Golgi proteomics : Identification of a novel cartilage-specific Golgi protein GoPro49

The Golgi complex is a central organelle of the secretory pathway, responsible for a range of post-translational modifications, as well as for membrane traffic to the plasma membrane and to the endosomal-lysosomal pathway. In addition, this organelle has roles in cell migration, in the regulation of traffic, and as a mitotic check point. The structure of the Golgi complex is highly dynamic and able to respond to the amount of cargo being transported and the stage of the cell cycle. The Golgi proteome reflects the functions and structure of this organelle, and can be divided into three major groups: the Golgi resident proteins (e.g. modification enzymes), the Golgi matrix proteins (involved in structure and tethering events), and trafficking proteins (e.g. vesicle coat proteins and Rabs). The Golgi proteome has been studied on several occasions, from both rat liver and mammary gland Golgi membranes using proteomic approaches, but still little more than half of the estimated Golgi proteome is known. Nevertheless, methodological improvements and introduction of shotgun proteomics have increased the number of identified proteins, and especially the number of identified transmembrane proteins. Cartilage, even though not a typical tissue in which to study membrane traffic, secretes large amounts of extracellular matrix proteins that are extensively modified, especially by amino acid hydroxylation, glycosylation and sulfation. Furthermore, the cartilage ECM contains several, large oligomeric proteins (such as collagen II) that are difficult to assemble and transport. Indeed, cartilage has been shown to be susceptible to changes both in secretory pathway (e.g. the COPII coat assembly) and in post-translational modifications (e.g. heparan sulfate formation). Dental follicle, and the periodontal ligament (PDL) that it forms, are another type of connective tissue, and they have a role in anchoring teeth to bone. This anchorage is achieved by numerous matrix fibres that connect the bone matrix with the cementum. These tissues have in common the secretion of large matrix molecules. In this study the Golgi proteome was analysed from purified, stacked Golgi membranes isolated from rat liver. The identified, extensive proteome included a protein similar to Ab2-095, or Golgi protein 49kDa (GoPro49), which was shown to localise to the Golgi complex as an EGFP fusion protein. Surprisingly, in situ hybridisation showed the GoPro49 expression to be highly restricted to different mesenchymal tissues, especially in cartilage, and this expression pattern was clearly developmentally regulated. In addition to cartilage, GoPro49 was also expressed in the dental follicle, but was not observed in the mature PDL. Importantly, GoPro49 is the first specific marker for the dental follicle. Endogenous GoPro49 protein co-localised with β-COP in both chondrosarcoma and primary dental follicle cell lines. The COPI staining in these cells was highly dynamic, showing a number of tubules. This may reflect the type of secretory cargo they secrete. Currently GoPro49 is the only Golgi protein with such a restricted expression pattern.

Mutations of mitochondrial DNA polymerase gamma: an important cause of neurological disorders

Thesis focuses on mutations of POLG1 gene encoding catalytic subunit polγ-α of mitochondrial DNA polymerase gamma holoenzyme (polG) and the association of mutations with different clinical phenotypes. In addition, particular defective mutant variants of the protein were characterized biochemically in vitro. PolG-holoenzyme is the sole DNA polymerase found in mitochondria. It is involved in replication and repair of the mitochondrial genome, mtDNA. Holoenzyme also includes the accessory subunit polγ-β, which is required for the enhanced processivity of polγ-α. Defective polγ-α causes accumulation of secondary mutations on mtDNA, which leads to a defective oxidative phosphorylation system. The clinical consequences of such mutations are variable, affecting nervous system, skeletal muscles, liver and other post-mitotic tissues. The aims of the studies included: 1) Determination of the role of POLG1 mutations in neurological syndromes with features of mitochondrial dysfunction and an unknown molecular cause. 2) Development and set up of diagnostic tests for routine clinical purposes. 3) Biochemical characterization of the functional consequences of the identified polγ-α variants. Studies describe new neurological phenotypes in addition to PEO caused by POLG1 mutations, including parkinsonism, premature amenorrhea, ataxia and Parkinson s disease (PD). POLG1 mutations and polymorphisms are both common and/or potential genetic risk factors at least among the Finnish population. The major findings and applications reported here are: 1) POLG1 mutations cause parkinsonism and premature menopause in PEO families in either a recessive or a dominant manner. 2) A common recessive POLG1 mutations (A467T and W748S) in the homozygous state causes severe adult or juvenile-onset ataxia without muscular symptoms or histological or mtDNA abnormalities in muscles. 3) A common recessive pathogenic change A467T can also cause a mild dominant disease in heterozygote carriers. 4) The A467T variant shows reduced polymerase activity due to defective template binding. 5) Rare polyglutamine tract length variants of POLG1 are significantly enriched in Finnish idiopathic Parkinson s disease patients. 6) Dominant mutations are clearly restricted to the highly conserved polymerase domain motifs, whereas recessive ones are more evenly distributed along the protein. The present results highlight and confirm the new role of mitochondria in parkinsonism/Parkinson s disease and describe a new mitochondrial ataxia. Based on these results, a POLG1 diagnostic routine has been set up in Helsinki University Central Hospital (HUSLAB).

Free and ATP-bound structures of Ap(4)A hydrolase from Aquifex aeolicus V5

Asymmetric diadenosine tetraphosphate (Ap(4)A) hydrolases degrade the metabolite Ap(4)A back into ATP and AMP. The three-dimensional crystal structure of Ap(4)A hydrolase (16 kDa) from Aquifex aeolicus has been determined in free and ATP-bound forms at 1.8 and 1.95 angstrom resolution, respectively. The overall three-dimensional crystal structure of the enzyme shows an alpha beta alpha-sandwich architecture with a characteristic loop adjacent to the catalytic site of the protein molecule. The ATP molecule is bound in the primary active site and the adenine moiety of the nucleotide binds in a ring-stacking arrangement equivalent to that observed in the X-ray structure of Ap(4)A hydrolase from Caenorhabditis elegans. Binding of ATP in the active site induces local conformational changes which may have important implications in the mechanism of substrate recognition in this class of enzymes. Furthermore, two invariant water molecules have been identified and their possible structural and/or functional roles are discussed. In addition, modelling of the substrate molecule at the primary active site of the enzyme suggests a possible path for entry and/or exit of the substrate and/or product molecule.

Conjunctival Melanoma in Finland 1967-2000: a population-based study

Eighty-five new cases of conjunctival melanoma (CM) were diagnosed in Finland between 1967 and 2000. The annual crude incidence of CM was 0.51 per million inhabitants. The average age-adjusted incidence of 0.54 doubled during the study period, analogous to the increase in the incidence of cutaneous malignant melanoma during this period, suggesting a possible role for ultraviolet radiation in its pathogenesis. Nonlimbal tumors were more likely than limbal ones to recur and they were associated with decreased survival. Increasing tumor thickness and recurrence of the primary tumor were other clinical factors related to death from CM. The histopathologic specimens of 85 patients with CM melanoma were studied for cell type, mitotic count, tumor-infiltrating lymphocytes and macrophages, mean vascular density, extravascular matrix loops and networks, and mean diameter of the ten largest nucleoli (MLN). The absence of epithelioid cells, increasing mitotic count and small MLN were associated with shorter time to recurrence according to the Cox univariate regression. None of the histopathologic variables was associated with mortality from CM. Four (5%) patients had a CM limited to the cornea without evidence of a tumor other than primary acquired melanosis of the conjunctiva. Because there are no melanocytes in the cornea, the origin of these melanomas most likely is the limbal conjunctiva. All four corneally displaced CM were limited to the epithelium, and none of the patients developed metastases. An anatomic sub-classification based on my patients and world literature was developed for corneally displaced CM. In 20 patients the metastatic pattern could be determined. Ten patients had initial systemic metastases detected, nine had initial regional metastases, and in one case the two types were detected simultaneously. The patients most likely to develop either type of initial metastases were those with nonlimbal conjunctival melanoma, those with a primary tumor more than 2 mm thick, and those with a recurrent conjunctival melanoma. Approximately two thirds of the patients had limbal CM, a location associated with good prognosis. One third, however, had a primary CM originating outside the limbus. In these patients the chance of developing local recurrences as well as systemic metastases was significantly higher than in patients with limbal CM. Each recurrence accompanies an increased risk of developing metastases, and recurrences contribute to death along with increasing tumor thickness and nonlimbal tumor location. In my data, an equal number of patients with initial locoregional and systemic metastasis existed. Patients with limbal primary tumors less than 2 mm in thickness rarely experienced metastases, unless the tumor recurred. Consequently, the patients most likely to benefit from sentinel lymph node biopsy are those who have nonlimbal tumors, CM that are over 2 mm thick, or recurrent CM. The histopathology of CM differs from that of uveal melanoma. Microvascular factors did not prove to be of prognostic importance, possibly due to the fact that CM at least as often disseminates first to the regional lymph nodes, unlike uveal melanoma that almost always disseminates hematogenously.

Performing immobility / protesting immobility: The risks & rewards of public space protest performances

Though there is much interest in mobilities and performing mobilities as a characteristic of modern, urban, social life today, this is not always matched by attention to immobilities, as the flipside of mobility in modern life. In this paper, I investigate public space performances designed to draw attention to precisely this counterpoint to current discourses of mobilities – performances about the socially produced immobilities many people with disabilities find a more fundamental feature of day-to-day life, the fight for mobility, and the freedom found when accommodations for alternative mobilities are made available. Although public policy is increasingly aligned with a social model of disability, which sees disability as socially constructed through systems, institutions and infrastructure deliberately designed to exclude specific bodies – stairs, curbs, queues and so forth – and although governments in the US, UK, and to a lesser degree Australia, New Zealand and other Commonwealth nations aim to address these inequalities, the experience of immobility is still every-present for many people. This often comes not just from pain, or from impairment, or event from lack of accommodations for alternative mobilities, but from fellow social performers’ antipathy to, appropriation of, or destruction of accommodations designed to facilitate access for a range of different bodies in public space, and thus the public sphere. The archetypal instance of this tension between the mobile, and those needing accommodations to allow mobility, is, of course, the antipathy many able bodied people feel towards the provision of disabled parking spaces. A cursory search online shows thousands of accounts of antagonism, vitriol, and even violence prompted by disputes which began when a disabled person asked an able person to exit a designated disabled parking space. For many, it seems, expecting them to pass by such parks so others can experience the mobility they take for granted is too much. In this paper, I examine a number of protest performances in public space in which activist present actions – for example, placing wheelchairs in every regular parking space in a precinct – to give bystanders, passersby and spectators, as well as antagonistic fellow social performers, a sense of what socially produced immobility feels like. I examine responses to such protest performances, and what they say about the potential social, political and ethical impacts of such protests, in terms of their potential to produce new attitudes to mobility, alternative mobility, and access to alternative modes of mobility.

Vector Evaluated Particle Swarm Optimization (VEPSO) of Supersonic Ejector for Hydrogen Fuel Cells

Fuel cells are emerging as alternate green power producers for both large power production and for use in automobiles. Hydrogen is seen as the best option as a fuel; however, hydrogen fuel cells require recirculation of unspent hydrogen. A supersonic ejector is an apt device for recirculation in the operating regimes of a hydrogen fuel cell. Optimal ejectors have to be designed to achieve best performances. The use of the vector evaluated particle swarm optimization technique to optimize supersonic ejectors with a focus on its application for hydrogen recirculation in fuel cells is presented here. Two parameters, compression ratio and efficiency, have been identified as the objective functions to be optimized. Their relation to operating and design parameters of ejector is obtained by control volume based analysis using a constant area mixing approximation. The independent parameters considered are the area ratio and the exit Mach number of the nozzle. The optimization is carried out at a particularentrainment ratio and results in a set of nondominated solutions, the Pareto front. A set of such curves can be used for choosing the optimal design parameters of the ejector.

Salt in the desert: A comparison of clinical versus serum-calculated osmolality determination of dehydration

Background: Clinicians frequently use their own judgement to assess patient’s hydration status although there is limited evidence for the diagnostic utility of any individual clinical symptom. Hence, the aim of this study was to compare the diagnostic accuracy of clinically assessed dehydration in older hospital patients (using multiple symptoms), versus dehydration measured using serum-calculated osmolality (CO) as the reference standard. Method: Participants were 44 hospital patients aged ≥ 60 years. Dehydration was assessed clinically and pathologically (CO) within 24 hours of admission and at study exit. Indicators of diagnostic accuracy were calculated. Results: Clinicians identified 27% of patients as dehydrated at admission, and 19% at exit, compared to 19% and 16% using CO. Agreement between the measures was fair at admission and poor at exit. Clinical assessment showed poor sensitivity for predicting dehydration with reasonable specificity. Conclusions: Compared to the use of CO, clinical assessment of dehydration in older patients was poor. Given that failure to identify dehydration in this population may have serious consequences, we recommend that clinicians do not rely upon their own assessments without also using the reference standard.

Associations between dehydration, cognitive impairment and frailty in older hospitalized patients: An exploratory study

An exploratory study was undertaken to assess the prevalence of dehydration in older patients (aged ≥60 years) with and without cognitive impairment (CI) admitted to hospital and to examine associations between dehydration, CI and frailty. Forty-four patients participated and dehydration was assessed within 24 hours of admission and at day 4 or at discharge, whichever occurred first (study exit). Patients’ cognitive function and frailty status were assessed using validated instruments. Twenty-seven (61%) patients had CI and 61% were frail. The prevalence of dehydration at admission was 29% (n=12) and 19% (n=6) at study exit and dehydration status did not differ according to either CI status or frailty status. Within the non-CI group, however, significantly more frail than fit patients were dehydrated at admission (p=0.03). These findings indicate that dehydration is common amongst older hospital patients, and that frailty may increase the risk for dehydration in cognitively intact older patients.

MECHANISMS AND EFFECTS OF MITOCHONDRIAL DNA INSTABILITY AND COPY NUMBER MANIPULATION

Defects in mitochondrial DNA (mtDNA) maintenance cause a range of human diseases, including autosomal dominant progressive external ophthalmoplegia (adPEO). This study aimed to clarify the molecular background of adPEO. We discovered that deoxynucleoside triphosphate (dNTP) metabolism plays a crucial in mtDNA maintenance and were thus prompted to search for therapeutic strategies based on the modulation of cellular dNTP pools or mtDNA copy number. Human mtDNA is a 16.6 kb circular molecule present in hundreds to thousands of copies per cell. mtDNA is compacted into nucleoprotein clusters called nucleoids. mtDNA maintenance diseases result from defects in nuclear encoded proteins that maintain the mtDNA. These syndromes typically afflict highly differentiated, post-mitotic tissues such as muscle and nerve, but virtually any organ can be affected. adPEO is a disease where mtDNA molecules with large-scale deletions accumulate in patients tissues, particularly in skeletal muscle. Mutations in five nuclear genes, encoding the proteins ANT1, Twinkle, POLG, POLG2 and OPA1, have previously been shown to cause adPEO. Here, we studied a large North American pedigree with adPEO, and identified a novel heterozygous mutation in the gene RRM2B, which encodes the p53R2 subunit of the enzyme ribonucleotide reductase (RNR). RNR is the rate-limiting enzyme in dNTP biosynthesis, and is required both for nuclear and mitochondrial DNA replication. The mutation results in the expression of a truncated form of p53R2, which is likely to compete with the wild-type allele. A change in enzyme function leads to defective mtDNA replication due to altered dNTP pools. Therefore, RRM2B is a novel adPEO disease gene. The importance of adequate dNTP pools and RNR function for mtDNA maintenance has been established in many organisms. In yeast, induction of RNR has previously been shown to increase mtDNA copy number, and to rescue the phenotype caused by mutations in the yeast mtDNA polymerase. To further study the role of RNR in mammalian mtDNA maintenance, we used mice that broadly overexpress the RNR subunits Rrm1, Rrm2 or p53R2. Active RNR is a heterotetramer consisting of two large subunits (Rrm1) and two small subunits (either Rrm2 or p53R2). We also created bitransgenic mice that overexpress Rrm1 together with either Rrm2 or p53R2. In contrast to the previous findings in yeast, bitransgenic RNR overexpression led to mtDNA depletion in mouse skeletal muscle, without mtDNA deletions or point mutations. The mtDNA depletion was associated with imbalanced dNTP pools. Furthermore, the mRNA expression levels of Rrm1 and p53R2 were found to correlate with mtDNA copy number in two independent mouse models, suggesting nuclear-mitochondrial cross talk with regard to mtDNA copy number. We conclude that tight regulation of RNR is needed to prevent harmful alterations in the dNTP pool balance, which can lead to disordered mtDNA maintenance. Increasing the copy number of wild-type mtDNA has been suggested as a strategy for treating PEO and other mitochondrial diseases. Only two proteins are known to cause a robust increase in mtDNA copy number when overexpressed in mice; the mitochondrial transcription factor A (TFAM), and the mitochondrial replicative helicase Twinkle. We studied the mechanisms by which Twinkle and TFAM elevate mtDNA levels, and showed that Twinkle specifically implements mtDNA synthesis. Furthermore, both Twinkle and TFAM were found to increase mtDNA content per nucleoid. Increased mtDNA content in mouse tissues correlated with an age-related accumulation of mtDNA deletions, depletion of mitochondrial transcripts, and progressive respiratory dysfunction. Simultaneous overexpression of Twinkle and TFAM led to a further increase in the mtDNA content of nucleoids, and aggravated the respiratory deficiency. These results suggested that high mtDNA levels have detrimental long-term effects in mice. These data have to be considered when developing and evaluating treatment strategies for elevating mtDNA copy number.

Studies on the nucleolus -II.The persisting nucleoli of Cicer arietinum Linn.

The behaviour of the nucleoli from mitotic prophase till the end of telophase is illustrated. Since at prophase, the SAT-threads appear embedded in the nucleolar matrix, their orientation in the mass and the rate of dissolution of the latter would determine whether this relationship would continue till meta- or ana-phase. The persistence of the nucleoli-SAT association often till anaphase indicates that the movement of such nucleoli on the spindle is governed by that of the SAT-chromosomes.

Integrin trafficking regulated by Rab21 is necessary for cytokinesis

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer.

Studies on the structure of the chromosomes - I. Allium cepa-Stain fixatives and their utility

The structure of the mitotic chromosomes of Allium cepa has been elucidated by controlling the temperature and time of exposure of fresh roots to stain fixatives. The details seen in material stained in N HCl-orcein for 8 min. at 60° C. and squashed after varying intervals of storage at room temperature were essentially similar to pictures obtained with 1% aceto-orcein and 1% aceto-orcein-N HCl (10:1) under identical conditions of handling. The chromosomes appear quadri-partite at metaphase and bi-partite at anaphase. A rare instance of the precocious assumption of a quadri-partite condition by two anaphase chromosomes is illustrated. Caduceus coiling of chromonemata was seen in chromosome bridges also. Chromosomes have material easily dissociable from the chromonemata and their removal does not affect the structural integrity of the chromosome.

Lightning-Induced Current and Voltage on a Rocket in the Presence of Its Trailing Exhaust Plume

This paper presents time-domain characteristics of induced current and voltage on a rocket in the presence of its exhaust plume when an electromagnetic (EM) wave generated by a nearby lightning discharge is incident on it. For the EM-field interaction with the rocket, the finite-difference time-domain technique has been used. The distributed electrical parameters, such as capacitance and inductance of the rocket and its exhaust plume, are computed using the method of moments technique. For the electrical characterization of the exhaust plume, the computational fluid dynamics technique has been used. The computed peak value of the electrical conductivity of the exhaust plume is 0.12 S/m near the exit plane and it reduces to 0.02 S/m at the downstream end. The relative permittivity varies from 0.91 to 0.99. The exhaust plume behaves as a good conductor for EM fields with frequencies less than 2.285 GHz. It has been observed that the peak value of the induced current on the rocket gets enhanced significantly in the presence of the conducting exhaust plume for the rocket and exhaust plume dimensions and parameters studied. The magnitude of the time-varying induced current at the tail is much more than that of any other section of the rocket.

Lightning-Induced Current and Voltage on a Rocket in the Presence of Its Trailing Exhaust Plume

This paper presents time-domain characteristics of induced current and voltage on a rocket in the presence of its exhaust plume when an electromagnetic (EM) wave generated by a nearby lightning discharge is incident on it. For the EM-field interaction with the rocket, the finite-difference time-domain technique has been used. The distributed electrical parameters, such as capacitance and inductance of the rocket and its exhaust plume, are computed using the method of moments technique. For the electrical characterization of the exhaust plume, the computational fluid dynamics technique has been used. The computed peak value of the electrical conductivity of the exhaust plume is 0.12 S/m near the exit plane and it reduces to 0.02 S/m at the downstream end. The relative permittivity varies from 0.91 to 0.99. The exhaust plume behaves as a good conductor for EM fields with frequencies less than 2.285 GHz. It has been observed that the peak value of the induced current on the rocket gets enhanced significantly in the presence of the conducting exhaust plume for the rocket and exhaust plume dimensions and parameters studied. The magnitude of the time-varying induced current at the tail is much more than that of any other section of the rocket.

Expression of cyclin E in endomitotic silk-gland cells from mulberry silkworm

The silk glands of mulberry silkworm Bombyx mori are endoreplicating tissues in which the genomic DNA undergoes multiple rounds of replication without mitosis and nuclear division. In the absence of normal mitotic division, the cell cycle essentially alternates between the G1 and S phases. Cyclin E is crucial for the G1/S transition in both mitotic and endoreplicating cycles. We have cloned and characterized cyclin E (cyclin box) from B. mori, which is nearly identical to the Drosophila cyclin E box except for an insertion of 21 amino acids. Two distinct cyclin E transcripts (1.7 and 2.1 kb) were detected in the silk-gland cells of B. mori and in the B. mori-derived embryonic cell line, BmN. Using anti Cyclin E antibodies two protein bands of 52 and 44 kDa were detected in silk glands and BmN cells at Comparable levels. Both BmN- and the silk-gland cells showed the presence of the interacting kinase Cdk2. Transcripts of the mitotic cyclin, cyclin B, were barely detectable in the endoreplicating silk-gland cells and amounted to only 4-7% of that seen in the mitotically dividing BmN cells. The near absence of cyclin B transcripts and the abundant expression of cyclin E in the silk glands correlate well with the alternation of only G1 and S phases without the intervening mitosis in these cells. (C) 2000 Elsevier Science B.V. All rights reserved.