-oxa-thia macrocycle, 5,14-dioxa-2,17-dithia[6](1,2)benzeno[6](2,6)pyridinophane, L1, the related smaller macrocycle 2,14-dithia-11-oxa-[3](1,2)benzeno[6](2,6)pyridinophane, L2, and the complexes with Pd(II) and Cu(II) of the macrocycle, L1, have been synthesized. The crystal structure of L2 and those of the two metal complexes have been determined. In the complexes, the metal ions adopt exclusively square planar geometry in which the pyridine nitrogen, two sulfurs and one chlorine atom are coordinated and there is no appreciable interaction with the oxygen donors. Thus, the `hard-soft acid-base' principle is illustrated by the behaviour of L1. The structures of both complexes are compared with the previously reported mixed aza-thia macrocycle, 2,5,14,17-tetrathia[6](1,2)benzeno[6](2,6)pyridinophane. The crystal structure of the smaller macrocycle, L2, is also discussed and due to the nature of its smaller cavity, attempts to make complexes with it have not been successful.