Identification of a novel nucleolin related protein (NRP) gene expressed during rat spermatogenesis

Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells.

Novel prognostic factors in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a malignant clonal blood disease that originates from a pluripotent hematopoietic stem cell. The cytogenetic hallmark of CML, the Philadelphia chromosome (Ph), is formed as a result of reciprocal translocation between chromosomes 9 and 22, which leads to a formation of a chimeric BCR-ABL fusion gene. The BCR-ABL protein is a constitutively active tyrosine kinase that changes the adhesion properties of cells, constitutively activates mitogenic signaling, enhances cell proliferation and reduces apoptosis. This results in leukemic growth and the clinical disease, CML. With the advent of targeted therapies against the BCR-ABL fusion protein, the treatment of CML has changed considerably during the recent decade. In this thesis, the clinical significance of different diagnostic methods and new prognostic factors in CML have been assessed. First, the association between two different methods for measuring CML disease burden (the RQ-PCR and the high mitotic index metaphase FISH) was assessed in bone marrow and peripheral blood samples. The correlation between positive RQ-PCR and metaphase FISH samples was high. However, RQ-PCR was more sensitive and yielded measurable transcripts in 40% of the samples that were negative by metaphase FISH. The study established a laboratory-specific conversion factor for setting up the International Scale when standardizing RQ-PCR measurements. Secondly, the amount of minimal residual disease (MRD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) was determined. For this, metaphase FISH was done for the bone marrow samples of 102 CML patients. Most (68%), had no residual cells during the entire follow-up time. Some (12 %) patients had minor (<1%) MRD which decreased even further with time, whereas 19% had a progressive rise in MRD that exceeded 1% or had more than 1% residual cells when first detected. Residual cells did not become eradicated spontaneously if the frequency of Ph+ cells exceeded 1% during follow-up. Next, the impact of deletions in the derivative chromosome 9, was examined. Deletions were observed in 15% of the CML patients who later received alloHSCT. After alloHSCT, there was no difference in the total relapse rate in patients with or without deletions. Nor did the estimates of overall survival, transplant-related mortality, leukemia-free survival and relapse-free time show any difference between these groups. When conventional treatment regimens are used, the der(9) status could be an important criterion, in conjunction with other prognostic factors, when allogeneic transplantation is considered. The significance of der(9) deletions for patients treated with tyrosine kinase inhibitors is not clear and requires further investigation. In addition to the der(9) status of the patient, the significance of bone marrow lymphocytosis as a prognostic factor in CML was assessed. Bone marrow lymphocytosis during imatinib therapy was a positive predictive factor and heralded optimal response. When combined with major cytogenetic response at three months of treatment, bone marrow lymphocytosis predicted a prognostically important major molecular response at 18 months of imatinib treatment. Although the validation of these findings is warranted, the determination of the bone marrow lymphocyte count could be included in the evaluation of early response to imatinib treatment already now. Finally, BCR-ABL kinase domain mutations were studied in CML patients resistant against imatinib treatment. Point mutations detected in the kinase domain were the same as previously reported, but other sequence variants, e.g. deletions or exon splicing, were also found. The clinical significance of the other variations remains to be determined.

Antizyme Inhibitor 2 : A Novel Regulator of Cellular Polyamine Homeostasis

Polyamines are organic polycations that participate in various physiological functions, including cell proliferation, differentiation and apoptosis. Cellular polyamines originate from endogenous biosynthesis and exogenous sources. Their subcellular pool is under strict control, achieved by regulating their uptake and metabolism. Polyamine-induced proteins called antizymes (AZ) act as key regulators of intracellular polyamine concentration. They regulate both the transport of polyamines and the activity and degradation of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. AZs themselves are negatively regulated by antizyme inhibitor (AZIN). AZIN functions as a positive regulator of cellular polyamine homeostasis, which by binding to AZs reactivates ODC and induces the uptake of polyamines. In various pathological conditions, including cancer, polyamine levels are misregulated. Polyamine homeostasis has therefore become an attractive target for therapeutic interventions and it is thus crucial to characterize the molecular basis underlying the homeostatic regulation. A novel human AZIN-resembling protein was previously identified in our group. The purpose of this study was to elucidate the function and distribution of this protein, termed as an antizyme inhibitor 2 (AZIN2). According to my results, AZIN2 functions as a novel regulator of polyamine homeostasis. It shows no enzymatic activity, but instead it binds AZs and negates their activity, which subsequently leads to reactivation of ODC and inhibition of its degradation. Expression of AZIN2 is restricted to terminally differentiated cells, such as mast cells (MC) and neurosecretory cells. In these actively secreting cell types, AZIN2 localizes to subcellular vesicles or granules where its function is important for the vesicle-mediated secretion. In MCs, AZIN2 localizes to the serotonin-containing subset of MC granules, and its expression is coupled to MC activation. The functional role of polyamines as potential mediators of MC activity was also investigated, and it was observed that the secretion of serotonin is selectively dependent on activation of ODC. In neurosecretory cells, AZIN2-positive vesicles localize mainly to the trans-Golgi network (TGN). Depletion of AZIN2 or cellular polyamines causes selective fragmentation of the TGN and retards secretion of proteins. Since addition of exogenous polyamines reverses these effects, the data indicate that AZIN2 and its downstream effectors, polyamines, are functionally implicated in the regulation of secretory vesicle transport. My studies therefore reveal a novel function for polyamines as modulators of both constitutive and regulated secretion. Based on the results, I propose that the role of AZIN2 is to act as a local in situ activator of polyamine biosynthesis.

New reagents 3: aluminium iodide - a highly regioselective ether-cleaving reagent with novel cleavage pattern

AlI3 is an easily accessible and versatile ether-cleaving reagent.

Novel insights on functions of myotilin/palladin family members

Poikkijuovaisen luuranko- ja syd��nlihaksen supistumisyksikk��, sarkomeeri, koostuu tarkoin j��rjestyneist�� aktiini- ja myosiinis��ikeist��. Rakenne eroaa muista solutyypeist��, joissa aktiinis��ikeist�� muovautuu jatkuvasti ja sen j��rjestyminen s����telee solun muotoa, solujakautumista, soluliikett�� ja solunsis��isten organellien kuljetusta. Myotilin, palladin ja myopalladin kuuluvat proteiiniperheeseen, jonka yhteispiirteen�� ovat immunoglobuliinin kaltaiset (Igl) domeenit. Proteiinit liittyv��t aktiinitukirankaan ja niiden arvellaan toimivan solutukirangan rakenne-elementtein�� ja s����telij��in��. Myotilinia ja myopalladinia ilmennet����n poikkijuovaisessa lihaksessa. Sen sijaan palladinin eri silmukointimuotoja tavataan monissa kudostyypeiss�� kuten hermostossa, ja eri muodoilla saattaa olla solutyypist�� riippuvia teht��vi��. Poikkijuovaisessa lihaksessa kaikki perheen j��senet sijaitsevat aktiinis��ikeit�� yhdist��v��ss�� Z-levyss�� ja ne sitovat Z-levyn rakenneproteiinia, ���-aktiniinia. Myotilingeenin pistemutaatiot johtavat periytyviin lihastauteihin, kun taas palladinin mutaatioiden on kuvattu liittyv��n periytyv����n haimasy��p����n ja lis����ntyneeseen syd��ninfarktin riskiin. T��ss�� tutkimuksessa selvitettin myotilinin ja pallainin toimintaa. Kokeissa l��ydettiin uusia palladinin 90-92kDa alatyyppiin sitoutuvia proteiineja. Yksi niist�� on aktiinidynamiikkaa s����telev�� profilin. Profilinilla on kahdenlaisia teht��vi��; se edesauttaa aktiinis��ikeiden muodostumista, mutta se voi my��s erist���� yksitt��isi�� aktiinimolekyylej�� ja edist���� s��ikeiden hajoamista. Solutasolla palladinin ja profilinin sijainti on yhtenev�� runsaasti aktiinia sis��lt��vill�� solujen reuna-alueilla. Palladinin ja profilinin sidos on heikko ja hyvin dynaaminen, joka sopii palladinin teht��v����n aktiinis��ideiden muodostumisen koordinoijana. Toinen palladinin sitoutumiskumppani on aktiinis��ikeit�� yhteensitova ���-aktiniini. ���-Aktiniini liitt���� solutukirangan solukalvon proteiineihin ja ankkuroi solunsis��isi�� viestint��molekyylej��. Sitoutumista v��litt��v�� alue on hyvin samankaltainen palladinissa ja myotilinissa. Luurankolihaksen liiallinen toistuva venytys muuttaa Z-levyjen rakennetta ja muotoa. Prosessin aikana syntyy uusia aktiinifilamenttej�� sis��lt��vi�� tiivistymi�� ja lopulta uusia sarkomeereja. L��yd��stemme perusteella myotilinin uudelleenj��rjestyminen noudattaa aktiinin muutoksia. T��m�� viittaa siihen, ett�� myotilin liitt���� yhteen uudismuodostuvia aktiinis��ikeit�� ja vakauttaa niit��. Myotilin saattaa my��s ankkuroida viesti- tai rakennemolekyylej��, joiden teht��v��n�� on edesauttaa Z-levyjen uudismuodostusta. Tulostemme perusteella arvelemme, ett�� myotilin toimii Z-levyjen rakenteen vakaajana ja aktiinis��ikeiden s����telij��n��. Palladinin puute johtaa siki��aikaiseen kuolemaan hiirill��, mutta myotilinin puutoksella ei ole samanlaisia vaikutuksia. Tuotettujen myotilin poistogeenisten hiirten todetiin syntyv��n ja kehittyv��n normaalisti eik�� niill�� esiintynyt rakenteellisia tai toiminnallisia h��iri��it��. Toisaalta aiemmissa kokeissa, joissa hiirille on siirretty ihmisen lihastautia aikaansaava myotilingeeni, n��hd����n samankaltaisia kuin sairailla ihmisill��. N��in ollen muuntunut myotilin n��ytt�� olevan lihaksen toiminnalle haitallisempi kuin myotilinin puute. Myotilinin ja palladinin yhteisvaikutusta selvitt����ksemme risteytimme myotilin poistegeenisen hiiren ja hiirilinjan, joka ilment���� puutteellisesti palladinin 200 kDa muotoa. Puutteellisesti 200 kDa palladinia ilment��vien hiirten syd��nlihaksessa todettiin v��h��isi�� hienorakenteen muutoksia, mutta risteytetyill�� hiirill�� tavattiin rakenteellisia ja toiminnallisia muutoksia my��s luurankolihaksessa. Tulosten perusteella voidaan todeta, ett�� palladinin 200 kDa muoto s����telee syd��nlihassolujen rakennetta. Luurankolihaksessa sen sijaan myotilinilla ja palladinilla n��ytt��isi olevan p����llekk��isi�� teht��vi��.

New Functional Biomaterials for Medicine and Healthcare

The book begins with an overview of the use of biomaterials in contemporary healthcare and the process of developing novel biomaterials; the key issues and challenges associated with the design of complex implantable systems are also highlighted. The book then reviews the main materials used in functional biomaterials, particularly their properties and applications. Individual chapters focus on both natural and synthetic polymers, metallic biomaterials, and bio-inert and bioactive ceramics.

Amide-catalysed isomerisation of 5,6-dihydroisoquinolines: a novel synthesis of 1,2-dihydroisoquinolines

Knoevenagel condensation of 2-acylcyclohexanones or 2-ethoxycarbonylcyclohexanone with either cyanoacetamide or malononitrile followed by silver salt alkylation gave the 5,6,7,8-tetrahydroisoquinolines (3a���i). Chromic acid oxidation of the 5,6,7,8-tetrahydroisoquinolines (3a���i) to the corresponding tetralones (4a���i) followed by sodium borohydride reduction and p-toluenesulphonic acid-catalysed dehydration of the resulting alcohols (5a���i) gave the 5,6-dihydroisoquinolines (6a���i). Reaction of 5,6-dihydroisoquinolines (6a���g) with potassium amide in liquid ammonia gave a mixture of the 1,3-dihydroisoquinolines (7a���g) and the isoquinolines (8a���g). The C-1 unsubstituted 1,2-dihydroisoquinoline (7c) was found to be very unstable. In the case of the 5,6-dihydroisoquinolines (6h and 6i), reaction of potassium amide in liquid ammonia resulted in a mixture of 1-aminoisoquinoline (9) and the isoquinolines (8h and 8i). All the above compounds have been characterised by spectral data. A probable pathway for the formation of the 1,2-dihydroisoquinolines (7a���g) and the isoquinolines (8a���i) is suggested.

Molecular structure of a cyclic tetrapeptide disulfide. A novel 310 helical conformation with an S-S bridge

The crystal structure of the cyclic peptide disulfide Boc-Cys-Pro-Aib-Cys-NHMe has been determined by X-ray diffraction. The peptide crystallizes in the space group P212121, with A = 8.646(1), B = 18.462(2), C = 19.678(3)�� and Z = 4. The molecules adopt a highly folded compact conformation, stabilized by two intramolecular 4��� 1 hydrogen bonds between the Cys (1) and Pro (2) CO groups and the Cys (4) and methylamide NH groups, respectively. The backbone conformational angles for the peptide lie very close to those expected for a 310 helix. The S-S bridge adopts a right handed twist with a dihedral angle of 82��. The structure illustrates the role of stereochemically constrained residues, in generating novel peptide conformations. Aib, ��-aminoisobutyric acid; Z, benzyloxycarbonyl; Boc, t-butyloxycarbonyl; OMe, methyl ester; OBz, benzyl ester; NHMe, N-methylamide; Tosyl, p-toluenesulfonyl.

A Novel Approach to the Study of Surface Oxidation States and Oxidation of Transition Metals by Auger Electron Spectroscopy

L$_{23}$ M$_{45}$ M$_{45}$/L$_{23}$ M$_{23}$ M$_{45}$, L$_{23}$ M$_{45}$ M$_{45}$/L$_{23}$ M$_{23}$ M$_{23}$ and L$_{23}$ M$_{23}$ M$_{45}$/L$_{23}$ M$_{23}$ M$_{23}$ Auger intensity ratios in transition metal oxides and sulphides are shown to be directly related to the number of valence electrons in the metal as well as to its oxidation state. The metal Auger intensity ratios provide a unique probe, independent of O (KLL) intensity, to study surface oxidation states of metals. These intensity ratios have been effectively employed to investigate surface oxidation of nickel, iron and copper. The oxidation studies have unravelled some interesting aspects of surface oxidation.

Novel role of the nitrite transporter NirC in Salmonella pathogenesis: SPI2-dependent suppression of inducible nitric oxide synthase in activated macrophages

Activation of macrophages by interferon gamma (IFN- ) and the subsequent production of nitric oxide (NO) are critical for the host defence against Salmonella enterica serovar Typhimurium infection. We report here the inhibition of IFN- -induced NO production in RAW264.7 macrophages infected with wild-type Salmonella. This phenomenon was shown to be dependent on the nirC gene, which encodes a potential nitrite transporter. We observed a higher NO output from IFN- -treated macrophages infected with a nirC mutant of Salmonella. The nirC mutant also showed significantly decreased intracellular proliferation in a NO-dependent manner in activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the gene in trans. Under acidified nitrite stress, a twofold more pronounced NO-mediated repression of SPI2 was observed in the nirC knockout strain compared to the wild-type. This enhanced SPI2 repression in the nirC knockout led to a higher level of STAT-1 phosphorylation and inducible nitric oxide synthase (iNOS) expression than seen with the wild-type strain. In iNOS knockout mice, the organ load of the nirC knockout strain was similar to that of the wild-type strain, indicating that the mutant is exclusively sensitive to the host nitrosative stress. Taken together, these results reveal that intracellular Salmonella evade killing in activated macrophages by downregulating IFN- -induced NO production, and they highlight the critical role of nirC as a virulence gene.

Research update: LANDMark study: Longitudinal assessment of neuropathy in diabetes using novel ophthalmic MARKers

The eye is a simple, non-invasive location for screening, diagnosing and follow up of diabetic peripheral neuropathy.

Novel intramolecular reductive C-C coupling during borohydride reduction of a bi-enone

Borohydride reduction of the bi-enone (1) gave the structurally and mechanistically interesting compounds (4) and (5a-c) resulting from intramolecular carbon-carbon coupling.

Pond apple (Annona glabra L.) - Investigating novel mechanical control options

Pond apple usually occurs in swampy areas, but mechanical control may be a viable option in some locations during drier periods. Two machines, the Positrack��� and the Tracksaw���, have been trialled for initial kill rate, amount of follow-up control required, safety to field operators, cost-efficiency and selectivity (effect on native vegetation), compared to other control options. The Positrack��� is a tracked bobcat with a slasher-type attachment that cuts individual trees off near ground level and mulches them. It has no on-board herbicide application capability and requires an additional on-ground operator to apply herbicide by hand. The Tracksaw��� is a tracked mini-excavator with a chainsaw bar and spray applicator on the boom that cuts individual trees off near ground level and applies chemical immediately to the cut stump, requiring only a single operator. Initial trials were done in infestations of similar sizes and densities at the Daintree (Positrack���) and in Innisfail (Tracksaw���) in late 2009. Kill rates to date are 83% for Positrack��� mechanical, 95% for Positrack��� mechanical plus herbicide, and 78% for the Tracksaw��� combined treatment. If ongoing comparison proves either of these machines to be more cost effective, selective, and safer than traditional control methods, mechanical control methods may become more widely used.