Escherichia coli murein-DD-endopeptidase insensitive to beta-lactam antibiotics.

A novel endopeptidase degrading the peptide cross-links in sacculi has been isolated from Escherichia coli and purified to homogeneity. The enzyme has a molecular weight of 30,000 and, in contrast to already known enzymes of similar specificity, remains fully active in the presence of beta-lactam antibiotics. In addition, it is exceptional in being inhibited by single-stranded deoxyribonucleic acid and by some polynucleotides. The possible role of the enzyme in cell division is discussed.     

Elektronenmikroskopische Untersuchungen über den Aufbau der sklera und der Cornea des Menschen

Zusammenfassung Es wurden acht normale Bulbi und drei Disci pathologisch veränderter Corneae (Trübung und Narben) elektronenmikroskopisch untersucht. Die Sklerafibrillen entsprechen weitgehend den Sehnenkollagenfibrillen. In der Cornea wurde neben den Fibrillen, die eine weitgehende Ähnlichkeit mit embryonalen Bindegewebsfibrillen besitzen, eine besondere Kittsubstanz morphologisch nachgewiesen, von der ein Teil zu den Substraten der Hyaluronidase gehört (Hyaluronsch wefelsäure). Die Dicke der nackten Fibrillen schwankt zwischen 25 und 33 m. Der Mittelwert beträgt 29 m. Die Fibrillen sind von einem Mantel von Kittsubstanz umgeben, der wesentlich dicker ist als beim Sehnen- und Sklerakollagen. Die Corneafibrillen liegen zu Bündeln zusammengefaßt und durch Kittsubstanz maskiert in den Lamellen. Die Dicke der Bündel schwankt zwischen 2,5 und 8 . Sie entsprechen den aus der Histologie bekannten Fibrillen. Das Problem der Durchsichtigkeit wurde an Hand der neuen Befunde diskutiert. Die Quellungs- und Entquellungstheorie konnte nicht bestätigt werden. Die Durchsichtigkeit der Cornea wird durch ein System feinster Fibrillen und einer besonderen, diese Fibrillen maskierenden Kittsubstanz erklärt. Veränderungen an den Fibrillen und der Kittsubstanz, bzw. Verschiebungen des Verhältnisses zwischen beiden führen zur Undurchsichtigkeit der Cornea, wie Befunde an den Narben zeigen. Diese nehmen in gewisser Hinsicht eine Zwischenstellung zwischen Cornea und Sklera ein. Weitere Untersuchungen auf diesem Gebiet sind erforderlich.     

Syntheses and evaluation of pyridazine and pyrimidine containing bioisosteres of (+/-)-pyrido[3.4-b]homotropane and pyrido-[3.4-b]tropane as novel nAChR ligands.

Bioisosteric replacement of the pyridine pharmacophoric element in (+/-)-pyrido[3.4-b]homotropane (PHT) and pyrido[3.4-b]tropane with the pyridazine and pyrimidine nucleus resulted in hitherto unknown nAChR ligands such as 5-8. Inverse type Diels-Alder reactions constitute the key steps in the new routes to the pyridazine- or pyrimidine-annulated bioisosteres. The enantiopure (+)-2-tropinone (11) from the 'chiral pool' is transformed to the ring-expanded silyl enol ether 12 and to the enamine 15. Both proved to be highly dienophilic species in the inverse type [4+2] cycloaddition reactions with the 1,2,4,5-tetrazines 13 and 16a,b or with the 1,3,5-triazine 19 to provide the enantiopure target compounds 5-7. In the same way the racemic pyrimidine-annulated species 8 was obtained from 3-tropanone 21. The new ligands were tested for their in vitro affinity for (alpha4)2(beta2)3 and alpha7* nAChR subtype. In comparison to PHT, well known to exhibit affinity for agonist binding sites in rat brain approximately equivalent to that of (+)-anatoxin-a (1), replacement of the pyridine by the bioisosteric pyridazine resulted in 30-fold lower affinity at the (alpha4)2(beta2)3 subtype. The annulated diazinotropanes 6-8, ligands with ferruginine-like structures more or less retained the affinity of (-)-norferruginine (3) except of compound 7. Remarkably, all of the novel ligands are devoid of affinity at the alpha7* subtype.     

Biology of advanced uveal melanoma and next steps for clinical therapeutics

Uveal melanoma is the most common intraocular malignancy although it is a rare subset of all melanomas. Uveal melanoma has distinct biology relative to cutaneous melanoma, with widely divergent patient outcomes. Patients diagnosed with a primary uveal melanoma can be stratified for risk of metastasis by cytogenetics or gene expression profiling, with approximately half of patients developing metastatic disease, predominately hepatic in location, over a 15‐yr period. Historically, no systemic therapy has been associated with a clear clinical benefit for patients with advanced disease, and median survival remains poor. Here, as a joint effort between the Melanoma Research Foundation's ocular melanoma initiative, CURE OM and the National Cancer Institute, the current understanding of the molecular and immunobiology of uveal melanoma is reviewed, and on‐going laboratory research into the disease is highlighted. Finally, recent investigations relevant to clinical management via targeted and immunotherpies are reviewed, and next steps in the development of clinical therapeutics are discussed.     

The Leber Congenital Amaurosis Protein AIPL1 and EB Proteins Co-Localize at the Photoreceptor Cilium

Purpose

The aim of this study was to investigate the interaction and co-localization of novel interacting proteins with the Leber congenital amaurosis (LCA) associated protein aryl hydrocarbon receptor interacting protein-like 1 (AIPL1).

Methods

The CytoTrapXR yeast two-hybrid system was used to screen a bovine retinal cDNA library. A novel interaction between AIPL1 and members of the family of EB proteins was confirmed by directed yeast two-hybrid analysis and co-immunoprecipitation assays. The localization of AIPL1 and the EB proteins in cultured cells and in retinal cryosections was examined by immunofluorescence microscopy and cryo-immunogold electron microscopy.

Results

Yeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, EB1 and EB3. EB1 and EB3 were specifically co-immunoprecipitated with AIPL1 from SK-N-SH neuroblastoma cells. In directed 1:1 Y2H analysis, the interaction of EB1 with AIPL1 harbouring the LCA-causing mutations A197P, C239R and W278X was severely compromised. Immunofluorescent confocal microscopy revealed that AIPL1 did not co-localize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous β-tubulin. Moreover, AIPL1 did not localize to primary cilia in ARPE-19 cells, whereas EB1 co-localized with the centrosomal marker pericentrin at the base of primary cilia. However, both AIPL1 and the EB proteins, EB1 and EB3, co-localized with centrin-3 in the connecting cilium of photoreceptor cells. Cryo-immunogold electron microscopy confirmed the co-localization of AIPL1 and EB1 in the connecting cilia in human retinal photoreceptors.

Conclusions

AIPL1 and the EB proteins, EB1 and EB3, localize at the connecting cilia of retinal photoreceptor cells, but do not co-localize in the cellular microtubule network or in primary cilia in non-retinal cells. These findings suggest that AIPL1 function in these cells is not related to the role of EB proteins in microtubule dynamics or primary ciliogenesis, but that their association may be related to a specific role in the specialized cilia apparatus of retinal photoreceptors.     

Electrical Impedance Tomography of Electrolysis

The primary goal of this study is to explore the hypothesis that changes in pH during electrolysis can be detected with Electrical Impedance Tomography (EIT). The study has relevance to real time control of minimally invasive surgery with electrolytic ablation. To investigate the hypothesis, we compare EIT reconstructed images to optical images acquired using pH-sensitive dyes embedded in a physiological saline agar gel phantom treated with electrolysis. We further demonstrate the biological relevance of our work using a bacterial E.Coli model, grown on the phantom. The results demonstrate the ability of EIT to image pH changes in a physiological saline phantom and show that these changes correlate with cell death in the E.coli model. The results are promising, and invite further experimental explorations.     

The Intimin periplasmic domain mediates dimerisation and binding to peptidoglycan

Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C‐terminal extracellular domain and a β‐barrel transmembrane domain, both proteins also contain a short N‐terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 μM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α‐helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM‐containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract.     

Conformational stability of the RNP domain controls fibril formation of PABPN1

The disease oculopharyngeal muscular dystrophy is caused by alanine codon trinucleotide expansions in the N‐terminal segment of the nuclear poly(A) binding protein PABPN1. As histochemical features of the disease, intranuclear inclusions of PABPN1 have been reported. Whereas the purified N‐terminal domain of PABPN1 forms fibrils in an alanine‐dependent way, fibril formation of the full‐length protein occurs also in the absence of alanines. Here, we addressed the question whether the stability of the RNP domain or domain swapping within the RNP domain may add to fibril formation. A variant of full‐length PABPN1 with a stabilizing disulfide bond at position 185/201 in the RNP domain fibrillized in a redox‐sensitive manner suggesting that the integrity of the RNP domain may contribute to fibril formation. Thermodynamic analysis of the isolated wild‐type and the disulfide‐linked RNP domain showed two state unfolding/refolding characteristics without detectable intermediates. Quantification of the thermodynamic stability of the mutant RNP domain pointed to an inverse correlation between fibril formation of full‐length PABPN1 and the stability of the RNP domain.