KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.     

Synthetic nanopores as a test case for ion channel theories: the anomalous mole fraction effect without single filing

The predictions of a theory for the anomalous mole fraction effect (AMFE) are tested experimentally with synthetic nanopores in plastic. The negatively charged synthetic nanopores under consideration are highly cation selective and 50 Å in diameter at their smallest point. These pores exhibit an AMFE in mixtures of Ca²⁺ and monovalent cations. An AMFE occurs when the conductance through a pore is lower in a mixture of salts than in the pure salts at the same concentration. For ion channels, the textbook interpretation of the AMFE is that multiple ions move through the pore in coordinated, single-file motion. However, because the synthetic nanopores are so wide, their AMFE shows that single filing is not necessary for the AMFE. It is shown that the AMFE in the synthetic nanopores is explained by a theory of preferential ion selectivity. The unique properties of the synthetic nanopores allow us to experimentally confirm several predictions of this theory. These same properties make synthetic nanopores an interesting new platform to test theories of ion channel permeation and selectivity in general.     

A gene-driven ENU-based approach to generating an allelic series in any gene

N-ethyl-N-nitrosourea (ENU) introduces mutations throughout the mouse genome at relatively high efficiency. Successful high-throughput phenotype screens have been reported and alternative screens using sequence-based approaches have been proposed. For the purpose of generating an allelic series in selected genes by a sequence-based approach, we have constructed an archive of over 4000 DNA samples from individual F₁ ENU-mutagenized mice paralleled by frozen sperm samples. Together with our previously reported archive, the total size now exceeds 6000 individuals. A gene-based screen of 27.4 Mbp of DNA, carried out using denaturing high-performance liquid chromatography (DHPLC), found a mutation rate of 1 in 1.01 Mbp of which 1 in 1.82 Mbp were potentially functional. Screening of whole or selected regions of genes on subsets of the archive has allowed us to identify 15 new alleles from 9 genes out of 15 tested. This is a powerful adjunct to conventional mutagenesis strategies and has the advantage of generating a variety of alleles with potentially different phenotypic outcomes that facilitate the investigation of gene function. It is now available to academic collaborators as a community resource.     

Novel Nano-Sized MR Contrast Agent Mediates Strong Tumor Contrast Enhancement in an Oncogene-Driven Breast Cancer Model

The current study was carried out to test the potential of a new nanomaterial (Spago Pix) as a macromolecular magnetic MR contrast agent for tumor detection and to verify the presence of nanomaterial in tumor tissue. Spago Pix, synthesized by Spago Nanomedical AB, is a nanomaterial with a globular shape, an average hydrodynamic diameter of 5 nm, and a relaxivity (r₁) of approximately 30 (mM Mn)⁻¹ s⁻¹ (60 MHz). The material consists of an organophosphosilane hydrogel with strongly chelated manganese (II) ions and a covalently attached PEG surface layer. In vivo MRI of the MMTV-PyMT breast cancer model was performed on a 3 T clinical scanner. Tissues were thereafter analyzed for manganese and silicon content using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The presence of nanomaterial in tumor and muscle tissue was assessed using an anti-PEG monoclonal antibody. MR imaging of tumor-bearing mice (n = 7) showed a contrast enhancement factor of 1.8 (tumor versus muscle) at 30 minutes post-administration. Contrast was retained and further increased 2–4 hours after administration. ICP-AES and immunohistochemistry confirmed selective accumulation of nanomaterial in tumor tissue. A blood pharmacokinetics analysis showed that the concentration of Spago Pix gradually decreased over the first hour, which was in good agreement with the time frame in which the accumulation in tumor occurred. In summary, we demonstrate that Spago Pix selectively enhances MR tumor contrast in a clinically relevant animal model. Based on the generally higher vascular leakiness in malignant compared to benign tissue lesions, Spago Pix has the potential to significantly improve cancer diagnosis and characterization by MRI.     

Assembly and channel opening in a bacterial drug efflux machine

Drugs and certain proteins are transported across the membranes of Gram-negative bacteria by energy-activated pumps. The outer membrane component of these pumps is a channel that opens from a sealed resting state during the transport process. We describe two crystal structures of the Escherichia coli outer membrane protein TolC in its partially open state. Opening is accompanied by the exposure of three shallow intraprotomer grooves in the TolC trimer, where our mutagenesis data identify a contact point with the periplasmic component of a drug efflux pump, AcrA. We suggest that the assembly of multidrug efflux pumps is accompanied by induced fit of TolC driven mainly by accommodation of the periplasmic component.     

Structural characterization of the O-specific polysaccharide from the lipopolysaccharide of the fish pathogen Aeromonas bestiarum strain P1S

The O-specific polysaccharide obtained by mild-acid degradation of lipopolysaccharide of Aeromonas bestiarum P1S was studied by sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy. The sequence of the sugar residues was determined using ¹H,¹H NOESY and ¹H,¹³C HMBC experiments. The O-specific polysaccharide was found to be a high-molecular-mass polysaccharide composed of tetrasaccharide repeating units of the structureSince small amounts of a terminal Quip3N residue were identified in methylation analysis, it was assumed that the elucidated structure also represented the biological repeating unit of the O-specific polysaccharide.     

Electron sweep across four b-hemes of cytochrome bc1 revealed by unusual paramagnetic properties of the Qi semiquinone intermediate

Dimeric cytochromes bc are central components of photosynthetic and respiratory electron transport chains. In their catalytic core, four hemes b connect four quinone (Q) binding sites. Two of these sites, Q_i sites, reduce quinone to quinol (QH₂) in a step-wise reaction, involving a stable semiquinone intermediate (SQ_i). However, the interaction of the SQ_i with the adjacent hemes remains largely unexplored. Here, by revealing the existence of two populations of SQ_i differing in paramagnetic relaxation, we present a new mechanistic insight into this interaction. Benefiting from a clear separation of these SQ_i species in mutants with a changed redox midpoint potential of hemes b, we identified that the fast-relaxing SQ_i (SQ_iF) corresponds to the form magnetically coupled with the oxidized heme b_H (the heme b adjacent to the Q_i site), while the slow-relaxing SQ_i (SQ_iS) reflects the form present alongside the reduced (and diamagnetic) heme b_H. This so far unreported SQ_iF calls for a reinvestigation of the thermodynamic properties of SQ_i and the Q_i site. The existence of SQ_iF in the native enzyme reveals a possibility of an extended electron equilibration within the dimer, involving all four hemes b and both Q_i sites. This substantiates the predicted earlier electron transfer acting to sweep the b-chain of reduced hemes b to diminish generation of reactive oxygen species by cytochrome bc₁. In analogy to the Q_i site, we anticipate that the quinone binding sites in other enzymes may contain yet undetected semiquinones which interact magnetically with oxidized hemes upon progress of catalytic reactions.     

The membrane action of antidiuretic hormone (ADH) on toad urinary bladder.

Radioactive tracer and electrical techniques were used to study the transport of nonelectrolytes and sodium, respectively, across toad urinary bladders in the presence and absence of ADH. The permeability of lipophilic molecules was roughly proportional to bulk phase oil/water partition coefficients both in the presence and absence of hormone; i.e., ADH elicited a general nonselective increase in the permeation of all nine solutes tested. The branched nonelectrolyte, isobutyramide, was less permeable than its straight-chain isomer, n-butyramide, in control tissues. ADH reduced the discrimination between these structural isomers. Hydrophilic solutes permeated more rapidly than expected. In the presence of hormone, there was no change in the permeation of large hydrophilic solutes considered to move via an extracellular pathway, but there was a marked increase in the permeability of water and other small hydrophilic solutes. Collectively, these results suggest that ADH acts to increase the motional freedom or fluidity of lipids in the cell membrane which is considered to be the preferred pathway for the permeation of lipophilic and small hydrophilic molecules. At concentrations of cAMP and ADH which elicit equivalent increments in the shortcircuit current, the effects of these agents on nonelectrolyte transport and membrane electrical conductance are divergent. Such observations suggest that some membrane effects of ADH may not be directly dependent upon cAMP. ADH in the mucosal solution increased the permeability of the toad bladder when the surface charge on the outer surface of the apical membrane was screened with the polyvalent cation, La-3+. These experiments emphasize that interaction of ADH with membranes of toad urinary bladder may account for at least some effects of this hormone.