Highly efficient synthesis of alternate alpha- and beta-(1-->3)-linked glucose hepta- and octasaccharides

Two heptasaccharides alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-1-OMP and beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp1-OMP, and two octasaccharides alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-1-OMP and beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp1-OMP were synthesized in a stereospecific way by remote control.     

The ratio of serum 24,25-dihydroxyvitamin D(3) to 25-hydroxyvitamin D(3) is predictive of 25-hydroxyvitamin D(3) response to vitamin D(3) supplementation

24,25-Dihydroxyvitamin D (24,25VD) is a major catabolite of 25-hydroxyvitamin D (25VD) metabolism, and may be physiologically active. Our objectives were to: (1) characterize the response of serum 24,25VD(3) to vitamin D(3) (VD(3)) supplementation; (2) test the hypothesis that a higher 24,25VD(3) to 25VD(3) ratio (24,25:25VD(3)) predicts 25VD(3) response. Serum samples (n=160) from wk 2 and wk 6 of a placebo-controlled, randomized clinical trial of VD(3) (28,000IU/wk) were analyzed for serum 24,25VD(3) and 25VD(3) by mass spectrometry. Serum 24,25VD(3) was highly correlated with 25VD(3) in placebo- and VD(3)-treated subjects at each time point (p<0.0001). At wk 2, the 24,25:25VD(3) ratio was lower with VD(3) than with placebo (p=0.035). From wk 2 to wk 6, the 24,25:25VD(3) ratio increased with the VD(3) supplement (p<0.001) but not with placebo, such that at wk 6 this ratio did not significantly differ between groups. After correcting for potential confounders, we found that 24,25:25VD(3) at wk 2 was inversely correlated to the 25VD(3) increment by wk 6 in the supplemented group (r=-0.32, p=0.02) but not the controls. There is a strong correlation between 24,25VD(3) and 25VD(3) that is only modestly affected by VD(3) supplementation. This indicates that the catabolism of 25VD(3) to 24,25VD(3) rises with increasing 25VD(3). Furthermore, the initial ratio of serum 24,25VD(3) to 25VD(3) predicted the increase in 25VD(3). The 24,25:25VD(3) ratio may therefore have clinical utility as a marker for VD(3) catabolism and a predictor of serum 25VD(3) response to VD(3) supplementation.     

Analogous antigenic alterations elicited in C3 by physiologic binding and by denaturation in the presence of sodium dodecylsulfate

The expression of three antigenic subsets of C3--the C3(S), the C3(N), and the C3(D) antigens--by soluble and target-bound forms of C3 was studied. The C3(S) subset is stable and is expressed by native as well as denatured C3 (exposure to sodium dodecyl sulphate (SDS) M greater than or equal to 10(-3)). The C3(N) and C3(D) subsets are labile and are expressed by native and denatured C3, respectively. Antisera to native C3, anti-C3(S-N), react with the C3(S) as well as the C3(N) subset. Antisera to isolated C3 subunits react exclusively with the C3(D) subset. A separation of anti-C3(S) and anti-C3(N) antibodies was accomplished by adsorbing the anti-C3(S-N) antiserum with insolubilized, denatured C3, anti-C3(N) antibodies remained unadsorbed. Anti-C3(S) antibodies were adsorbed and subsequently eluted from the denatured C3. Agglutination studies with EAC1423b cells showed significant agglutination with anti-C3(S) and anti-C3(D) antisera but reduced agglutination with anti-C3(N) antisera. Agglutination by anti-C3(D) antisera was unaffected in the presence of EDTA serum containing converted or unconverted C3. These data suggest an antigenic modification of C3b-b' upon binding that mirrors the antigenic transition associated with SDS denaturation of C3.     

The F subunit of Thermus thermophilus V1-ATPase promotes ATPase activity but is not necessary for rotation

V(1)-ATPase from the thermophilic bacterium Thermus thermophilus is a molecular rotary motor with a subunit composition of A(3)B(3)DF, and its central rotor is composed of the D and F subunits. To determine the role of the F subunit, we generated an A(3)B(3)D subcomplex and compared it with A(3)B(3)DF. The ATP hydrolyzing activity of A(3)B(3)D (V(max) = 20 s(-1)) was lower than that of A(3)B(3)DF (V(max) = 31 s(-1)) and was more susceptible to MgADP inhibition during ATP hydrolysis. A(3)B(3)D was able to bind the F subunit to form A(3)B(3)DF. The C-terminally truncated F((Delta85-106)) subunit was also bound to A(3)B(3)D, but the F((Delta69-106)) subunit was not, indicating the importance of residues 69-84 of the F subunit for association with A(3)B(3)D. The ATPase activity of A(3)B(3)DF((Delta85-106)) (V(max) = 24 s(-1)) was intermediate between that of A(3)B(3)D and A(3)B(3)DF. A single molecule experiment showed the rotation of the D subunit in A(3)B(3)D, implying that the F subunit is a dispensable component for rotation itself. Thus, the F subunit binds peripherally to the D subunit, but promotes V(1)-ATPase catalysis.     

Chemical characterization of acidic oligosaccharides in milk of the red kangaroo (Macropus rufus)

In the milk of marsupials, oligosaccharides usually predominate over lactose during early to mid lactation. Studies have shown that tammar wallaby milk contains a major series of neutral galactosyllactose oligosaccharides ranging in size from tri- to at least octasaccharides, as well as β(1-6) linked N-acetylglucosamine-containing oligosaccharides as a minor series. In this study, acidic oligosaccharides were purified from red kangaroo milk and characterized by (1)H-nuclear magnetic resonance spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to be as follows: Neu5Ac(α2-3)Gal(β1-4)Glc (3'-SL), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3'-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc. These acidic oligosaccharides were shown to be sialylated or sulfated in the non-reducing ends to the major linear and the minor branched series of neutral oligosaccharides of tammar wallaby milk.     

Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.     

Chemical characterization of milk oligosaccharides of the common brushtail possum (Trichosurus vulpecula)

Structural characterizations of marsupial milk oligosaccharides have been performed in only three species: the tammar wallaby, the red kangaroo and the koala. To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, 21 oligosaccharides of the milk carbohydrate fraction of the common brushtail possum were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of mid-lactation milk and characterized by ¹H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the 7 neutral oligosaccharides were Gal(β1-3)Gal(β1-4)Glc (3’-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3”, 3’-digalactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I), Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)Gal(β1-4)Glc (galactosyl lacto-N-novopentaose II). The structures of the 14 acidic oligosaccharides detected were Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3’-galactosyllactose), Gal(β1-3)(O-3-sulfate)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate a) Gal(β1-3)[Gal(β1-4)(O-3-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)(?3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)[Gal(β1-4)(?3-O-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(?3-O-sulphate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(?3-O-sulphate)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)(?3-O-sulphate)GlcNAc(β1-6)]Gal(β1-4)Glc and Gal(β1-3)Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl sialyl lacto-N-novopentaose b). No fucosyl oligosaccharides were detected. Galactosyl lacto-N-novopentaose II, lacto-N-novopentaose I sulfate a, lacto-N-novopentaose I sulfate b and galactosyl sialyl lacto-N-novopentaose b are novel oligosaccharides. The results are compared with those of previous studies on marsupial milk oligosaccharides.     

Fast biphasic regulation of type 3 inositol trisphosphate receptors by cytosolic calcium

In cytosol-like medium (CLM) with a free [Ca(2+)] of 200 nm, a supramaximal concentration of inositol 1,4,5-trisphosphate (IP(3)) (30 microm) evoked (45)Ca(2+) release from type 3 IP(3) receptors only after a latency of 48 +/- 6 ms; this latency could not be reduced by increasing the IP(3) concentration. In CLM containing a low free [Ca(2+)] ( approximately 4 nm), 300 microm IP(3) evoked (45)Ca(2+) release after a latency of 66 +/- 11 ms; this was reduced to 14 +/- 3 ms when the [Ca(2+)] was 1 mm. Preincubation with CLM containing 100 microm Ca(2+) caused a rapid (half-time = 33 +/- 9 ms), complete, and fully reversible inhibition that could not be overcome by a high concentration of IP(3) (300 microm). Hepatic (type 2) IP(3) receptors were not inhibited by Ca(2+) once they had bound IP(3), but 100 microm Ca(2+) rapidly inhibited type 3 IP(3) receptors whether it was delivered before addition of IP(3) or at any stage during a response to IP(3). Ca(2+) increases the affinity of IP(3) for hepatic receptors by slowing IP(3) dissociation, but Ca(2+) had no effect on IP(3) binding to type 3 receptors. The rate of inhibition of type 3 IP(3) receptors by Ca(2+) was faster than the rate of IP(3) dissociation, and occurred at similar rates whether receptors had bound a high (adenophostin) or low affinity (3-deoxy-3-fluoro-IP(3)) agonist. Dissociation of agonist is not therefore required for Ca(2+) to inhibit type 3 IP(3) receptors. We conclude that type 2 and 3 IP(3) receptors are each biphasically regulated by Ca(2+), but by different mechanisms. For both, IP(3) binding causes a stimulatory Ca(2+)-binding site to be exposed allowing Ca(2+) to bind and open the channel. IP(3) binding protects type 2 receptors from Ca(2+) inhibition, but type 3 receptors are inhibited by Ca(2+) whether or not they have IP(3) bound. Increases in cytosolic [Ca(2+)] will immediately inhibit type 3 receptors, but inhibit type 2 receptors only after IP(3) has dissociated.     

Growth, thermal and spectral properties of Er3+-Doped and Er3+/Yb3+-Codoped Li3Ba2La3(WO4)8 crystals

This paper reports the growth and spectral properties of Er(3+)-doped and Er(3+)/Yb(3+)-codoped Li(3)Ba(2)La(3)(WO(4))(8) crystals. The Er(3+): Li(3)Ba(2)La(3)(WO(4))(8) crystal with dimensions of 56 mm × 28 mm × 9 mm and Er(3+)/Yb(3+): Li(3)Ba(2)La(3)(WO(4))(8) crystal with dimensions of 52 mm × 24 mm × 8 mm were obtained by the top-seeded solution growth (TSSG) method. Thermal expansion coefficients and thermal conductivity of both crystals were measured. The spectroscopic characterizations of both crystals were investigated. The spectroscopic analysis reveals that the Er(3+)/Yb(3+): Li(3)Ba(2)La(3)(WO(4))(8) crystal has much better optical properties than the Er(3+): Li(3)Ba(2)La(3)(WO(4))(8) crystal, thus it may become a potential candidate for solid-state laser gain medium material.     

Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ～0.3 and ～1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.     

The mechanism of 1,25-dihydroxyvitamin D(3) autoregulation in keratinocytes

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) from its precursor, 25-dihydroxyvitamin D(3) (25(OH)D(3)), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase). It has been generally assumed that 1,25(OH)(2)D(3) inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)(2)D(3) reduced the apparent conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) while stimulating the conversion of 1,25(OH)(2)D(3) and 25(OH)D(3) to 1,24,25(OH)(3)D(3) and 24,25(OH)(2)D(3), respectively. However, 1,25(OH)(2)D(3) failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)(2)D(3) was blocked with a specific inhibitor of the 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1alpha-hydroxylase activity by 1,25(OH)(2)D(3) was reversed. Thus, the apparent reduction in 1alpha-hydroxylase activity induced by 1,25(OH)(2)D(3) is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.     

IP3 receptor/Ca2+ channel: from discovery to new signaling concepts

Inositol 1,4,5-trisphosphate (IP(3)) is a second messenger that induces the release of Ca(2+) from the endoplasmic reticulum (ER). The IP(3) receptor (IP(3)R) was discovered as a developmentally regulated glyco-phosphoprotein, P400, that was missing in strains of mutant mice. IP(3)R can allosterically and dynamically change its form in a reversible manner. The crystal structures of the IP(3)-binding core and N-terminal suppressor sequence of IP(3)R have been identified. An IP(3) indicator (known as IP(3)R-based IP(3) sensor) was developed from the IP(3)-binding core. The IP(3)-binding core's affinity to IP(3) is very similar among the three isoforms of IP(3)R; instead, the N-terminal IP(3) binding suppressor region is responsible for isoform-specific IP(3)-binding affinity tuning. Various pathways for the trafficking of IP(3)R have been identified; for example, the ER forms a meshwork upon which IP(3)R moves by lateral diffusion, and vesicular ER subcompartments containing IP(3)R move rapidly along microtubles using a kinesin motor. Furthermore, IP(3)R mRNA within mRNA granules also moves along microtubules. IP(3)Rs are involved in exocrine secretion. ERp44 works as a redox sensor in the ER and regulates IP(3)R1 activity. IP(3) has been found to release Ca(2+), but it also releases IRBIT (IP(3)R-binding protein released with IP(3)). IRBIT is a pseudo-ligand for IP(3) that regulates the frequency and amplitude of Ca(2+) oscillations through IP(3)R. IRBIT binds to pancreas-type Na, bicarbonate co-transporter 1, which is important for acid-base balance. The presence of many kinds of binding partners, like homer, protein 4.1N, huntingtin-associated protein-1A, protein phosphatases (PPI and PP2A), RACK1, ankyrin, chromogranin, carbonic anhydrase-related protein, IRBIT, Na,K-ATPase, and ERp44, suggest that IP(3)Rs form a macro signal complex and function as a center for signaling cascades. The structure of IP(3)R1, as revealed by cryoelectron microscopy, fits closely with these molecules.     

Effects of polyamines and analogs on staphylococcal nuclease.

The promoting activity of polyamine analogs (IV approximately XV) on staphylococcal nuclease with DNA as the substrate was compared with that of natural polyamines (I APPROXIMATELY III): I. NH2(CH2)3NH(CH2)4NH(CH2)3NH2(spermine); II. NH2(CH2)3NH(CH2)3NH(CH2)3NH2(thermine); III. NH2(CH2)4NH2 (putrescine); IV. CN(CH2)2NH(CH2)4NH(CH2)2CN; V. HOOC(CH2)2NH(CH2)4NH(CH2)2COOH; VI. C2H5OOC(CH2)2NH(CH2)4NH(CH2)2COOC2H5; VII. HO(CH2)3NH(CH2)4HH(CH2)3OH; VIII. CH3COHH(CH2)3NH(CH2)4NH(CH2)3NHCOCH3; IX. C2H5NH(CH2)3NH(CH2)4NH(CH2)3NHC2H5; X. NH2(CH2)3S(CH2)4S(CH2)3NH2; XI. NH2(CH2)3NH(CH2)2O(CH2)2NH(CH2)3NH2; XII. NH2(CH2)3NCH3(CH2)4HCH3(CH2)3NH2; XIII. CN(CH2)2NCH3(CH2)4NCH3(CH2)2CN; XIV. (CH3)2N(CH2)3NCH3(CH2)4NCH3(CH2)3N(CH3)2; XV. NH2(CH2)2O(CH2)2NH2 Replacement of the terminal groups by CN, COOH, COOEt, NHAc, NHEt, or N(CH3)2 remarkably decreased the activity. The compound VII with terminal hydroxyl groups had a lower promoting activity at low concentrations, but revealed higher activity at higher concentrations and, in contrast to spermine, no inhibition at all even at very high concentrations. Replacement of both internal amino groups by sulfur or NCH3 decreased the activity. The introduction of an ether bond into the internal methylene groups (compound XI) highly decreased the activity. Based upon these findings the possible relationship between structure and activity is discussed.     

The type 2 inositol (1,4,5)-trisphosphate (InsP3) receptor determines the sensitivity of InsP3-induced Ca2+ release to ATP in pancreatic acinar cells

Calcium release through inositol (1,4,5)-trisphosphate receptors (InsP(3)R) is the primary signal driving digestive enzyme and fluid secretion from pancreatic acinar cells. The type 2 (InsP(3)R2) and type 3 (InsP(3)R3) InsP(3)R are the predominant isoforms expressed in acinar cells and are required for proper exocrine gland function. Both InsP(3)R2 and InsP(3)R3 are positively regulated by cytosolic ATP, but InsP(3)R2 is 10-fold more sensitive than InsP(3)R3 to this form of modulation. In this study, we examined the role of InsP(3)R2 in setting the sensitivity of InsP(3)-induced Ca(2+) release (IICR) to ATP in pancreatic acinar cells. IICR was measured in permeabilized acinar cells from wild-type (WT) and InsP(3)R2 knock-out (KO) mice. ATP augmented IICR from WT pancreatic cells with an EC(50) of 38 mum. However, the EC(50) was 10-fold higher in acinar cells isolated from InsP(3)R2-KO mice, indicating a role for InsP(3)R2 in setting the sensitivity of IICR to ATP. Consistent with this idea, heterologous expression of InsP(3)R2 in RinM5F cells, which natively express predominately InsP(3)R3, increased the sensitivity of IICR to ATP. Depletion of ATP attenuated agonist-induced Ca(2+) signaling in WT pancreatic acinar cells. This effect was more profound in acinar cells prepared from InsP(3)R2-KO mice. These data suggest that the sensitivity of IICR to ATP depletion is regulated by the particular complement of InsP(3)R expressed in an individual cell. The effects of metabolic stress on intracellular Ca(2+) signals can therefore be determined by the relative amount of InsP(3)R2 expressed in cells.     

Inositol 1,4,5-trisphosphate receptor type 1 is a substrate for caspase-3 and is cleaved during apoptosis in a caspase-3-dependent manner

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), an IP(3)-gated Ca(2+) channel located on intracellular Ca(2+) stores, modulates intracellular Ca(2+) signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP(3)R type 1 (IP(3)R1) was found to be cleaved. IP(3)R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH(2)DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP(3)R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP(3)R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is (1888)DEVD*(1892)R (mouse IP(3)R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP(3)R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP(3)R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-alpha- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP(3)R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP(3)R1 degradation upon apoptotic stimuli. Therefore IP(3)R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP(3)R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP(3)R1 was digested, indicating that caspase-3 inactivates IP(3)R1 channel functions.     

Molecular cloning of mouse type 2 and type 3 inositol 1,4,5-trisphosphate receptors and identification of a novel type 2 receptor splice variant

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R2 and IP(3)R3, respectively) from mouse lung and found a novel alternative splicing segment, SI(m2), at 176-208 of IP(3)R2. The long form (IP(3)R2 SI(m2)(+)) was dominant, but the short form (IP(3)R2 SI(m2)(-)) was detected in all tissues examined. IP(3)R2 SI(m2)(-) has neither IP(3) binding activity nor Ca(2+) releasing activity. In addition to its reticular distribution, IP(3)R2 SI(m2)(+) is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca(2+) ionophore stimulation, most of the IP(3)R2 SI(m2)(+) is in clusters. IP(3)R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca(2+) ionophore stimulation. IP(3)R2 SI(m2)(-) does not form clusters in either resting or stimulated cells. IP(3) binding-deficient site-directed mutants of IP(3)R2 SI(m2)(+) and IP(3)R3 fail to form clusters, indicating that IP(3) binding is involved in the cluster formation by these isoforms. Coexpression of IP(3)R2 SI(m2)(-) prevents stimulus-induced IP(3)R clustering, suggesting that IP(3)R2 SI(m2)(-) functions as a negative coordinator of stimulus-induced IP(3)R clustering. Expression of IP(3)R2 SI(m2)(-) in CHO-K1 cells significantly reduced ATP-induced Ca(2+) entry, but not Ca(2+) release, suggesting that the novel splice variant of IP(3)R2 specifically influences the dynamics of the sustained phase of Ca(2+) signals.     

A simple procedure for synthesis of diastereoisomers of thymidine cyclic 3',5'-phosphate derivatives

Diastereoisomeric thymidine cyclic (3',5')-methanephosphonates (3a), cyclic (3',5')-phosphoranilidates (3b) and cyclic (3',5')-phosphoranilidothioates (3c) were prepared by treatment of diastereoisomerically pure thymidine 3'-O-[O-(4-nitrophenyl)methanephosphonates] (2a), 3'-O-[O-(4-nitrophenyl)phosphoranilidates] (2b) or 3'-O-[O-(4-nitrophenyl)phosphoranilidothioates] (2c), respectively, with sodium hydroxide in dioxane-water solution.     

Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c₁) and 3-methyl-dihydro-2(3H)-furanone (4c₂) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.     

Primary structure of ten galactosides formed by transglycosylation during lactose hydrolysis by Bifidobacterium bifidum

Oligosaccharides formed by a transgalactosylation reaction during lactose hydrolysis with Bifidobacterium bifidum were separated into eight fractions by gel-permeation chromatography and their structures studies determined by trimethylsilylation analysis, methylation analysis, f.a.b.-m.s., g.l.c.-m.s. and enzymic hydrolysis as beta-D-Galp-(1----3)-D-Glc, beta-D-Galp-(1----6)-D-Glc, beta-D-Galp-(1----6)-D-Gal, beta-D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, beta-D-Galp-(1----6)[beta-D-Galp-(1----4)]-D-Glc, beta-D-Galp-(1----2)[beta-D-Galp-(1----6)]-D-Glc, beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Ga lp- (1----4)-D-Glc, beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-DGalp-(1----3)-beta -D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, and beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Ga lp-(1----3)-beta-D-G-alp-(1----3) beta-D-Galp-(1----4)-D-Glc.