The purification and properties of cyclohexanone oxygenase from Nocardia globerula CL1 and Acinetobacter NCIB 9871.

1. Cyclohexanone oxygenases from Norcardia globerula CL1 and Acinetobacter NCIB 9871 have been purified 12-fold and 35-fold respectively and each gives a single symmetrical sedimentation peak in the ultracentrifuge and a single protein band on 2.25 nm average pore radius polyacrylamide gels. 2. The enzyme from N. globerula has a molecular weight of 53000 while that from Acinetobacter has a molecular weight of about 59000. Each is a single polypeptide chain with one mole of bound FAD per mole of protein that does not dissociate during purification. Acidification of the Acinetobacter enzyme in the presence of (NH4)2SO4 releases the bound FAD and yields native apoenzyme from which the active holoenzyme can be reconstituted. The apparent dissociation constant for the FAD is 40 nM.     

The activation of amino acid analogues by phenylalanyl- and tyrosyl-tRNA synthetases from plants

Partially purified preparations of Phe- and Tyr-tRNA synthetases were obtained from seed or seedlings of Phaseolus aureus, Delonix regia and Caesalpinia tinctoria, and the ability of a variety of structural analogues of Phe or Tyr to act as alternative substrates or inhibitors was tested. 3-Hydroxymethylphenylalanine, a natural product of C. tinctoria, formed a particularly effective substrate for the Tyr-tRNA synthetase from P. aureus. The structural features commensurate with substrate activity in an analogue molecule are discussed.     

A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety.

1. 4-(N-2-Aminoethyl2'-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole (compound I) was synthesized and evaluated as a fluorescent labelling reagent for thiol groups. 2. The design of compound (I) as one example of a general type of reporter group delivery reagent (2-pyridyl-S-S-X, where X contains an environmentally sensitive spectroscopic probe) is discussed. 3. The electronic absorption spectrum of compound (I) was determined over a wide range of pH and the spectral changes that accompany its reaction with low-molecular-weight thiols, e.g. L-cysteine, and with papain (EC 3.4.22.2) and bovine serum albumin are discussed. 4. A new value of epsilon343 for 2-thiopyridone (Py-2-SH) was determined as 8.08 X 10(3) +/- 0.08 X 10(3)M-1-cm-1. 5. Spectral analysis of the reactions of compound (I) with L-cysteine and with papain (in the pH range 3.5-8.0) showed that even under equimolar conditions the reaction (thiol-disulphide interchange to release Py-2-SH) is essentially stoicheimoetric and probably proceeds by specific attack at the sulphur atom distal from the pyridyl ring of compound (I). 6. The fluorescence-emission spectra of compound (I) and of the products of its reaction with papain and with ficin (EC 3.4.22.3) were determined. Compound (I) is highly fluorescent in aqueous solution. Excitation within the intense visible absorption band (lambda max. 481 nm, epsilon max. 2.52 X 10(4)M-1-cm-1) provides green fluorescence with an emission maximum at 540 nm. Both papain and ficin labelled by reaction with compound (I) are characterized by fluorescence-emission maxima (535 nm and 530 nm respectively) of even higher intensity. The fluorescence emission of the product of the reaction of papain with compound (I) was shown to be 25 times more intense than that of the product of the reaction of papain with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride). 7. The second-order rate constants (k2) for the reactions of compound (I) and of Nbd chloride with GSH, papain, albumin, ficin, 2-benzimidazolylmethanethiol and 2-benzimidazolylethanethiol were determined at 25.0 degrees C and various pH values. At pH4 the values of k2(compound I)/k2(Nbd chloride) are: GSH, 288; albumin, 36; papain 3 X 10(3); ficin, 3 X 10(4). 8. The pH-k2 profiles for the reactions of compound (I) and of Nbd chloride with the two 2-benzimidazolylalkanethiols were determined. Of the four profiles only that for the reaction of compound (I) with 2-benzimidazolylmethanethiol is characterized by a striking rate maximum in acidic media.     

Ultrastructure of the compound eye of the diploid female beetle,Xyleborus ferrugineus

Summary The compound eye of female (diploid) Xyleborus ferrugineus beetles was examined with scanning and transmission electron microscopy. The eye is emarginate, and externally consists of roughly 70–100 facets. Each ommatidium is composed of a thickly biconvex lenslet with about 50 electron dense and rare layers. The lens facet overlies a crystalline cone of the acone type which is roughly hourglass-shaped. Pigment cells envelop the entire ommatidium, and pigment granules also are abundant throughout the cytoplasm of the 8 retinular cells. The rhabdomeres of 2 centrally situated photoreceptor cells effectively fuse into a rhabdom that extends from the base of the crystalline cone deeply into the ommatidium. Six distal peripheral retinular cells encircle the 2 central cells, and their rhabdomeres join laterally to form a rhabdomeric ring around the central rhabdom. The rhabdom and rhabdomeric ring are effectively separated by the cytoplasm of the two central retinular cells which contains the usual organelles and an abundance of shielding pigment granules. Eight axons per ommatidium gather in a tracheae-less fascicle before exiting the eye through the fenestrate basement membrane. No tracheation was observed among the retinular cells. Each Semper cell of each observed crystalline cone contained an abundance of virus-like particles near the cell nucleus. The insect is laboratory reared, and the visual system seems very amenable to photoreceptor investigations.This research was supported by the Director of the Research Division, C.A.L.S., University of Wisconsin, Madison; and in part by research grant No. RR-00779 from the Division of Research Resources, National Institutes of Health and by funds from the Schoenleber Foundation, Milwaukee, WI to D.M.N.     

DNA replication in Escherichia coli is initiated by membrane detachment of oriC. A model

An adequate model for the initiation of chromosome replication in Escherichia coli should explain why the introduction of multiple copies of the chromosomal origin of replication, oriC, does not perturb cells seriously and why such multiple origins are replicated synchronously; it should explain why the key initiator protein, DnaA, is activated in vitro by binding specifically to acidic phospholipids and why the Dam methyltransferase is essential for the correct timing of initiation; it should explain why phospholipid synthesis and fluidity are necessary for initiation. In the detachment model, presented here, cyclical changes in the phospholipid composition of the cytoplasmic membrane activate initiator proteins such as DnaA protein and cause origins to detach; this detachment allows torsional stresses to open 13mer sequences in oriC; DnaA assists in the serial opening of these sequences and guides the entry of the helicase to form a pre-priming complex and trigger initiation; the greater affinity of hemi-methylated origin for membrane is re-interpreted as a mechanism for preventing re-initiation.     

Acidithiobacillus ferrianus sp. nov.: an ancestral extremely acidophilic and facultatively anaerobic chemolithoautotroph

Strain MG, isolated from an acidic pond sediment on the island of Milos (Greece), is proposed as a novel species of ferrous iron- and sulfur-oxidizing Acidithiobacillus. Currently, four of the eight validated species of this genus oxidize ferrous iron, and strain MG shares many key characteristics with these four, including the capacities for catalyzing the oxidative dissolution of pyrite and for anaerobic growth via ferric iron respiration. Strain MG also grows aerobically on hydrogen and anaerobically on hydrogen coupled to ferric iron reduction. While the 16S rRNA genes of the iron-oxidizing Acidi-thiobacillus species (and strain MG) are located in a distinct phylogenetic clade and are closely related (98–99% 16S rRNA gene identity), genomic relatedness indexes (ANI/dDDH) revealed strong genomic divergence between strain MG and all sequenced type strains of the taxon, and placed MG as the first cultured representative of an ancestral phylotype of iron oxidizing acidithiobacilli. Strain MG is proposed as a novel species, Acidithiobacillus ferrianus sp. nov. The type strain is MG^T (= DSM 107098^T = JCM 33084^T). Similar strains have been found as isolates or indicated by cloned 16S rRNA genes from several mineral sulfide mine sites.

     

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.     

The short‐ and long‐term fitness consequences of natal dispersal in a wild bird population

Dispersal is a key process in population and evolutionary ecology. Individual decisions are affected by fitness consequences of dispersal, but these are difficult to measure in wild populations. A long‐term dataset on a geographically closed bird population, the Mauritius kestrel, offers a rare opportunity to explore fitness consequences. Females dispersed further when the availability of local breeding sites was limited, whereas male dispersal correlated with phenotypic traits. Female but not male fitness was lower when they dispersed longer distances compared to settling close to home. These results suggest a cost of dispersal in females. We found evidence of both short‐ and long‐term fitness consequences of natal dispersal in females, including reduced fecundity in early life and more rapid aging in later life. Taken together, our results indicate that dispersal in early life might shape life history strategies in wild populations.     

Acid Ceramidase Promotes Nuclear Export of PTEN through Sphingosine 1-Phosphate Mediated Akt Signaling

The tumor suppressor PTEN is now understood to regulate cellular processes at the cytoplasmic membrane, where it classically regulates PI3K signaling, as well as in the nucleus where multiple roles in controlling cell cycle and genome stability have been elucidated. Mechanisms that dictate nuclear import and, less extensively, nuclear export of PTEN have been described, however the relevance of these processes in disease states, particularly cancer, remain largely unknown. We investigated the impact of acid ceramidase on the nuclear-cytoplasmic trafficking of PTEN. Immunohistochemical analysis of a human prostate tissue microarray revealed that nuclear PTEN was lost in patients whose tumors had elevated acid ceramidase. We found that acid ceramidase promotes a reduction in nuclear PTEN that is dependent upon sphingosine 1-phosphate-mediated activation of Akt. We were further able to show that sphingosine 1-phosphate promotes formation of a complex between Crm1 and PTEN, and that leptomycin B prevents acid ceramidase and sphingosine 1-phosphate mediated loss of nuclear PTEN, suggesting an active exportin-mediated event. To investigate whether the tumor promoting aspects of acid ceramidase in prostate cancer depend upon its ability to export PTEN from the nucleus, we used enforced nuclear expression of PTEN to study docetaxel-induced apoptosis and cell killing, proliferation, and xenoengraftment. Interestingly, while acid ceramidase was able to protect cells expressing wild type PTEN from docetaxel, promote proliferation and xenoengraftment, acid ceramidase had no impact in cells expressing PTEN-NLS. These findings suggest that acid ceramidase, through sphingosine 1-phosphate, promotes nuclear export of PTEN as a means of promoting tumor formation, cell proliferation, and resistance to therapy.