On D-modules related to the b-function and Hamiltonian flow

Let f be a quasi-homogeneous polynomial with an isolated singularity. We compute the length of the D-modules $Df^c/Df^{c+1}$ generated by complex powers of f in terms of the Hodge filtration on the top cohomology of the Milnor fiber. For 1/f we obtain one more than the reduced genus of the singularity. We conjecture that this holds without the quasi-homogeneous assumption. We also deduce that the aforementioned quotient is nonzero when c is a root of the b-function of f (which Saito recently showed fails to hold in the inhomogeneous case). We obtain these results by comparing these D-modules to those defined by Etingof and the second author which represent invariants under Hamiltonian flow.Comment: 15 pages, final version. All comments welcom

Deficiency of RgpG causes major defects in cell division and biofilm formation, and deficiency of LytR-CpsAPsr family proteins leads to accumulation of cell wall antigens in culture medium by Streptococcus mutans

ABSTRACT Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG , encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, “squarish” dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation ( P &lt; 0.01). Double and triple mutants with deficiency in brpA and/or psr , genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using the rgpG mutant. There were no major differences in growth rates between the wild-type strain and the rgpG brpA and rgpG psr double mutants, but the growth rate of the rgpG brpA psr triple mutant was reduced drastically ( P &lt; 0.001). Under transmission electron microscopy, both double mutants resembled the rgpG mutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, the rgpG mutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and the brpA and psr single mutants. These results suggest that RgpG in S. mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope. IMPORTANCE Streptococcus mutans , a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans , indicative of a significant role of RGP in cell division and biofilm formation in S. mutans . These results are novel not only in S. mutans , but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans . This study reveals new potential targets to develop anticaries therapeutics. </jats:p

Biogenesis of iron-sulfur clusters and intracellular iron metabolism

Iron-sulfur ([Fe-S]) clusters represent one of natures most diverse and ubiquitous protein prosthetic groups. [Fe-S] proteins are integral for diverse biological processes. Reconstitution of apo-proteins can occur by exogenously adding excess iron and sulfide. However, due to the toxicity of iron and sulfide, it is most likely that proteins coordinate [Fe-S] cluster assembly in cells. The first part of this dissertation addressed the iron donor for [Fe-S] cluster assembly. Recently, it has been shown that IscA is capable of binding iron with an association constant of 3.0 x 1019M-1. In addition, iron-loaded IscA is capable of donating iron to the proposed scaffold IscU for nascent [Fe-S] cluster assembly. We show that hIscA, the human homolog of E. coli IscA, functions as an iron chaperone for the assembly of [Fe-S] clusters in E. coli IscU. hIscA’s iron binding ability is similar to E. coli IscA. Moreover, hIscA is able to donate iron to IscU in the presence of 100-fold excess citrate, a metabolite capable of binding iron. This comparison signifies that [Fe-S] cluster assembly is conserved from bacteria to humans. The second part of this dissertation determined the participation of Ferritin A (FtnA) in [Fe-S] cluster assembly. FtnA, the major iron storage protein in E. coli, could serve as an iron reservoir when intracellular iron is depleted. We have shown that FtnA is capable of buffering iron when oxidative stress disrupts nascent [Fe-S] clusters and alleviates the production of hydroxyl radicals. Moreover, when physiological conditions return, IscA is able to retrieve iron from FtnA for [Fe-S] cluster assembly. The final part of this dissertation corroborated the interrelatedness of the oxidative and nitrosative stress response pathways. NsrR, a nitric oxide (NO) sensitive transcriptional repressor, is shown to coordinate a redox active [2Fe-2S] cluster with a midpoint redox potential of -346 ± 7 mV. The NsrR [2Fe-2S] cluster reacts with NO more quickly than other [Fe-S] proteins, signifying its role as a NO sensor. Finally, modification of the NsrR [2Fe-2S] cluster by NO results in the formation of a protein-bound dinitrosyl iron complex, relieving its DNA binding ability as a repressor

Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies

Progressive Structural Defects in Canine Centronuclear Myopathy Indicate a Role for HACD1 in Maintaining Skeletal Muscle Membrane Systems

Mutations in HACD1/PTPLA cause recessive congenital myopathies in humans and dogs. Hydroxyacyl-coA dehydratases are required for elongation of very long chain fatty acids, and HACD1 has a role in early myogenesis, but the functions of this striated muscle-specific enzyme in more differentiated skeletal muscle remain unknown. Canine HACD1 deficiency is histopathologically classified as a centronuclear myopathy (CNM). We investigated the hypothesis that muscle from HACD1-deficient dogs has membrane abnormalities in common with CNMs with different genetic causes. We found progressive changes in tubuloreticular and sarcolemmal membranes and mislocalized triads and mitochondria in skeletal muscle from animals deficient in HACD1. Furthermore, comparable membranous abnormalities in cultured HACD1-deficient myotubes provide additional evidence that these defects are a primary consequence of altered HACD1 expression. Our novel findings, including T-tubule dilatation and disorganization, associated with defects in this additional CNM-associated gene provide a definitive pathophysiologic link with these disorders, confirm that dogs deficient in HACD1 are relevant models, and strengthen the evidence for a unifying pathogenesis in CNMs via defective membrane trafficking and excitation-contraction coupling in muscle. These results build on previous work by determining further functional roles of HACD1 in muscle and provide new insight into the pathology and pathogenetic mechanisms of HACD1 CNM. Consequently, alterations in membrane properties associated with HACD1 mutations should be investigated in humans with related phenotypes

LEKTI proteolytic processing in human primary keratinocytes, tissue distribution and defective expression in Netherton syndrome

SPINK5, encoding the putative multi-domain serine protease inhibitor LEKTI, was recently identified as the defective gene in the severe autosomal recessive ichthyosiform skin condition, Netherton syndrome (NS). Using monoclonal and polyclonal antibodies, we show that LEKTI is a marker of epithelial differentiation, strongly expressed in the granular and uppermost spinous layers of the epidermis, and in differentiated layers of stratified epithelia. LEKTI expression was also demonstrated in normal differentiated human primary keratinocytes (HK) through detection of a 145 kDa full-length protein and a shorter isoform of 125 kDa. Both proteins are N-glycosylated and rapidly processed in a post-endoplasmic reticulum compartment into at least three C-terminal fragments of 42, 65 and 68 kDa, also identified in conditioned media. Processing of the 145 and 125 kDa precursors was prevented in HK by treatment with a furin inhibitor. In addition, in vitro cleavage of the recombinant 145 kDa precursor by furin generated C-terminal fragments of 65 and 68 kDa, further supporting the involvement of furin in LEKTI processing. In contrast, LEKTI precursors and proteolytic fragments were not detected in differentiated HK from NS patients. Defective expression of LEKTI in skin sections was a constant feature in NS patients, whilst an extended reactivity pattern was observed in samples from other keratinizing disorders, demonstrating that loss of LEKTI expression in the epidermis is a diagnostic feature of NS. The identification of novel processed forms of LEKTI provides the basis for future functional and structural studies of fragments with physiological relevanc

Bernstein-Sato theory modulo pmp^m

For fixed prime integer $p > 0$ we develop a notion of Bernstein-Sato polynomial for polynomials with $\mathbb{Z} / p^m$-coefficients, compatible with existing theory in the case $m = 1$. We show that the ``roots" of such polynomials are rational and we show that the negative roots agree with those of the mod-$p$ reduction. We give examples to show that, surprisingly, roots may be positive in this context. Moreover, our construction allows us to define a notion of ``strength" for roots by measuring $p$-torsion, and we show that ``strong" roots give rise to roots in characteristic zero through mod-$p$ reduction.Comment: Comments welcom