Prenatal developmental toxicity evaluation of 2',3'-dideoxyinosine (ddI) and 2',3'-didehydro-3'-deoxythymidine (d4T) co-administered to Swiss Albino (CD-1) mice

BACKGROUND: In pregnant women, antiretroviral drugs improve maternal health and reduce vertical transmission of human immunodeficiency virus to the infant. However, few nonclinical studies have examined the potential for adverse drug interactions. METHODS: On gestational days (GD) 6-16, mice were dosed with vehicle, ddI (360, 1440, or 2,880 mg/kg/day, p.o.), d4T (60, 240, or 480), or ddI/d4T combinations (360/60, 1,440/240, or 2,880/480). Daily doses were divided into two equal parts that were administered >or=6-hr apart. Body weight, clinical signs, and feed consumption were monitored. Pregnancies (22-24/group) were confirmed at necropsy. Maternal liver and gravid uterine weights (GUW), uterine implants (resorption, live or dead fetus), fetal body weight, gender, and morphologic anomalies (external, visceral, skeletal) were recorded. RESULTS: Maternal body weight, clinical signs, and GUW were unaffected. Maternal weight change corrected for GUW was greater than controls at 60 and 480 d4T. Relative feed consumption during treatment was increased relative to controls at 1,440 and 2,880 ddI and 2,880/480 ddI/d4T. Relative maternal liver weight was elevated above controls at 240 and 480 d4T and 2,880/480 ddI/d4T, and above the constituent dose of ddI at 1,440/240 and 2,880/480 ddI/d4T. Liver weight was not affected by ddI and there was no significant drug interaction. Prenatal mortality and morphologic anomalies were not increased. Fetal body weight showed only a decreasing trend for ddI/d4T, no effect for ddI or d4T, and no statistically significant drug interaction. CONCLUSIONS: In pregnant mice, ddI/d4T combinations were not associated with well-defined developmental toxicity or adverse drug interactions.     

Hydroxylation of naringin by Trichoderma harzianum to dramatically improve its antioxidative activity

Transforming naringin using the mycelium of Trichoderma harzianum CGMCC 1523 produces two metabolites, 3′,4′,5,7-tetrahydroxy flavanone-7-rhamnoglucoside (3′-OHN) and 3′,4′,5′,5,7-pentahydroxy flavanone-7-rhamnoglucoside (3′,5′-DOHN), both of which were characterized by ESI–MS, ¹H NMR and ¹³C NMR analyses. The time course of the biotransformation by T. harzianum showed that 3′-OHN and 3′,5′-DOHN appeared simultaneously at 6 h, and the conversion yield (32.6%) of 3′,5′-DOHN was higher (10.6%) than that of 3′-OHN at 56 h. The optimal biotransformation temperature was 30 °C, the optimal pH was 5.0, and the optimal concentration of naringin was 400 mg/l. The bigger volume of biotransformation mixture and lower shaking speed did not favor hydroxylation reactions. The radical scavenging activity of naringin at 2000 μM was 11.1%, whereas activity of 3′-OHN at 100 μM could reach 38.4%, which is 68.6 times more than naringin. Antioxidative activity of 3′,5′-DOHN was increased 13.5% at 100 μM compared to 3′-OHN.     

Purification of human erythrocyte glucose 6-phosphate dehydrogenase and glutathione reductase enzymes using 2',5'-ADP Sepharose 4B affinity column material in single chromatographic step

The enzymes of glucose 6-phosphate dehydrogenase and glutathione reductase were purified from human erythrocytes in one chromatographic step consisting of the use of the commercially available resin 2',5'-ADP Sepharose 4B by using different washing buffers. Ammonium sulfate (30-70%) precipitation was performed on the hemolysate before applying to the affinity column. Using this procedure, G6PG, having the specific activity of 22.9 EU/mg proteins, was purified with a yield of 43% and 9150-fold; GR, having the specific activity of 20.7 EU/mg proteins, was purified with a yield of 26% and 8600-fold. The purity of the enzymes was checked on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and each purified enzyme showed a single band on the gel. This procedure has advantages of preventing of enzyme denaturation, short experimental duration, and use of less chemical materials for purification of the enzymes.     

1′,2′-Dideacetylboronolide, an α-pyrone from Iboza riparia

The structural elucidation of 1′,2′-dideacetylboronolide, 5,6-dihydro-6-(3′-acetoxy-1′,2′-dihydroxyheptyl)2-pyrone, a new α-pyrone isolated from the leaves of Iboza riparia has been performed. Additionally, three sterols, sitosterol, stigmasterol and campesterol, have been identified in this species.     

Inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl during greening of groundnut leaves

The site of inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl was found to be at the level of conversion of chlorophyllide (672 nm) to chlorophyll (678 nm) during greening of groundnut leaves. This inhibition was partially reversed by certain divalent cations.     

1-[2-Hydroxy-3-octadecan-1′-oate]propyl-2″,2″,5″,5″-tetramethyl Pyrolidine-N-oxyl-3″-carboxylate as a potential spin probe for membrane structure studies

The synthesis of a new minimum steric perturbing proxyl nitroxide, which is a derivative of glycerol and contains a stearic acid moiety, has been carried out. Its localization in model membrane -α-dipalmitoyl phosphatidyl choline (DPPC) was ascertained with the help of ESR, DSC, ¹H and ³¹P NMR techniques. The nitroxide was used for detecting the changes in the phase transition temperature of the model membranes in the presence and absence of drugs. The permeation of the vasodilating drug epinephrine has also been studied using this spin label. The results prove the potential applicability of the new spin probe in the spin labeling of biomembranes.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Biochemical characterization of a novel beta-1--3, 1--4 glucan 4-glucanohydrolase from Thermomonospora sp. having a single active site for lichenan and xylan

A bifunctional high molecular weight (Mr, 64,500 Da) beta-1-3, 1-4 glucan 4-glucanohydrolase was purified to homogeneity from Thermomonospora sp., exhibiting activity towards lichenan and xylan. A kinetic method was used to analyze the active site that hydrolyzes lichenan and xylan. The experimental data was in agreement with the theoretical values calculated for a single active site. Probing the conformation and microenvironment at active site of the enzyme by fluorescent chemo-affinity label, OPTA resulted in the formation of an isoindole derivative with complete inactivation of the enzyme to hydrolyse both lichenan and xylan confirmed the results of kinetic method. OPTA forms an isoindole derivative by cross-linking the proximal thiol and amino groups. The modification of cysteine and lysine residues by DTNB and TNBS respectively abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating the participation of cysteine and lysine in the formation of isoindole complex.     

DNA-binding geometry dependent energy transfer from 4′,6-diamidino-2-phenylindole to cationic porphyrins

The circular and linear dichroism (CD and LD) spectral properties of the meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP)–DNA complex at a [porphyrin]/[DNA] ratio below 0.015 showed that TMPyP intercalates between DNA base pairs. Contrarily, when cis–bis(N-methylpyridinium-4-yl)porphyrin (BMPyP) is associated with DNA, no CD spectrum was induced and a bisignate LD spectrum was observed. These spectral properties of both the TMPyP and BMPyP were essentially retained when the minor groove of the DNA was saturated with 4′,6-diamidino-2-phenylindole (DAPI). The fluorescence of the DNA-bound DAPI was effectively quenched by BMPyP and TMPyP. The quenching by BMPyP can be described through a pure static mechanism while TMPyP quenching produced an upward bending curve in the Stern–Volmer plot. Quenching efficiency was by far greater than predicted by the “sphere of action model”, suggesting that the DNA provides some additional processes for an effective energy transfer.     

Antinoceptive effect of triterpenoid α,β-amyrin in rats on orofacial pain induced by formalin and capsaicin

The effects of α,β-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with α,β-amyrin (10, 30, and 100 mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 μl, 1.5%) or capsaicin (20 μl, 1.5 μg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0–5 min) and a late second phase (10–20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10–20 min. Treatment with α,β-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, α,β-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, α,β-amyrin produced much greater antinociceptive effect at 100 mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that α,β-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.     

Nuclear Location of a Diadenosine 5′,5′”-P1,P4Tetraphosphate (Ap4A) Hydrolase in Tomato Cells Grown in Suspension Cultures1

Diadenosine 5′,5′”-P¹,P⁴-tetraphosphate (Ap₄A) cleaving enzymes are assumed to regulate intracellular levels of Ap₄A, a compound known to affect cell proliferation and stress responses. From plants an Ap₄A hydrolase was recently purified using tomato cells grown in suspension. It was partially sequenced and a peptide antibody was prepared (Feussner et al., 1996). Using this polyclonal monospecific antibody, an abundant nuclear location of Ap₄A hydrolase in 4-day-old cells of atomato cell suspension culture is demonstrated here by means of immunocytochemical techniques using FITC (fluorescein-5-isothiocyanate) labeled secondary antibodies. The microscopic analysis of the occurrence of Ap₄A hydrolase performed for different stages of the cell cycle visualized by parallel DAPI (4,6-diamidino-2-phenylindole) staining revealed that the protein accumulates within nuclei of cells in the interphase, but is absent in the nucleus as well as cytoplasm during all stages of mitosis. This first intracellular localization of an Ap₄A degrading enzyme within the nucleus and its pattern of appearance during the cell cycle is discussed in relation to the suggested role of Ap₄A in triggering DNA synthesis and cell proliferation.     

Modeling of α(1–4) chain arrangements in α(1–4)(1–6) glucans: The action and outcome of β-amylase and Pseudomonas stutzeri amylase on an α(1–4)(1–6) glucan model

Simulated enzymic debranching of a β-limit dextrin model, prepared from a computed construct made by random extension and branching, and given the CCL value of w-maize amylopectin (and equal amounts of external chains with ECL values of 2 and 3) has been related to experimental chromatograms of the debranched β-limit dextrin of the amylopectin. The profile was similar to those from gel chromatograms and IEC-PAD chromatography.The equivalent lengths in glucosyl units of grid-links (g-links) of internal and external chains in constructs were calculated from the ICL and ECL values of amylopectin and models produced from the constructs with the appropriate lengths for internal and external chains. These derived models were subjected to simulated hydrolysis by Pseudomonas stutzeri amylase and the products compared with those of the experimental distribution from w-maize amylopectin. With the model the amounts of maltotetraose and maltodextrins released were similar to the experimental values but the distribution of branched maltodextrins was quite different. Unlike w-maize amylopectin – a polymer with the cluster structure – which has given a profile of molecular sizes of maltodextrins with low amounts of single and small numbers of internal chains and with a peak at a M_W of about 14,000 (13 chains), in the model the proportion of maltodextrin with one internal chain was high and as d.p. increased the amounts decreased exponentially. This would be expected if the distribution of internal chains in the core was random. It is suggested that in the core of a model prepared from a construct made with alternating probabilities of extension – one in which this probability is high relative to branching, and a second in which it is low – may give clusters of branched maltodextrins with short internal chains which are joined by longer chains; more closely approximating the distribution of internal chains of different lengths in amylopectin.An arrangement for amylopectin molecules in the starch granule has been proposed. In this, they have a wafer-like, discoidal shape, composed of the amorphous zone overlain with the double helical, crystalline region. The flat macromolecules are concentrically layered with the former on the inside and the latter oriented to the outside of the granule.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Integrin alphavbeta6 mediates HT29-D4 cell adhesion to MMP-processed fibrinogen in the presence of Mn2+

Mn(2+) was found to induce adhesion of HT29-D4 adenoma carcinoma cells to fibrinogen (Fb). This was independent of the expression of the beta3 integrin subunit and involved endogenous alphavbeta6 but not alphavbeta5 integrin. Thus, addition of Mn(2+) led to a change in integrin alphavbeta6 specificity. Furthermore, Mn(2+) was found to strongly activate the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in the HT29-D4 cell line. As a MAPK inhibitor strongly reduced the Mn(2+)-induced cell adhesion to Fb, it is suggested that a link between MAPK activation and cell adhesion to Fb exists. Both expression and activity of matrix metalloproteinase-9 (MMP-9) were enhanced by Mn(2+) and this led to Fb processing. MMP inhibitors prevented Mn(2+)-mediated cell adhesion to Fb, leading us to suggest that Mn(2+) promoted convergent changes in integrin alphavbeta6 conformation and Fb structure through activation of ERK/MAPK and MMP-9. Finally, we found that Mn(2+) and activators of the ERK pathway cooperated in HT29-D4 cell adhesion to Fb. Such a process may be involved in bone metastasis of some cancer cells.     

Monoclonal antibodies specific for type 3 and type 4 chain-based blood group determinants: Relationship to the A1 and A2 subgroups

Two monoclonal antibodies, specific for A type 3 and A type 4 blood group determinants, are described. These antibodies recognized A1 but not A2 erythrocytes. A third monoclonal antibody showing specificity for A type 3 and A type 4, and also for H type 3 and H type 4, did not discriminate between A1 and A2 erythrocytes. On red cells these three antibodies recognized glycosphingolipids and binding to glycoproteins could not be demonstrated. On paraffin-embedded tissue sections the three antibodies labelled a supranuclear area, characteristic of the Golgi apparatus, of all cells producing A antigens. This labelling occurred irrespective of the A1, A2 status.The results suggest that glycolipids of erythrocytes and possibly of other cell types bear the A type 3/4 determinant specific for the A1 subgroup and that A type 3/4 determinants of glycoproteins might be present in both A1 and A2 subgroups on short oligosaccharide chains which are only detectable at the level of the Golgi apparatus.     

Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.     

Beta-1 integrins mediate substrate dependent effects of 1α,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. β₁ integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1α,25(OH)₂D₃ in a surface-dependent manner. To determine if β₁ has a role in mediating osteoblast response, we silenced β₁ expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human β₁ to block ligand binding. β₁-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor β₁, prostaglandin E₂, and osteoprotegerin in comparison with control cells. Moreover, β₁-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1α,25(OH)₂D₃. Anti β₁ antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1α,25(OH)₂D₃ on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that β₁ plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1α,25(OH)₂D₃. The results also show that β₁ mediates, in part, the synergistic effects of surface roughness and 1α,25(OH)₂D₃.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.