Structure of murine and human renal type II Na+-phosphate cotransporter genes (Npt2 and NPT2).

Na+-phosphate (Pi) cotransport across the renal brush border membrane is the rate limiting step in the overall reabsorption of filtered Pi. Murine and human renal-specific cDNAs (NaPi-7 and NaPi-3, respectively) related to this cotransporter activity (type II Na+-Pi cotransporter) have been cloned. We now report the cloning and characterization of the corresponding mouse (Npt2) and human (NPT2) genes. The genes were cloned by screening mouse genomic and human chromosome 5-specific libraries, respectively. Both genes are approximately 16 kb and are comprised of 13 exons and 12 introns, the junctions of which conform to donor and acceptor site consensus sequences. Putative CAAT and TATA boxes are located, respectively, at positions -147 and -40 of the Npt2 gene and -143 and -51 of the NPT2 gene, relative to nucleotide 1 of the corresponding cDNAs. The translation initiation site is within exon 2 of both genes. The first 220 bp of the mouse and human promoter regions exhibit 72% identity. Two transcription start sites (at positions -9 and - 10 with respect to nucleotide 1 of NaPi-7 cDNA) and two polyadenylylation signals were identified in the Npt2 gene by primer extension, 5' and 3' rapid amplification of cDNA ends (RACE). A 484-bp 5' flanking region of the Npt2 gene, comprising the CAAT box, TATA box, and exon 1, was cloned upstream of a luciferase reporter gene; this construct significantly stimulated luciferase gene expression, relative to controls, when transiently transfected into OK cells, a renal cell line expressing type II Na+ -Pi cotransporter activity. The present data provide a basis for detailed analysis of cis and trans elements involved in the regulation of Npt2/NPT2 gene transcription and facilitate screening for mutations in the NPT2 gene in patients with autosomally inherited disorders of renal Pi reabsorption.

I. Nickel-Exchanged Zincosilicate Catalysts for the Oligomerization of Propylene and II. Organic SDA-Free Catalysts for the Methanol-to-Olefins Reaction

Nickel-containing catalysts are developed to oligomerize light olefins. Two nickel-containing zincosilicates (Ni-CIT-6 and Ni-Zn-MCM-41) and two nickel-containing aluminosilicates (Ni-HiAl-BEA and Ni-USY) are synthesized as catalysts to oligomerize propylene into C_3n (C₆ and C₉) products. All catalysts oligomerize propylene, with the zincosilicates demonstrating higher average selectivities to C_3n products, likely due to the reduced acidity of the Zn heteroatom.

To test whether light alkanes can be incorporated into this oligomerization reaction, a supported homogeneous catalyst is combined with Ni-containing zincosilicates. The homogeneous catalyst is included to provide dehydrogenation/hydrogenation functions. When this tandem catalyst system is evaluated using a propylene/n-butane feed, no significant integration of alkanes are observed.

Ni-containing zincosilicates are reacted with 1-butene and an equimolar propylene/1-butene mixture to study other olefinic feeds. Further, other divalent metal cations such as Mn²⁺, Co²⁺, Cu²⁺, and Zn²⁺ are exchanged onto CIT-6 samples to investigate stability and potential use for other reactions. Co-CIT-6 oligomerizes propylene, albeit less effectively than Ni-CIT-6. The other M-CIT-6 samples, while not able to oligomerize light olefins, may be useful for other reactions, such as deNO_x.

Molecular sieves are synthesized, characterized, and used to catalyze the methanol-to-olefins (MTO) reaction. The Al concentration in SSZ-13 samples is varied to investigate the effect of Al number on MTO reactivity when compared to a SAPO-34 sample with only isolated Si Brønsted acid sites. These SSZ-13 samples display reduced transient selectivity behavior and extended reaction lifetimes as Si/Al increases; attributable to fewer paired Al sites. MTO reactivity for the higher Si/Al SSZ-13s resembles the SAPO-34 sample, suggesting that both catalysts owe their stable reaction behavior to isolated Brønsted acid sites.

Zeolites CHA and RHO are prepared without the use of organic structure-directing agents (OSDAs), dealuminated by steam treatments (500°C-800°C), and evaluated as catalysts for the MTO reaction. The effects of temperature and steam partial pressure during steaming are investigated. X-ray diffraction (XRD) and Ar physisorption show that steaming causes partial structural collapse of the zeolite, with degradation increasing with steaming temperature. ²⁷Al MAS NMR spectra of steamed materials reveal the presence of tetrahedral, pentacoordinate, and hexacoordinate aluminum.

Proton forms of as-synthesized CHA (Si/Al=2.4) and RHO (Si/Al=2.8) rapidly deactivate under MTO testing conditions (400°C, atmospheric pressure). CHA samples steamed at 600°C performed best among samples tested, showing increased olefin selectivities and catalyst lifetime. Acid washing these steamed samples further improved activity. Reaction results for RHO were similar to CHA, with the RHO sample steamed at 800°C producing the highest light olefin selectivities. Catalyst lifetime and C₂-C₃ olefin selectivities increase with increasing reaction temperature for both CHA-type and RHO-type steamed samples.

I. Synthetic Studies Toward the Total Synthesis of Polycyclic Natural Products – Communesin F, Perophoramidine and Ineleganolide. II. Nickel Catalyzed Intramolecular C–O Bond Formation

Expedient synthetic approaches to the highly functionalized polycyclic alkaloids communesin F and perophoramidine are described using a unified approach featuring a key decarboxylative allylic alkylation to access a crucial and highly congested 3,3-disubstituted oxindole. Described are two distinct, stereoselective alkylations that produce structures in divergent diastereomeric series possessing the critical vicinal all-carbon quaternary centers needed for each synthesis. Synthetic studies toward these challenging core structures have revealed a number of unanticipated modes of reactivity inherent to these complex alkaloid scaffolds. Finally, a previously unknown mild and efficient deprotection protocol for the o-nitrobenzyl group is disclosed – this serendipitous discovery permitted a concise endgame for the formal syntheses of both communesin F and perophoramidine.

In addition, the atroposelective synthesis of PINAP ligands has been accomplished via a palladium-catalyzed C–P coupling process through dynamic kinetic resolution. These catalytic conditions allow access to a wide variety of alkoxy- and benzyloxy-substituted PINAP ligands in high enantiomeric excess.

An efficient and exceptionally mild intramolecular nickel-catalyzed carbon–oxygen bond-forming reaction between vinyl halides and primary, secondary, and tertiary alcohols has been achieved. This operationally simple method allows direct access to cyclic vinyl ethers in high yields in a single step.

Finally, synthetic studies toward polycyclic ineleganolide are described. The entire fragmented carbon framework has been constructed from this work. Highly (Z)-selective olefination was achieved by the method by the Ando group.

External pressure in the hardening of phosphate in tribofilm on iron surfaces

Purpose: In the present work we consider our (in progress) spectroscopy study of zinc and iron phosphates under the influence external high pressure to determine zinc ion change coordination from tetrahedral to octahedral (or hexahedral) structure.----- Design/methodology/approach: The standard equipment is the optical high pressure cell with diamond (DAC). The DAC is assembled and then vibrational or electronic spectra are collected by mounting the cell in an infrared, Raman, EXAFS or UV-visible spectrometer.----- Findings: Mechanism by which zinc and iron methaphosphate material is transformed to glassy meta-phosphate is enhancing mechanical properties of tribofilm. The two decades of intensive study demonstrates that Zn (II) and Fe (III) ions participate to cross-link network under friction, hardening the phosphate.----- Research limitations/implications: Transition metal atoms with d orbital have flexible coordination numbers, for example zinc acts as a cross-linking agent increasing hardness, by changing coordination from tetrahedral to octahedral. Perhaps the external pressure effect on the [Zn–(O-P-)4 ] complex causes a transformation to an [Zn –(O-P-)6] grouping.----- Originality/value: This paper analyses high-pressure spectroscopy which has been applied for the investigation of 3D transition metal ions in solids. When studying pressure effects on coordination compounds structure, we can expect changes in ground electronic state (spin-crossovers), electronic spectra due to structural distortions (piezochromism), and changes in the ligand field causing shifts in the electronic transitions.

Renal bioenergetics during early gram-negative mammalian sepsis and angiotensin II infusion

Purpose: To measure renal adenosine triphosphate (ATP) (bioenergetics) during hypotensive sepsis with or without angiotensin II (Ang II) infusion. Methods: In anaesthetised sheep implanted with a renal artery flow probe and a magnetic resonance coil around one kidney, we induced hypotensive sepsis with intravenous Escherichia coli injection. We measured mean arterial pressure (MAP), heart rate, renal blood flow RBF and renal ATP levels using magnetic resonance spectroscopy. After 2 h of sepsis, we randomly assigned sheep to receive an infusion of Ang II or vehicle intravenously and studied the effect of treatment on the same variables. Results: After E. coli administration, the experimental animals developed hypotensive sepsis (MAP from 92 ± 9 at baseline to 58 ± 4 mmHg at 4 h). Initially, RBF increased, then, after 4 h, it decreased below control levels (from 175 ± 28 at baseline to 138 ± 27 mL/min). Despite decreased RBF and hypotension, renal ATP was unchanged (total ATP to inorganic phosphate ratio from 0.69 ± 0.02 to 0.70 ± 0.02). Ang II infusion restored MAP but caused significant renal vasoconstriction. However, it induced no changes in renal ATP (total ATP to inorganic phosphate ratio from 0.79 ± 0.03 to 0.80 ± 0.02). Conclusions:During early hypotensive experimental Gram-negative sepsis, there was no evidence of renal bioenergetic failure despite decreased RBF. In this setting, the addition of a powerful renal vasoconstrictor does not lead to deterioration in renal bioenergetics.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(l-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/GF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

A phase II study of the modulation of 5-fluorouracil and folinic acid with high-dose infusional hydroxyurea in metastatic colorectal carcinoma

Background: Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, may potentiate the activity of 5-fluorouracil (5-FU) and folinic acid (FA) by reducing the deoxyribonucleotide pool available for DNA synthesis and repair. However as HU may inhibit the formation of 5-fluoro-2-deoxyuridine-5- monophosphate (FdUMP), one of the principal active metabolites of 5-FU, the scheduling of HU may be critical. In vitro experiments suggest that administration of HU following 5-FU, maintaining the concentration in the region of I mM for six or more hours, significantly enhances the efficacy of 5-FU. Patients and methods: 5-FU/FA was given as follows: days 1 and 2 - FA 250 mg/m 2 (max. 350 mg) over two hours followed by 5-FU 400 mg/m 2 by intravenous bolus (ivb) over 15 minutes and subsequently 5-FU 400 mg/m 2 infusion (ivi) over 22 hours. HU was administered on day 3 immediately after the 5-FU with 3 g ivb over 15 minutes followed by 12 g ivi over 12 hours. Results: Thirty patients were entered into the study. Median survival was nine months (range 1-51 + months). There were eight partial responses (28%, 95% CI: 13%-47%). The median duration of response was 6.5 (range 4-9 months). Grade 3-4 toxicities included neutropenia (grade 3 in eight patients and grade 4 in five), anaemia (grade 3 in one patient) and diarrhoea (grade 3 in two patients). Neutropenia was associated with pyrexia in two patients. Phlebitis at the infusion site occurred in five patients. The treatment was complicated by pulmonary embolism in one patient and deep venous thrombosis in another. Conclusion: HU administered in this schedule is well tolerated. Based on these results and those of other phase II studies, a randomised phase III study of 5-FU, FA and HU versus 5-FU and FA using the standard de Gramont schedule is recommended.

Highly sensitive electrochemical sensor for nitric oxide using the self-assembled monolayer of 1,8,15,22-tetraaminophthalo-cyanatocobalt(II) on glassy carbon electrode

Glassy carbon (GC) electrode modified with a self-assembled monolayer (SAM) of 1,8,15,22-tetraaminophthalocyanatocobalt(II) (4α-CoIITAPc) was used for the selective and highly sensitive determination of nitric oxide (NO). The SAM of 4α-CoIITAPc was formed on GC electrode by spontaneous adsorption from DMF containing 1 mM 4α-CoIITAPc. The SAM showed two pairs of well-defined redox peaks corresponding to CoIII/CoII and CoIIIPc−1/CoIIIPc−2 in 0.2 M phosphate buffer (PB) solution (pH 2.5). The SAM modified electrode showed excellent electrocatalytic activity towards the oxidation of nitric oxide (NO) by enhancing its oxidation current with 310 mV less positive potential shift when compared to bare GC electrode. In amperometric measurements, the current response for NO oxidation was linearly increased in the concentration range of 3×10−9 to 30×10−9 M with a detection limit of 1.4×10−10 M (S/N=3). The proposed method showed a better recovery for NO in human blood serum samples.

Amino group position dependent orientation of self-assembled monomolecular films of tetraaminophthalocyanatocobalt(II) on Au surfaces

Self-assembled monomolecular films of 1,8,15,22-tetraaminophthalocyanatocobalt(II) (4α-CoIITAPc) and 2,9,16,23-tetraaminophthalocyanatocobalt(II) (4β-CoIITAPc) on Au surfaces were prepared by spontaneous adsorption from solution. These films were characterized by cyclic voltammetry and Raman spectroscopy. Both the surface coverage (Γ) and intensity of the in-plane stretching bands obtained from Raman studies vary for these monomolecular films, indicating different orientations adopted by them on Au surfaces. The 4α-CoIITAPc-modified electrode exhibits an E1/2 of 0.35 V, while the 4β-CoIITAPc-modified electrode exhibits an E1/2 of 0.19 V, corresponding to the CoII/CoIII redox couple in 0.1 M H2SO4. The Γ estimated from the charge associated with the oxidation of Co(II) gives (2.62 ± 0.10) × 10-11 mol cm-2 for 4α-CoIITAPc and (3.43 ± 0.14) × 10-10 mol cm-2 for 4β-CoIITAPc. In Raman spectral studies, the intensity ratio between in-plane phthalocyanine (Pc) stretching and the Au−N stretching was found to be 6.6 for 4β-CoIITAPc, while it was 1.6 for 4α-CoIITAPc. The obtained lower Γ and intensity ratio values suggest that 4α-CoIITAPc adopts nearly a parallel orientation on the Au surface, while the higher Γ and intensity ratio values suggest that 4β-CoIITAPc adopts a perpendicular orientation. The electrochemical reduction of dioxygen was carried out using these differently oriented Pc's in phosphate buffer solution (pH 7.2). Both the Pc's catalyze the reduction of dioxygen; however, the 4α-CoIITAPc-modified electrode greatly reduces the dioxygen reduction overpotential compared to 4β-CoIITAPc-modified and bare Au electrodes.

Synthesis of some copper(II)-chelating (dialkylamino)pyridine amphiphiles and evaluation of their esterolytic capacities in cationic micellar media

Three new (dialkylamino)pyridine (DAAP)-based ligand amphiphiles 3-5 have been synthesized. All of the compounds possess a metal ion binding subunit in the form of a 2,6-disubstituted DAAP moiety. In addition, at least one ortho-CH2OH substituent is present in all the ligands. Complex formation by these ligands with various metal ions were examined under micellar conditions, but only complexes with Cu(II) ions showed kinetically potent esterolytic capacities under micellar conditions. Complexes with Cu(II) were prepared in host comicellar cetyltrimethylammonium bromide (CTABr) media at pH 7.6. Individual complexes were characterized by UV-visible absorption spectroscopy and electron paramagnetic resonance spectroscopy. These metallomicelles speed the cleavage of the substrates p-nitrophenyl hexanoate or p-nitrophenyl diphenyl phosphate. To ascertain the nature of the active esterolytic species, the stoichiometries of the respective Cu(II) complexes were determined from the kinetic version of Job's plot. In all the instances, 2:1 complex ligand/Cu(II) ion are the most kinetically competent species. The apparent pK(a) values of the Cu(II)-coordinated hydroxyl groups of the ligands 3, 4, and 5, in the comicellar aggregate, are 7.8, 8.0, and 8.0, respectively, as estimated from the rate constant vs pH: profiles of the ester cleavage reactions. The nucleophilic metallomicellar reagents and the second-order "catalytic" rate constants toward esterolysis of the substrate p-nitrophenyl hexanoate (at 25 degrees C, pH 7.6) are 37.5 for 3, 11.4 for 4, and 13.8 for 5. All catalytic systems comprising the coaggregates of 3, 4, or 5 and CTABr demonstrate turnover behavior in the presence of excess substrate.

A Theoretical Investigation of the Ni(II)-Catalyzed Hydrovinylation of Styrene

We report a detailed and full computational investigation on the hydrovinylation reaction of styrene with the Ni(II)-phospholane catalytic system, which was originally presumed to proceed through a cationic mechanism involving a nickel hydride intermediate. The following general features emerge from this study on a specific catalyst complex that was found to give quantitative yield and moderate selectivity: (a) the activation barrier for the initiation (18.8 kcal/mol) is higher than that for the reaction due to a low-lying square-planar pentenyl chelate intermediate originating from a Ni(II)-allyl catalyst precursor. Consequently there is an induction period for the catalysis; (b) the exit of product from the catalyst is via a β-H-transfer step instead of the usual β-H elimination pathway, which has a very high activation energy due to a trans effect of the phospholane ligand; (c) the turnover-limiting and enantio- determining transition state is also the β-H-transfer; (d) because of the absence of a hydride intermediate, the unwanted isomerization of the product is prevented; (e) since the enantio-discrimination is decided at the H-transfer stage itself, the configuration of the product in a catalytic cycle influences the enantioselectivity in the subsequent cycle; (f) the trans effect of the sole strong ligand in the d8 square-planar Ni(II), the stability of the η3-benzyl intermediate, and the availability of three coordination sites enable regioselective hydrovinylation over the possible oligomerization/polymerization of the olefin substrates and linear hydrovinylation. This work has also confirmed the previously recognized role of the hemilabile group at various stages in the mechanism.

X-Ray Structure Of A Ternary Metal-Complex - Copper(Ii) Inosine 5'-Monophosphate Imidazole - Metal-Binding To The N(7) Atom Of The Nucleotide In A Mixed-Ligand System Containing An Aromatic Amine

A ternary metal-nucleotide complex, Na2[Cu(5’-IMP)2(im)o,8(H20)l,2(H20)2h]as~ 1be2e.n4 pHr2ep0a,r ed and its structure analyzed by X-ray diffraction (5’-IMP = inosine 5’-monophos hate; im = imidazole). The complex crystallizes in space group C222, with a = 8.733 (4) A, b = 23.213 (5) A, c = 21.489 (6) 1, and Z = 4. The structure was solved by the heavy-atom method and refined by full-matrix least-squares technique on the basis of 2008 observed reflections to a final R value of 0.087. Symmetry-related 5’-IMP anions coordinate in cis geometry through the N(7) atoms of the bases. The other cis positions of the coordination plane are statistically occupied by nitrogen atoms of disordered im groups and water oxygens with occupancies 0.4 and 0.6, respectively. Water oxygens in axial positions complete the octahedral coordination of Cu(I1). The complex is isostructural with C~S-[P~(S’-IMP),(NH~)~a] m”,o del proposed for Pt(I1) binding to DNA. The base binding observed in the present case is different from the typical ”phosphate only” binding shown from earlier studies on metal-nucleotide complexes containing various other ?r-aromatic amines.

Nutrition and bone metabolism : In vivo effects of inorganic phosphate on rats and in vitro effects of bioactive tripeptides on human osteoblasts

Nutrition affects bone health throughout life. To optimize peak bone mass development and maintenance, it is important to pay attention to the dietary factors that enhance and impair bone metabolism. In this study, the in vivo effects of inorganic dietary phosphate and the in vitro effects of bioactive tripeptides, IPP, VPP and LKP were investigated. Dietary phosphate intake is increased through the use of convenience foods and soft drinks rich in phosphate-containing food additives. Our results show that increased dietary phosphate intake hinders mineral deposition in cortical bone and diminishes bone mineral density (BMD) in the aged skeleton in a rodent model (Study I). In the growing skeleton (Study II), increased phosphate intake was observed to reduce bone material and structural properties, leading to diminished bone strength. Studies I and II revealed that a low Ca:P ratio has negative effects on the mature and growing rat skeleton even when calcium intake is sufficient. High dietary protein intake is beneficial for bone health. Protein is essential for bone turnover and matrix formation. In addition, hydrolysis of proteins in the gastrointestinal tract produces short peptides that possess a biological function beyond that of being tissue building blocks. The effects of three bioactive tripeptides, IPP, VPP and LKP, were assessed in short- and long-term in vitro experiments. Short-term treatment (24 h) with tripeptide IPP, VPP or LKP influenced osteoblast gene expression (Study III). IPP in particular, regulates genes associated with cell differentiation, cell growth and cell signal transduction. The upregulation of these genes indicates that IPP enhances osteoblast proliferation and differentiation. Long-term treatment with IPP enhanced osteoblast gene expression in favour of bone formation and increased mineralization (Study IV). The in vivo effects of IPP on osteoblast differentiation might differ since eating frequency drives food consumption, and protein degradation products, such as bioactive peptides, are available periodically, not continuously as in this study. To sum up, Studies I and II raise concern about the appropriate amount of dietary phosphate to support bone health as excess is harmful. Studies III and IV in turn, support findings of the beneficial effects of dietary protein on bone and provide a mechanistic explanation since cell proliferation and osteoblast function were improved by treatment with bioactive tripeptide IPP.

Interaction of palladium (II) with DNA

The nature of interaction of palladium(II) with calf thymus DNA was studied using viscometry, ultraviolet, visible and infrared spectrophotometry and optical rotatory disperison and circular dichroism measurements. The results indicate that Pd(II) interacts with both the phosphate and bases of DNA. The ORD/CD data indicate that the binding of Pd(II) to DNA brings about considerable conformational changes in DNA.

Amino Acid Based MOFs: Synthesis, Structure, Single Crystal to Single Crystal Transformation, Magnetic and Related Studies in a Family of Cobalt and Nickel Aminoisophthales

Four new 5-aminoisophthalates of cobalt and nickel have been prepared employing hydro/solvothermal methods: [Co2(C8H5NO4)2(C4H4N2)(H2O)2]·3H2O (I), [Ni2(C8H5NO4)2(C4H4N2)(H2O)2]·3H2O (II), [Co2(H2O)(μ3-OH)2(C8H5NO4)] (III), and [Ni2(H2O)(μ3-OH)2(C8H5NO4)] (IV). Compounds I and II are isostructural, having anion-deficient CdCl2 related layers bridged by a pyrazine ligand, giving rise to a bilayer arrangement. Compounds III and IV have one-dimensional M−O(H)−M chains connected by the 5-aminoisophthalate units forming a three-dimensional structure. The coordinated as well as the lattice water molecules of I and II could be removed and inserted by simple heating−cooling cycles under the atmospheric conditions. The removal of the coordinated water molecule is accompanied by changes in the coordination environment around the M2+ (M = Co, Ni) and color of the samples (purple to blue, Co; green to dark yellow, Ni). This change has been examined by a variety of techniques that include in situ single crystal to single crystal transformation studies and in situ IR and UV−vis spectroscopic studies. Magnetic studies indicate antiferromagnetic behavior in I and II, a field-induced magnetism in III, and a canted antiferromagnetic behavior in IV.