Studies on chromophore coupling in isolated phycobiliproteins: III. Picosecond excited state kinetics and time-resolved fluorescence spectra of different allophycocyanins from Mastigocladus laminosus

The excited state kinetics of three different allophycocyanin (AP) complexes has been studied by picosecond fluorescence spectroscopy. Both the fluorescence kinetics and the decay-associated fluorescence spectra of the different complexes can be understood on the basis of a structural model for AP which uses (a) an analogy to the known x-ray determined structure of C-phycocyanin, (b) the biochemical analogies of AP and C-phycocyanin, and (c) the biochemical composition of AP-B (AP-681). A model is developed that describes the excited state kinetics as a mixture of internal conversion processes within a coupled exciton pair and energy transfer processes between exciton pairs. We found excited state relaxation times in the range of 13 ps (AP with linker peptide) up to 66 ps (AP-B). The trimeric aggregates AP 660 and AP 665 show one fast relaxation component each, as was expected on the basis of their symmetry properties. The lower symmetry of AP-B (AP-681) gives rise to two fast lifetime components (τ₁ = 23 ps and τ₂ = 66 ps) which are attributed to internal conversion and/or energy transfer between excitonic states formed by the coupling of symmetrically and spectrally nonequivalent chromophores. It is proposed that the internal conversion between exciton states of strongly coupled chromophores fulfills the requirements of the small energy gap limit. Thus, internal conversion rates in the order of tens of picoseconds are feasible. The influence of the interaction of the linker peptide on the properties of the AP trimer are manifested in the fluorescence kinetics. Lack of the linker peptide in AP 660 gives rise to a heterogeneity in the chromophore conformations and chromophore-chromophore interactions.     

Is urinary flow rate a major regulator of prostaglandin E excretion in man?

Urinary PGE₂ excretion is enhanced in several polyuric states in man suggesting that PGE₂ synthesis could be a mediator of diuresis. To explore the alternate hypothesis that polyuria is the cause of the increased PGE₂ excretion, we increased urine flow rate by intravenous administration of dextrose and water with different magnesium, calcium and potassium solutions in four normal males. Urinary PGE excretion rose in parallel with urine volume (r = 0.65 p < 0.01) independently of the electrolyte solution. To determine the effects of chronic alterations in water balance in 5 female subjects, we sequentially regulated oral water intake to induce 1, 2, 4 and 8 liters of urine volume/day. During low (40 mEq) sodium diets, PGE increased from 540 ± 50 to 4880 ± 1240 ng/d with increasing urinary volume (r = 0.81, p < 7.01). Similarly, for 200 mEq sodium intake PGE paralleled urinary volume (from 630 ± 100 to 4740 ± 800 ng/d, r = 0.61, p <0.01). In vitro sample dilution studies demonstrated no interference from method blank, and the addition of thin layer chromatography prior to Sephadex chromatography failed to alter assay measurements. We conclude that extreme increases in urinary flow rate may directly enhance PGE excretion in man.     

Effects of High Doses of Cholecalciferol in Normal Subjects: A Randomized Double-Blinded,Placebo-Controlled Trial

Background

Vitamin D repletion with high doses of vitamin D is often recommended to patients and healthy subjects. The safety, especially concerning changes in urinary calcium excretion is of great importance.

Methods

In a double-blinded, placebo-controlled study in 40 healthy volunteers, we examined the changes in mineral metabolism during supplementation with 3000 IU of oral cholecalciferol daily during 4 months.

Results

Both 25(OH)vitamin D and 1,25(OH)₂vitamin D increased significantly in the active treated group as compared to the placebo group (186% versus 14% (P<0.001) and 28% versus – 8% (P<0.001)). No change was observed in urinary calcium excretion in the active group compared to the placebo group (P = 0.891). Fibroblast growth factor 23 increased significantly by 10% (P<0.018) in the active group. However, there was no difference in changes in FGF23 between treatment groups (P = 0.457).

Conclusion

High dose cholecalciferol significantly increases 25(OH)vitamin D and 1,25(OH)₂vitamin D levels compared to placebo. No changes in urinary calcium excretion or other measured components of the mineral metabolism were found between groups.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00952562","term_id":"NCT00952562"}}NCT00952562.     

Genetic association study of adaptor protein complex 4 with cerebral palsy in a Han Chinese population

Adaptor protein complex 4 (AP-4) plays a key role in vesicle formation, trafficking, and sorting processes that are critical for brain development and function. AP-4 consists of four subunits encoded by the AP4E1, AP4B1, AP4M1, and AP4S1 genes. A number of studies have pointed to the involvement of AP-4–mediated vesicular trafficking pathways in the etiology of cerebral palsy (CP), the most notable of which are the causative mutations that have recently been identified in each of the AP-4 genes in different CP families. We postulated, therefore, that variations in AP-4 genes might influence an indivual’s susceptibility to CP. In the present study, 16 SNPs were genotyped among 517 CP patients and 502 healthy controls from the Han Chinese population. We systematically analyzed the association of the AP4E1, AP4B1, AP4M1, and AP4S1 genes with CP on the basis of clinical characteristics. No significant associations were found between these variants and the overall risk of CP. Subgroup analysis showed that rs1217401 of AP4B1 was significantly associated with CP as a sequela of hypoxic-ischemic encephalopathy (HIE) (CP + HIE) (allele: p = 0.042151; genotype: p = 4.46 × 10^?6). Our results indicate that the 16 variants studied in the genes of the four subunits of AP-4 have no detectable effects on the overall susceptibility to CP, but AP4B1 appears to be a susceptibility gene for CP + HIE in the Han Chinese population.     

Competitive protein-binding radioassay of 24,25-dihydroxyvitamin D in sera from normal and anephric subjects

A competitive protein-binding radioassay for 24,25-dihydroxyvitamin D [24,25-(OH)₂D] in human serum has been developed. Whereas small amounts of [³H]24,25-(OH)₂D must be biosynthesized in order to trace the efficiency of the extraction and chromatographic procedures, tritiated 25-hydroxyvitamin D₃ ([³H]25-OHD₃) can be used as the assay tracer. Since 25-OHD₃ and 24,25-(OH)₂D₃ are equipotent in their competitive displacement of [³H]25-OHD₃ from rat serum, 25-OHD₃ can be used as the assay standard. Liquid-gel partition chromatography on small columns of Sephadex LH-20 can reliably isolate 24,25-(OH)₂D by batch elution. The purity of biosynthesized [³H]24,25-(OH)₂D₃ and the 24,25-(OH)₂D fraction isolated from serum was confirmed by high-pressure chromatography on 0.2 × 50 cm columns of 10-μm silica. Serum 24,25-(OH)₂D levels averaged 16% of the serum 25-OHD concentrations in normal subjects. Since chronic hemodialysis patients, without kidneys, had normal serum 24,25-(OH)₂D levels, significant extrarenal 25-hydroxycalciferol 24-hydroxylase activity occurs in these subjects. Since the present assay represents a reasonably simple extension of 25-OHD assay methodology, it should prove to be a useful technique in the analysis of clinical disorders of vitamin D metabolism.     

Effects of 1,25(OH)2D3 and vitamin D receptor on peripheral CD4+/CD8+ double‐positive T lymphocytes in a mouse model of systemic lupus erythematosus

This study aims to explore effects of 1,25(OH)₂D₃ and vitamin D receptor (VDR) on peripheral CD4⁺/CD8⁺ double‐positive (DP) T lymphocytes in systemic lupus erythematosus (SLE). MRL‐LPr/LPr mice with SLE (n = 20) and normal MRL mice (n = 20) were assigned into the control group (normal mice, without feeding with 1,25(OH)₂D₃), the 1,25(OH)₂D₃ group (SLE mice, feeding with 1,25(OH)₂D₃), the VDR‐knock‐in + 1,25(OH)₂D₃ group (SLE mice, VDR‐knock‐in, feeding with 1,25(OH)₂D₃) and the VDR‐knockout group (normal mice, VDR‐knockout, without feeding with 1,25(OH)₂D₃) (n = 10 per group). Levels of T lymphocytes were measured by flow cytometry. The mRNA and proteins expressions of inflammatory factors were measured by qRT‐PCR and ELISA. Extracellular signal‐regulated kinase‐1/2 (ERK1/2) expression was measured by Western blotting. Compared with normal mice, SLE mice showed reduced levels of CD4⁺, CD4⁺/CD8⁺ ratio, and DP lymphocytes. The levels of SLE‐related indicators all increased significantly, followed with severe skin ulcers and urinary system infection. With the increase in time, skin ulcers and urinary system infection were significantly improved, levels of CD4⁺, CD4⁺/CD8⁺ ratio, and DP lymphocytes increased, and levels of SLE‐related indicators all decreased in the 1,25(OH)₂D₃ group. There were no significant changes in bioindicators in the control and the VDR‐knock‐in + 1,25(OH)₂D₃ groups. The symptoms of SLE gradually occurred in the VDR‐knockout group. This study demonstrates that VDR and 1,25(OH)₂D₃ could elevate CD4⁺/CD8⁺ DP T lymphocytes and reduce expressions of inflammatory factors, thus inhibiting the development and progression of SLE.     

Altered Calcium and Vitamin D Homeostasis in First-Time Calcium Kidney Stone-Formers

Background

Elevated serum 1,25-dihydroxyvitamin D (1,25(OH)₂D) concentrations have been reported among cohorts of recurrent calcium (Ca) kidney stone-formers and implicated in the pathogenesis of hypercalciuria. Variations in Ca and vitamin D metabolism, and excretion of urinary solutes among first-time male and female Ca stone-formers in the community, however, have not been defined.

Methods

In a 4-year community-based study we measured serum Ca, phosphorus (P), 25-hydroxyvitamin D (25(OH)D), 1,25(OH)₂D, 24,25-dihydroxyvitamin D (24,25(OH)₂D), parathyroid hormone (PTH), and fibroblast growth factor-23 (FGF-23) concentrations in first-time Ca stone-formers and age- and gender frequency-matched controls.

Results

Serum Ca and 1,25(OH)₂D were increased in Ca stone-formers compared to controls (P = 0.01 and P = 0.001). Stone-formers had a lower serum 24,25(OH)₂D/25(OH)D ratio compared to controls (P = 0.008). Serum PTH and FGF-23 concentrations were similar in the groups. Urine Ca excretion was similar in the two groups (P = 0.82). In controls, positive associations between serum 25(OH)D and 24,25(OH)₂D, FGF-23 and fractional phosphate excretion, and negative associations between serum Ca and PTH, and FGF-23 and 1,25(OH)₂D were observed. In SF associations between FGF-23 and fractional phosphate excretion, and FGF-23 and 1,25(OH)₂D, were not observed. 1,25(OH)₂D concentrations associated more weakly with FGF-23 in SF compared with C (P <0.05).

Conclusions

Quantitative differences in serum Ca and 1,25(OH)₂D and reductions in 24-hydroxylation of vitamin D metabolites are present in first-time SF and might contribute to first-time stone risk.     

Seedling responses to band applied NH4OH rates and to N form in two maize hybrids

Growth of maize seedlings can be improved by enhanced ammonium nutrition, but placing fertilizer anhydrous ammonia close to seedlings introduces the risk of ammonia toxicity. In this study, growth and root elongation response to rates of closely placed NH₄OH bands were investigated in two contrasting maize hybrids. Seven rates of NH₄OH, ranging from 0 to 200 mg N kg^-1 soil were injected into the center of each pot. A single rate of Ca(NO₃)₂-N was included to compare hybrids for N form preference at a moderate N rate. Three seedlings per pot were planted 5.7 cm from the injection point.Hybrid B73×LH51 produced a quadratic response in shoot growth to NH₄OH rates, whereas LH74×LH123 exhibited a significant linear decline in response to NH₄OH rate. Root length density sampled from the fertlized zone declined linearly in response to NH₄OH rate while a slight increase in root length density in unfertilized zones was observed at intermediate NH₄OH rates. Hybrids did not differ in root length density in either zone.The hybrid with greater tolerance of NH₄OH rates (B73×LH51) also showed a preference in shoot growth for NH₄-over NO₃-N at 66.7 mg N kg^-1 compared to LH74×LH123. On average across hybrids, nitrate concentrations of xylem exudate collected from detopped plants were 14.5 mmol g^-1 for Ca(NO₃)₂ treatments and 1.5 mmol g^-1 for NH₄OH treatments, indicating that contrasting N-form nutrition resulted from fertilizer treatments. Malate concentrations were higher in the NH₄OH treatment indicating that this organic acid anion may substitute for the negative charge of nitrate during enhanced ammonium nutrition in maize.The results suggest that potentially useful genetic variation exists in maize for N form preference and for tolerance to increasing ammonical-N rates.     

Cloning and expression of a plant homologue of the small subunit of the Golgi-associated clathrin assembly protein AP19 from Camptotheca acuminata

Clathrin-coated vesicles (CCVs) are involved in selective protein transport in eukaryotes. AP-1 and AP-2 are protein complexes found in the CCVs of the Golgi apparatus and the plasma membrane respectively. AP19 is the smallest polypeptide chain components of AP-1. We have identified a cDNA clone (CAP19) encoding a putative homologue for the assembly protein AP19 from the Chinese medicinal tree, Camptotheca acuminata. The deduced polypeptide contains 161 amino acids and has a predicted M _r of 18 820. DNA blot analysis suggests that the AP19s of C. acuminata are encoded by a small gene family. CAP19 was expressed ubiquitously throughout the plant suggesting that it may be involved in general Golgi-mediated secretion.     

AP1/2β-mediated exocytosis of tapetum-specific transporters is required for pollen development in Arabidopsis thaliana

AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1 and β2 and α for AP-2), a medium subunit μ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of β subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both β adaptins is lethal in plants. We identified a critical role of β adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, β adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types.

Arabidopsis AP1/2β adaptins are shared by the AP-1 and AP-2 complexes and required for pollen development by mediating the trafficking of ABCG transporters in tapetal cells.

IN A NUTSHELL Background: Adaptor protein (AP) complexes are critical for the recruitment of cargo proteins during vesicle trafficking. AP-1 and AP-2 mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 regulates trafficking to the plasma membrane (exocytosis) and vacuole, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1, β2, and α for AP-2), a medium subunit μ, and a small subunit σ. The general roles of some AP-1 and AP-2 adaptins in vegetative and reproductive development have been characterized in plants. However, the function of the large β subunits and whether they are shared by the two AP-1 and AP-2 complexes in plants is currently unknown. Questions: Are β adaptins shared by AP-1 and AP-2 complexes in Arabidopsis thaliana? What function do they play in plant development? Findings: We found that the two putative Arabidopsis AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization to the TGN and the plasma membrane. However, in tapetal cells of developing anthers, AP1/2β adaptins localize almost exclusively to TGN. Mutations in AP1/2β adaptins result in collapsed pollen grains with abnormal walls and reduced pollen germination due to impaired exocytosis of the tapetum-specific plasma membrane transporters ABCG9 and ABCG16, highlighting the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types. Next steps: We will investigate the mechanism by which AP1/2β adaptins recognize cargo proteins and their role in female gametophyte and embryonic development.     

Urinary excretion of immunoreactive prostaglandin F2α in healthy children and adults

The 24-hours urinary excretion of immunoreactive prostaglandin F_2α (U-iPGF_2α) in normal children on a free diet was not significantly different in 30 boys (aged 3–15 years; geometric mean 589 ng/24 h) compared to 27 girls (aged 4–14 years; mean 473 ng/24 h). In both sexes this excretion rose with age until adolescence where it reached a plateau.In normal adults the men had significantly higher (p < 0.001) excretions of U-iPGF_2α than the women; also body weight and urinary creatinine excretion were higher in men (p < 0.001).In the children, as well as in the total population, U-iPGF_2α correlated best with body weight (r = 0.44 and r = 0.48 respectively; p < 0.001) and the urinary creatinine excretion (r = 0.53 and 0.57 respectively; p < 0.001); both body weight and urinary creatinine excretion are reflections of total body development. After the correction for urinary creatinine excretion or for body weight, the sex difference in the adult U-iPGF_2α totally disappeared.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1

Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1–AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN.Membrane trafficking in plants shares many fundamental features with those in yeast and animals (Bassham et al., 2008). In general, vacuolar proteins are synthesized on the rough endoplasmic reticulum and then transported to vacuoles via the Golgi apparatus (Xiang et al., 2013; Robinson and Pimpl, 2014). The vacuolar trafficking in plants has been studied by monitoring the transport of reporter proteins to lytic vacuoles in vegetative cells and tissues (Jin et al., 2001; Pimpl et al., 2003; Miao et al., 2008; Niemes et al., 2010). Recently, seed storage proteins became a model cargo for monitoring the transport of endogenous vacuolar proteins in plants (Shimada et al., 2003a; Sanmartín et al., 2007; Isono et al., 2010; Pourcher et al., 2010; Uemura et al., 2012; Shirakawa et al., 2014). During seed maturation, a large amount of storage proteins are synthesized and sorted to specialized vacuoles, the protein storage vacuoles (PSVs). To properly deliver vacuolar proteins, sorting receptors play a critical role in recognizing the vacuole-targeting signal of the proteins. VACUOLAR PROTEIN SORTING10 and Man-6-P receptor function as sorting receptors for vacuolar/lysosomal proteins in the trans-Golgi network (TGN) of yeast and mammals, respectively. The best-characterized sorting receptors in plants are VACUOLAR SORTING RECEPTOR (VSR) family proteins (De Marcos Lousa et al., 2012). VSRs have been shown to function in sorting both storage proteins to PSVs (Shimada et al., 2003a; Fuji et al., 2007) and lytic cargos to lytic vacuoles (Zouhar et al., 2010).To sort the receptors in the TGN into vacuoles/lysosomes, the adaptor protein (AP) complex binds the cytosolic domain of the receptors. The AP complexes form evolutionarily conserved machinery that mediates the post-Golgi trafficking in eukaryotic cells (Robinson, 2004). There are five types of AP complexes, AP-1 to AP-5. The functions of AP-1, AP-2, and AP-3 have been established. AP-1 appears to be involved in trafficking between the TGN and endosomes (Hirst et al., 2012), AP-2 is involved in clathrin-mediated endocytosis (McMahon and Boucrot, 2011), and AP-3 is involved in protein trafficking from the TGN/endosomes to the vacuole/lysosomes (Dell’Angelica, 2009). However, little is known about AP-4 and AP-5. Mammalian AP-4 may be involved in basolateral sorting in polarized cells and in the transport of specific cargo proteins, such as the amyloid precursor protein APP, from the TGN to endosomes (Burgos et al., 2010). The fifth AP complex, AP-5, was recently identified, and its orthologs are widely conserved in the eukaryotic genomes (Hirst et al., 2011). The AP complexes exist as heterotetrameric proteins that consist of two large subunits (β1-5 and one each of ɣ/α/δ/ε/ζ), one medium subunit (µ1-5), and one small subunit (σ1-5). The sorting mechanism is best characterized for the medium (µ) subunit, which is known to recognize the Tyr-based YXXФ motif (where Ф represents Leu, Ile, Phe, Met, or Val) that is present in the cytosolic domains of cargo proteins (Ohno et al., 1995). Mutations of the YXXФ motif abolish the interaction with µ and alter the subcellular localization of the cargo proteins.The genome of Arabidopsis (Arabidopsis thaliana) contains all five sets of putative AP genes (Bassham et al., 2008; Hirst et al., 2011). The function of AP-4 in membrane trafficking and its physiological roles in plants are largely unknown. In this study, we identified and characterized the AP-4 complex in Arabidopsis. Mutants lacking the AP-4 subunits exhibited defects in VSR1-mediated vacuolar sorting of storage proteins in seeds. Our results provide new insights into the receptor-mediated vacuolar trafficking in post-Golgi pathways.     

The effect of extracellular calcium on colonocytes: evidence for differential responsiveness based upon degree of cell differentiation

Calcium supplementation decreases the incidence of colon cancer in animal models and may prevent colon cancer in man. Potential mechanisms include binding of mitogens and direct effects of calcium on colonic epithelial cells. In this study, the effects of extracellular calcium on epithelial cell growth and differentiation were studied in three colon carcinoma and two colonic adenoma cell lines. The characteristics studied included morphology, cell cycle kinetics, [Ca²⁺]_IC (intracellular calcium concentration), proliferation, and expression of differentiation markers such as carcinoembryonic antigen (CEA) and alkaline phosphatase (AP). Sodium butyrate (NaB) and 1,25-dihydroxyvitamin D₃ were used as controls in the latter three assays as these two agents are known differentiating agents. Alteration of [Ca⁺²]_EC (extracellular calcium concentration) did not affect carcinoembryonic antigen (CEA) or alkaline phosphatase (AP) expression. NaB enhanced the expression of AP three-fold and CEA five-fold. This effect was augmented by increasing [Ca²⁺]_EC. The exposure of cells to 1,25-(OH)₂-Vitamin D₃ increased CEA but not AP. [Ca²⁺]_IC increased in response to 1,25-(OH)₂-vitamin D₃ and NaB but not with variation in [Ca²⁺]_EC. Increased [Ca²⁺]_EC inhibited proliferation of well-differentiated cells, but had no effect on poorly-differentiated cells. Morphological studies showed that extracellular calcium was necessary for normal cell—cell interactions. These studies have demonstrated direct effects of calcium on colonic epithelial cells which may contribute to the protective effects of dietary calcium against colon cancer. Loss of responsivess to the antiprotective effects of [Ca²⁺]_EC with de-differentiation suggests that calcium supplementation may be most beneficial prior to the development of neoplastic changes in colonic epithelium.     

Impaired Systemic Tetrahydrobiopterin Bioavailability and Increased Dihydrobiopterin in Adult Falciparum Malaria: Association with Disease Severity,Impaired Microvascular Function and Increased Endothelial Activation

Tetrahydrobiopterin (BH₄) is a co-factor required for catalytic activity of nitric oxide synthase (NOS) and amino acid-monooxygenases, including phenylalanine hydroxylase. BH₄ is unstable: during oxidative stress it is non-enzymatically oxidized to dihydrobiopterin (BH₂), which inhibits NOS. Depending on BH₄ availability, NOS oscillates between NO synthase and NADPH oxidase: as the BH₄/BH₂ ratio decreases, NO production falls and is replaced by superoxide. In African children and Asian adults with severe malaria, NO bioavailability decreases and plasma phenylalanine increases, together suggesting possible BH₄ deficiency. The primary three biopterin metabolites (BH₄, BH₂ and B₀ [biopterin]) and their association with disease severity have not been assessed in falciparum malaria. We measured pterin metabolites in urine of adults with severe falciparum malaria (SM; n=12), moderately-severe malaria (MSM, n=17), severe sepsis (SS; n=5) and healthy subjects (HC; n=20) as controls. In SM, urinary BH₄ was decreased (median 0.16 ¼mol/mmol creatinine) compared to MSM (median 0.27), SS (median 0.54), and HC (median 0.34)]; p<0.001. Conversely, BH₂ was increased in SM (median 0.91 ¼mol/mmol creatinine), compared to MSM (median 0.67), SS (median 0.39), and HC (median 0.52); p<0.001, suggesting increased oxidative stress and insufficient recycling of BH2 back to BH4 in severe malaria. Overall, the median BH₄/BH₂ ratio was lowest in SM [0.18 (IQR: 0.04-0.32)] compared to MSM (0.45, IQR 0.27-61), SS (1.03; IQR 0.54-2.38) and controls (0.66; IQR 0.43-1.07); p<0.001. In malaria, a lower BH₄/BH₂ ratio correlated with decreased microvascular reactivity (r=0.41; p=0.03) and increased ICAM-1 (r=-0.52; p=0.005). Decreased BH4 and increased BH₂ in severe malaria (but not in severe sepsis) uncouples NOS, leading to impaired NO bioavailability and potentially increased oxidative stress. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.     

Cyrstal structure of a complex of α-cyclodextrin with 2-fluoro-4-nitrophenol · 3H2O

The crystal structure of a complex of α-cyclodextrin (α-CD) with 2-fluoro-4-nitrophenol · 3H₂O has been determined by the X-ray diffraction technique. The complex crystallizes in space group P2₁2₁2₁ with cell dimensions: a = 13.431(3), b = 15.299(4), c = 24.780(5) Å. The structure was solved by direct methods and refined to R = 6.7% for 4483 reflections. The crystal structure is isomorphous to the α-CD-4-nitrophneol · 3H₂O complex. The phenyl group is inside the cavity, so that the O-4 hexagon of the α-CD is distorted in a systematic manner: the longest diagonal [O-4(G2) O-4(G5)] is in the direction of the benzene ring. The phenolic OH group protrudes from the secondary OH side of the cavity and the NO₂ group is situated on the primary OH side. The hydrophobic F atom is statitically disordered over two sites and is located in the hydrophilic space, just beyond the rim of the secondary OH side of the cavity.     

A theoretical study on the hydrogen-bonding interactions between flavonoids and ethanol/water

Ethanol and water are the solvents most commonly used to extract flavonoids from propolis. Do hydrogen-bonding interactions exist between flavonoids and ethanol/water? In this work, this question was addressed by using density functional theory (DFT) to provide information on the hydrogen-bonding interactions between flavonoids and ethanol/water. Chrysin and Galangin were chosen as the representative flavonoids. The investigated complexes included chrysin–H₂O, chrysin–CH₃CH₂OH, galangin–H₂O and galangin–CH₃CH₂OH dyads. Molecular geometries, hydrogen-bond binding energies, charges of monomers and dyads, and topological analysis were studied at the B3LYP/M062X level of theory with the 6?31++G(d,p) basis set. The main conclusions were: (1) nine and ten optimized hydrogen-bond geometries were obtained for chrysin–H₂O/CH₃CH₂OH and galangin–H₂O/CH₃CH₂OH complexes, respectively. (2) The hydrogen atoms except aromatic H1 and H5 and all of the oxygen atoms can form hydrogen-bonds with H₂O and CH₃CH₂OH. Ethanol and water form strong hydrogen-bonds with the hydroxyl, carbonyl and ether groups in chrysin/galangin and form weak hydrogen-bonds with aromatic hydrogen atoms. Except in structures labeled A and B, chrysin and galangin interact more strongly with H₂O than CH₃CH₂OH. (3) When chrysin and galangin form hydrogen-bonds with H₂O and CH₃CH₂OH, charge transfers from the hydrogen-bond acceptor (H₂O and CH₃CH₂OH in structures A, B, G, H, I, J) to the hydrogen-bond donor (chrysin and galangin in structure A, B, G, H, I, J). The stronger hydrogen-bond makes the hydrogen-bond donor lose more charge (A> B> G> H> I> J). (4) Most of the hydrogen-bonds in chrysin/galangin?H₂O/CH₃CH₂OH complexes may be considered as electrostatic dominant, while C?O2···H in structures labeled E and C?O5···H in structures labeled J are hydrogen-bonds combined of electrostatic and covalent characters. H9, H7, and O4 are the preferred hydrogen-bonding sites.     

Sorting of the v-SNARE VAMP7 in Dictyostelium discoideum: a role for more than one Adaptor Protein (AP) complex

Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptors (SNAREs) participate in the specificity of membrane fusions in the cell. The mechanisms of specific SNARE sorting are still however poorly documented. We investigated the possible role of Adaptor Protein (AP) complexes in sorting of the Dictyostelium discoideum v-SNARE VAMP7. In live cells, GFP-VAMP7 is observed in the membrane of endocytic compartments. It is also observed in the plasma membrane of a small proportion of the cells. Mutation of a potential dileucine motif dramatically increases the proportion of cells with GFP-VAMP7 in their plasma membrane, strongly supporting the participation of an AP complex in VAMP7 sorting to the endocytic pathway. A partial increase occurs in knockout cells for the medium subunits of AP-2 and AP-3 complexes, indicating a role for both AP-2 and AP-3. VAMP7, as well as its t-SNAREs partners syntaxin 8 and Vti1, are co-immunoprecipitated with each of the medium subunits of the AP-1, AP-2, AP-3 and AP-4 complexes. This result supports the conclusion that VAMP7 directly interacts with both AP-2 and AP-3. It also raises the hypothesis of an interaction with AP-1 and AP-4. GFP-VAMP7 is retrieved from the endocytic pathway at and/or before the late post-lysosomal stage through an AP-independent mechanism.     

Preparation and characterization of (9-methyladenine)triammineplatinum(II) and trans-dihydroxo(9-methyladenine)triammineplatinum(IV) complexes

The preparation is reported of [(NH₃)₃Pt(9- MeA)] X₂ (9-MeA = 9-methyladenine) with XCl (1a) and XClO₄ (1b) and of trans-[(OH)₂Pt(NH₃)₃- (9-MeA)]X₂ with XCl (2a) and XClO₄ (2b), and the crystal structure of 1b. [(NH₃)₃Pt(C₆H₇N₅)](ClO₄)₂ crystallizes in space group P2₁/n with a = 20.810(7) Å, b = 7.697(3) Å, c = 10.567(4) Å, β = 91.57(6)°, Z = 4. The structure was refined to R = 0.054, R_w = 0.063. In all four compounds Pt coordination is through N7 of 9-MeA, as is evident from ³J coupling between H8 of the adenine ring and ¹⁹⁵Pt. Pt(II) and Pt(IV) complexes can be differentiated on the basis of different ³J values, larger for Pt(II) than for Pt(IV) by a factor of 1.57 (av). In Me₂SO-d₆, hydrogen bonding occurs between Cl^? and C(8)H of 9-MeA as weil as between Cl^? and the NH₃ groups in the case of the Pt(II) complex 1a. Protonation of the 9-MeA ligands was followed using ¹H NMR spectroscopy and pK_a values for the N1 protonated 9-MeA ligands were determined in D₂O. They are 1.9 for 1a and 1.8 for 2a, which compares with 4.5 for the non-platinated 9-MeA. Possible consequences for hydrogen bonding with the complementary bases thymine or uracil are discussed briefly. Protonation of the OH groups in the Pt(IV) complexes has been shown not to occur above pH 1.