PENGAWASAN RUANG TERBUKA HIJAU SEBAGAI KAWASAN PERUMAHAN BERDASARKAN PERATURAN DAERAH KOTA MADIUN NOMOR 06 TAHUN 2011 TENTANG RENCANA TATA RUANG WILAYAH KOTA MADIUN 2010-2030

Dalam skripsi ini penulis mengenai masalah pengawasan ruang terbuka hijau sebagai kawasan perumahan yang diatur dalam Peraturan Daerah Kota Madiun Nomor 06 Tahun 2011 Tentang Rencana Tata Ruang Wilayah Kota Madiun 2010-2030. Hal ini yang dilatarbelakangi banyaknya penyalahgunaan kawasan yang seharusnya diperuntukan sebagai kawasan ruang terbuka hijau dialihfungsikan menajadi kawasan perumahan ataupun kawasan pertokoan tanpa ada kontrol yang jelas dari pemerintah kota madiun. permasalahan inilah yang membuat penulis melakukan penelitian pengawasan ruang terbuka hijau sebagai kawasan perumahan.Dalam penelitian ini penulis mengunakan metode yuridis sosiologis. Dimana dalam pendekatan ini mengkaji mengenai reaksi dan interaksi yang terjadi ketika sistem norma bekerja di dalam masyarakat.Berdasarkan hasil penelitian, penulis memperoleh jawaban atas permasalahan yang ada bahwa pengawasan ruang terbuka hijau sebagai kawasan perumahan menurut Peraturan Daerah Kota Madiun Nomor 06 Tahun 2011 Tentang Rencana Tata Ruang Wilayah Kota Madiun 2010-2030 hanya pengawasan secara umum tanpa ada pengawasan lanjutan, ini yang menjadi kelemahan pada peraturan daerah yang berlaku dan menjadikan pengawasan berjalan kurang optimal. Selain itu dari sisi kelembagaan, ada lembaga yang tidak dilibatkan dalam pegawasan. Dari segi teknis, pengawasan hanya mengandalkan peran Satpol PP sehingga menjadi tidak maksimal dalam pengawasannya. Masyarakat Kota Madiun sendiri banyak yang acuh atau tidak peduli dengan adanya ruang terbuka hijau. Hal-hal di atas yang menjadikan pengawasan ruang terbuka hijau Kota Madiun menjadi kurang maksimal.Solusi terkait hambatan-hambatan terkait permasalahan pengawasan ruang terbuka hijau Kota Madiun dengan menambah aturan khusus terkait pengawasan ruang terbuka hijau. Selain itu dari pihak Badan Perencanaan Pembangunan Daerah Kota Madiun untuk lebih meningkatkan lagi pemngawasan pemberian advice planning pada setiap pemohon ijin. Dan bagi masyarakat agar lebih meningkatkan pemahaman akan pentingnya ruang terbuka hijau

Expression, Purification and Characterization of Soluble Red Rooster Laforin as a Fusion Protein in Escherichia Coli

BACKGROUND: The gene that encodes laforin, a dual-specificity phosphatase with a carbohydrate-binding module, is mutated in Lafora disease (LD). LD is an autosomal recessive, fatal progressive myoclonus epilepsy characterized by the intracellular buildup of insoluble, hyperphosphorylated glycogen-like particles, called Lafora bodies. Laforin dephosphorylates glycogen and other glucans in vitro, but the structural basis of its activity remains unknown. Recombinant human laforin when expressed in and purified from E. coli is largely insoluble and prone to aggregation and precipitation. Identification of a laforin ortholog that is more soluble and stable in vitro would circumvent this issue. RESULTS: In this study, we cloned multiple laforin orthologs, established a purification scheme for each, and tested their solubility and stability. Gallus gallus (Gg) laforin is more stable in vitro than human laforin, Gg-laforin is largely monomeric, and it possesses carbohydrate binding and phosphatase activity similar to human laforin. CONCLUSIONS: Gg-laforin is more soluble and stable than human laforin in vitro, and possesses similar activity as a glucan phosphatase. Therefore, it can be used to model human laforin in structure-function studies. We have established a protocol for purifying recombinant Gg-laforin in sufficient quantity for crystallographic and other biophysical analyses, in order to better understand the function of laforin and define the molecular mechanisms of Lafora disease

Structural Mechanism of Laforin Function in Glycogen Dephosphorylation and Lafora Disease

Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease

“Pengawasan Ruang Terbuka Hijau Sebagai Kawasan Perumahan Menurut Peraturan Daerah Kota Madiun Nomor 06 Tahun 2011 Tentang Rencana Tata Ruang Wilayah Kota Madiun 2010-2030 ”,.

Dalam Skripsi Ini Penulis Mengenai Masalah Pengawasan Ruang Terbuka Hijau Sebagai Kawasan Perumahan Yang Diatur Dalam Peraturan Daerah Kota Madiun Nomor 06 Tahun 2011 Tentang Rencana Tata Ruang Wilayah Kota Madiun 2010-2030. Hal Ini Yang Dilatarbelakangi Banyakanya Penyalahgunaan Kawasan Yang Seharusnya Peruntukan Sebagai Kawasan Ruang Terbuka Hijau Dialihfungsikan Menajadi Kawasan Perumahan Ataupun Kawasan Pertokoan Tanpa Ada Kontrol Yang Jelas Dari Pemerintah Kota Madiun. Permasalahan Inilah Yang Membuat Penulis Melakukan Penelitian Pengawasan Ruang Terbuka Hijau Sebagai Kawasan Perumahan. Dalam Penelitiaan Ini Penulis Mengunakan Metode Yuridis Sosiologis. Dimana Dalam Pendekatan Ini Mengkaji Mengenai Reaksi Dan Interaksi Yang Terjadi Ketika Sistem Norma Bekerja Di Dalam Masyarakat. Berdasarkan Hasil Penelitian, Penulis Memperoleh Jawaban Atas Permasalahan Yang Ada Bahwa Pengawasan Ruang Terbuka Hijau Sebagai Kawasan Perumahan Menurut Peraturan Daerah Kota Madiun Nomor 06 Tahun 2011 Tentang Rencana Tata Ruang Wilayah Kota Madiun 2010-2030 Hanya Pengawasan Secara Umum Tanpa Ada Pengawasan Lanjutan, Ini Yang Menjadi Kelemahan Pada Peraturan Daerah Yang Berlaku Dan Menjadikan Pengawasan Berjalan Kurang Optimal. Selain Itu Dari Sisi Kelembagaan, Ada Lembaga Yang Tidak Dilibatkan Dalam Pegawasan. Dari Segi Teknis, Pengawasan Hanya Mengandalkan Peran Satpol Pp Sehingga Menjadi Tidak Maksimal Dalam Pengawasannya. Masyarakat Kota Madiun Sendiri Banyak Yang Acuh Atau Tidak Peduli Dengan Adanya Ruang Terbuka Hijau. Hal-Hal Di Atas Yang Menjadikan Pegawasan Ruang Terbuka Hijau Kota Madiun Menjadi Kurang Maksimal. Solusi Terkait Hambatan-Hambatan Terkait Permasalahan Pengawasan Ruang Terbuka Hijau Kota Madiun Dengan Menambah Aturan Khusus Terkait Pengawasan Ruang Terbuka Hijau. Selain Itu Dari Pihak Badan Perencanaan Pembangunan Daerah Kota Madiun Untuk Lebih Meningkatkan Lagi Pengawasan Pemberian Advice Planning Pada Setiap Pemohon Ijin. Dan Bagi Masyarakat Agar Lebih Meningkatkan Pemahaman Akan Pentingnya Ruang Terbuka Hija

Tinjauan Yuridis Pemungutan Pajak Penghasilan Atas Transaksi Jual Beli Hak Atas Tanah dan/atau Bangunan

Penelitian ini mengnalisis pemungutan Pajak Penghasilan (PPh) atas transaksi jual beli hak atas tanah dan/atau bangunan di Kota Malang yang tergantung hasil verifikasi lapangan pajak BPHTB dan mengetahui dasar hukum verifikasi lapangan dalam menentukan besaran tarif pemungutan Pajak Penghasilan (PPh) atas transaksi jual beli hak atas tanah dan/atau bangunan di Kota Malang. Metode penelitian yang digunakan adalah yuridis empiris dengan pendekatan yuridis sosiologis. Hasil penelitian ini menunjukkan bahwa Tarif pemungutan pajak penghasilan (pph) atas transaksi jual beli hak atas tanah dan/atau bangunan ditentukan oleh hasil verifikasi lapangan PPh BPHTB tetap pada besaran yang telah ditetapkan dalam PP Nomor 34 Tahun 2016 yaitu hanya sebesar 2,5% tanpa harus dihitung kemudian dengan hasil verifikasi lapangan seperti yang terjadi di Kota Malang. Dasar hukum dalam melakukan verifikasi lapangan dalam menentukan besaran tarif pemungutan pajak penghasilan (pph) atas transaksi jual beli hak atas tanah dan/atau bangunan di Kota Malang tidak ada sama sekali, baik di dalam Perda maupun Perwal sehingga kegiatan verifikasi lapangan yang selama ini dijadikan dasar untuk menentukan besaran PPh BPHTB tidak memiliki kepastian hukum This study analyzes the collection of Income Tax (PPh) on the sale and purchase transactions of land and / or building rights in Malang which depend on the verification result of BPHTB tax field and to know the legal basis of field verification in determining the tariff rate of Income Tax (PPh) on sale and purchase transactions rights to land and / or buildings in Malang. The research method used is empirical juridical with sociological juridical approach. The results of this study indicate that the tariff of income tax (pph) on the sale and purchase transactions of land and / or building rights is determined by the verification result of the field of fixed income tax on the amount specified in Government Regulation Number 34 Year 2016 which is only 2.5% must be calculated later with the results of field verification as happened in Malang. The legal basis for conducting field verification in determining the amount of income tax collection tariff (PPh) on the sale and purchase transaction of land and / or building in Malang is not present at all, either in Perda or Perwal so that field verification activity which has been used as the basis to determine the amount of PPh BPHTB does not have legal certainty

Dimerization of the glucan phosphatase laforin requires the participation of cysteine 329

11 páginas, 5 figuras.Laforin, encoded by a gene that is mutated in Lafora Disease (LD, OMIM 254780), is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin's oligomerization.This work was supported by a grant from the Spanish Ministry of Education and Science (SAF2011-27442), a grant from la Fundacio´ La Marato de TV3 (ref. 100130) and a grant from Generalitat Valenciana (Prometeo 2009/051) to PS as well as National Institutes of Health Grants P20RR020171 and R01NS070899 and University of Kentucky College of Medicine startup funds to MSGPeer reviewe

Structure of the Arabidopsis glucan phosphatase like sex four2 reveals a unique mechanism for starch dephosphorylation

Starch is a water-insoluble, Glc-based biopolymer that is used for energy storage and is synthesized and degraded in a diurnal manner in plant leaves. Reversible phosphorylation is the only known natural starch modification and is required for starch degradation in planta. Critical to starch energy release is the activity of glucan phosphatases; however, the structural basis of dephosphorylation by glucan phosphatases is unknown. Here, we describe the structure of the Arabidopsis thaliana starch glucan phosphatase like sex four2 (LSF2) both with and without phospho-glucan product bound at 2.3Å and 1.65Å, respectively. LSF2 binds maltohexaose-phosphate using an aromatic channel within an extended phosphatase active site and positions maltohexaose in a C3-specific orientation, which we show is critical for the specific glucan phosphatase activity of LSF2 toward native Arabidopsis starch. However, unlike other starch binding enzymes, LSF2 does not possess a carbohydrate binding module domain. Instead we identify two additional glucan binding sites located within the core LSF2 phosphatase domain. This structure is the first of a glucan-bound glucan phosphatase and provides new insights into the molecular basis of this agriculturally and industrially relevant enzyme family as well as the unique mechanism of LSF2 catalysis, substrate specificity, and interaction with starch granules

Laforin-C329S is monomeric.

<p>(A) I<i>n vitro</i> dimerization assay. Recombinant proteins expressed in bacteria were purified and subjected to non-reducing, non-denaturing electrophoresis and were immunodetected using anti-laforin antibody. All mutants could form dimers with the exception of C329S (left panel). WT laforin and C329S were further analyzed by incubation in the presence of 10 mM DTT or 10 mM H₂O₂ and analyzed for the presence of monomeric and dimeric species (right panel). (B) Laforin C329S exclusion chromatography analysis. A single peak is observed in an elution volume corresponding to 32.5 kDa (molecular size markers are indicated). (C) <i>In vitro</i> phosphatase activity assay of the same sample showed that the C329S mutant displayed a catalytic activity comparable to the wild type protein. (D) <i>In vitro</i> dimerization assay of laforin-C329S expressed in mammalian cells. Crude lysates from HEK293 cells transfected with myc-laforin (wt, wild type) or myc-C329S plasmids were analyzed by Western blot. The electrophoresis was carried out in non-reducing, non-denaturing conditions, and the immunodetection was performed using anti-laforin antibody. The position of the monomeric and dimeric forms of laforin is indicated.</p

Human laforin contains nine cysteines.

<p>(A) Multiple sequence alignment of laforin orthologs. Sequences of several vertebrate (indicated by the brackets) and two invertebrate (<i>N. vectensis</i> and <i>T. gondii</i>) organisms were used (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069523#pone.0069523.s001" target="_blank">Table S1</a> for details). The different cysteines are highlighted in yellow, and the position of cysteines in human laforin is marked (catalytic cysteine C266 appears in red). (B) Schematic of laforin domains (CBM: carbohydrate binding module; DSPD: dual-specificity phosphatase domain). The location of the nine cysteines is shown (blue numbers). (C) Tertiary structure prediction of the CBM (left) and DSPD (right) laforin domains. Homology models were created using the structures of glucoamylase (PDB: 1ACZ) and SEX4 (PDB: 3NME) as templates for the CBM and DSPD, respectively. The models were used to estimate the possible location of the cysteines in the tertiary structure. The cysteines studied in this work appear in yellow in the CBM and in orange in the DSPD; in grey, known tryptophans responsible of the carbohydrate binding.</p

Laforin-C329S and malin physically interact and form a functional complex in mammalian cells.

<p>(A) Yeast two-hybrid analysis. THY-AP4 yeast strain was transformed with pACT2-malin and LexA-laforin (WT or C329S) and the interaction was assessed by measuring the β-galactosidase activity. (B) Co-immunoprecipitation assay. HEK293 cells were co-transfected with plasmids myc-laforin (WT or C329S) and HA-malin. Cells were lysed and total lysates were incubated with anti-myc antibody and protein A/G beads. After washing, beads were boiled in loading buffer and purified proteins analyzed by SDS-PAGE and Western blot using anti-myc or anti-HA antibodies. (C) Ubiquitination analysis of R5/PTG by the laforin-malin complex. Overexpression of 6xHis-ubiquitin, pCMV-HA-malin, pCMV-myc-R5/PTG and pCMV-HA-laforin (wild type or C329S) in HEK293 cells, followed by lysis in presence of guanidinium chloride and purification of the ubiquitinated proteins by affinity chromatography using a cobalt resin. The result of the purification was analyzed using Western blot with anti-myc antibodies. Bound: proteins retained in the resin; crude extracts (50 µgr, C.E.) were immunodetected with anti-HA antibodies. *: polyubiquitinated forms.</p