Expedient syntheses of neoglycoproteins using phase transfer catalysis and reductive amination as key reactions

Starting from peracetylated chloro- or bromo-glycosyl donors ofN-acetylneurmainic acid,N-acetylglucosamine, glucose and lactose, the correspondingp-formylphenyl glycosides were synthesized stereospecifically under phase transfer catalysed conditions at room temperature in yields of 38–67%. After Zemplén de-O-acetylation, the formyl groups were directly and chemoselectively coupled to the lysine residues of bovine serum albumin by reductive amination using sodium cyanoborohydride. The conjugation reactions were followed as a function of time and under a series of different molar ratios of the reactants to provide glycoconjugates of varying degree of antigenicities. Thus, carbohydrate protein conjugates were made readily available using essentially two key reactions.Presented in part at the 15th International Carbohydrate Symposium, Yokohama, Japan, August 12–17, 1990.     

Seasonal variations in the pituitary response to LHRH in the brown hare (Lepus europaeus)

In the brown hare, fertile mating takes place from the beginning of December to September. Pituitary and ovarian response to a monthly i.v. injection of 5 micrograms LHRH was studied from September 1983 to October 1984 in 2 groups of 6 hares. The basal concentrations of LH remained undetectable until the end of January, rose from 0.23 +/- 0.14 ng/ml from February to a maximum of 1.44 +/- 0.57 ng/ml in July. LHRH injection was always followed by a release of LH. Between September and December, the LH value peaked 15 min after injection and returned to basal concentrations 2 h later. From January, this pattern altered and a second peak of LH appeared 2 h after injection. Peak levels 15 min after LHRH were around 10 ng/ml between September and December, increased from 47.0 +/- 8.0 ng/ml in January to 106 +/- 33 ng/ml in July and decreased in August (69.4 +/- 10.6 ng/ml). The values of the second peak rose from 11.0 +/- 2.2 ng/ml in January to 90.6 +/- 12.4 ng/ml between March and July and decreased in August (24.5 +/- 5.1 ng/ml). The LH surge induced by LHRH was always followed by a transient rise in progesterone. During the breeding season, this progesterone secretion increased considerably. Ovulation was possible between January and August and the number of ovulating females was maximum between March and July. The amount and duration of progesterone secretion during the resulting pseudopregnancies increased during the breeding season.     

Peroxidase-catalyzed O-demethylation reactions. Quinone-imine formation from 9-methoxyellipticine derivatives

Despite numerous reports on the N-demethylation reactions catalyzed by peroxidases, to our knowledge, O-demethylation reactions with the same enzymes seem to be still a questionable matter. Unexpectedly, a peroxidase system (horseradish peroxidase and hydrogen peroxide) is able to effect the O-demethylation of the cytotoxic agents 9-methoxyellipticine and N2-methyl-9-methoxyellipticinium acetate. The reaction leads directly to the formation of the corresponding quinone-imine derivatives with the concomitant formation of one molecule of methanol per molecule of methoxy compound. One hydrogen peroxide molecule is consumed during the process. Experiments in H218O-enriched water clearly indicate that 18O is nearly quantitatively incorporated in the carbonyl group of the generated quinone-imine compound with the concomitant elimination of the methoxy group as methanol. So this peroxidase-catalyzed apparent O-demethylation in fact implies an oxidative demethoxylation step. This enzymatic reaction exhibits normal Michaelis-Menten saturation kinetics. Like the 9-hydroxylated ellipticines, both the 9-methoxylated ellipticines show a good affinity for the peroxidase itself (Km approximately 10 microM) but are slowly transformed to the corresponding quinone-imines. The Vmax values for methoxylated ellipticines are 10(-1)-10(-3) lower than those for hydroxylated compounds. This new route for the in vitro formation of electrophilic derivatives from the cytotoxic 9-methoxyellipticine and N2-methyl-9-methoxyellipticinium might be considered as a novel possible metabolic pathway for these drugs, especially if we bear in mind the "bio-oxidative alkylation" process previously described for at least one of the corresponding hydroxylated ellipticine derivatives (see Bernadou, J., Meunier, B., Meunier, G., Auclair, C., and Paoletti, C. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1297-1301; and Monsarrat, B., Maftouh, M., Meunier, G., Dugué, B., Bernadou, J., Armand, J. P., Picard-Fraire, C., Meunier, B., and Paoletti, C. (1983) Biochem. Pharmacol. 32, 3887-3890).     

On the mechanical characterization of compact bone structure using the homogenization theory

In a previous paper (Crolet et al., 1993, J. Biomechanics 26, 677–687), a modelling of the mechanical behavior of compact bone was presented, in which the homogenization theory was the basic tool of computation. In this simulation, approximations were used for the modelling of the lamellae and the osteons: the lamella and the osteon were divided into cylindrical sectors, each sector being approximated as a parallelepiped having a periodic structure (fibrous composite for the lamella, superimposition of plates for the osteon). The present study deals with a new model without these approximations. First, it can be proved that the homogenized elasticity tensor for a lamella, which has a non-periodic structure, is obtained at each geometrical point as a homogenized tensor of a periodic problem. Similarly, for the osteonal structure, the components of the homogenized tensor are determined at each point as the result of a periodic homogenization.

The software OSTEON, which is the computational method associated with this model, allows one to obtain a better understanding of the effects of many bony parameters. The obtained results are in accordance with experimental data.     

Possible involvement of inhibin in altered follicle-stimulating hormone (FSH) secretion during dissociated luteinizing hormone (LH) and FSH release: unilateral castration and experimental cryptorchidism

Male rats were either unilaterally or bilaterally castrated, or were rendered cryptorchid when they were either 15 or 45 days old. Subsequently, blood was sampled over the next several weeks and plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and immunoreactive inhibin-alpha (irI alpha) levels were measured by specific radioimmunoassays (RIAs). At the end of the experiment, gonadal expression of inhibin-alpha, inhibin-beta A, and inhibin-beta B subunits was measured by S1 nuclease analysis and in situ hybridization. In both age groups, bilateral castration (BC) produced the expected marked (p less than or equal to 0.01) increases in plasma LH and FSH levels, and concomitant decreases in T and irI alpha secretion within 1 - 2 days after surgery. In 15-day-old animals, unilateral castration (UC) significantly increased FSH and decreased circulating levels of irI alpha, but did not measurably alter LH or androgen production. At 7 days after surgery, the level of inhibin mRNA in the remaining testis was unchanged. In 45-day-old animals, UC caused a measurable increase in FSH, with little or no changes in the circulating levels of irI alpha. Plasma T levels were lowered (p less than or equal to 0.05) by UC; however, there were no statistical changes in LH levels in these UC rats. Finally, T administration markedly reversed UC-induced increase in FSH secretion in both age groups. Androgen therapy also interfered with inhibin release in 45-day-old, but not in 15-day-old rats. In rats 15 days old at the time of surgery, cryptorchidism produced a small but measurable increase (p less than or equal to 0.05) in LH release at Week 6 only, which was accompanied by a significant (p less than or equal to 0.01) decline in T secretion. Plasma FSH levels were elevated at all times in cryptorchid rats, and at 2, 4, and 6 wk, these levels were not statistically distinguishable (p greater than 0.05) from those of castrated animals. In this group of rats, cryptorchidism caused a transient increase (p less than or equal to 0.05) in irI alpha values 1 wk after surgery, but no changes at later times. Finally, measurement of testicular inhibin-alpha subunit messenger RNA (mRNA) levels showed an approximately 2-fold increase compared to total RNA levels in the testis. However, because of the significant decrease in total RNA levels per testis caused by cryptorchidism, the absolute change in inhibin-alpha subunit mRNA levels per testis corresponded to an approximately 3-fold decrease.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cleavage of double-stranded DNA by manganese tris(methylpyridiniumyl)porphyrin linked to 3′-spermine oligonucleotides

 Cleavage of double-stranded DNA was performed with cationic manganese porphyrin complexes linked via a spermine tether to the 3′- or 5′-side of triple-helix-forming oligonucleotides (cleaver-TFO conjugates). The targeted sequence was a 15-polypurine sequence present in the env gene of HIV-1 (positions 7301–7315). The presently used TFOs contain only thymine and 5-methylcytosine residues and one adenine at the 3′-end in order to be able to easily introduce a 3′-polyamine linker by reductive amination of the corresponding 3′-apurinic polypyrimidine oligonucleotides. With this method we prepared these TFO-cleaver conjugates in 45% yield with only two equivalents of the Mn-TrisMPyP-COOH precursor. These new metalloporphyrin-TFO conjugates were able to cleave a complementary 45-mer duplex at 10 nM concentration with only ten equivalents of TFO-cleaver. Conjugates without spermine, without 5-methylcytosine, with a random sequence or with the managanese porphyrin-spermine entity on the 5′-end of TFOs were synthesized for comparative studies. Received: 6 December 1995 / Accepted: 5 February 1996     

In vitro influence of zinc and magnesium on the deformability of red blood cells artificially hardened by heating

Trace elements have been shown to improve red blood cell (RBC) deformability: zinc in sickle cell disease and magnesium in an in vitro model of chemically rigidified erythrocytes. In this study, we investigated the effect and the influence of incubation time of zinc or magnesium on an in vitro model of rigidified RBCs by heating. Erythrocyte rigidity was determined by viscosimetry at high shear rate by a falling ball viscosimeter MT 90. In the first part of the study, six normal volunteers participated. Viscosimetry was performed on native blood before and after heating the sample for 10 min at 50°C. Therefore, increasing concentrations of zinc gluconate (final concentration: 0.5–4 g/L) or isotonic NaCl as control medium were added to the sample. Heating induced a twofold increase in all indices of RBC rigidity (p<0.05). At all these concentrations of zinc, a highly significant, dose-related fluidifying effect was observed (40–70%): this effect was immediately obtained and did not change over 60 min. Even at the highest concentration, recovery was not complete. In the second part of the study, we studied magnesium’s effects on blood. In a first protocol, whole blood was rigidified by heating at 56°C for 10 min, and the correcting effect of 5 min of incubation at 37°C of RBCs in 150 mmol/L NaCl, MgSO₄, magnesium acetate, and magnesium gluconate was investigated. In a second protocol, the same incubation with NaCl and magnesium salts was made on blood that had not been previously heated. In a third protocol, the correcting effect of magnesium gluconate on heated red blood cells was tested at four concentrations (75, 150, 225, and 300 mmol/L) over 1 h, for evaluating the effects of both concentration and time. Erythrocyte rigidity by heating is corrected by the three salts employed in protocol 1 (compared to sodium). In protocol 2, the deformability of normal (nonheated) red cells is not modified by magnesium. In protocol 3, no marked modification over 1 h is observed. The correcting effect is not complete for 75 mmol/L Mg, but remains the same at the three other concentrations. This study shows that zinc and magnesium at supraphysiological concentration are able to reverse RBC’s rigidification induced by heating, but that magnesium does not modify the flexibility of normal RBCs. This article suggests that zinc and magnesium may be studied in vivo as potential pharmacologic tools for improving hemorheologic disturbances.     

Effects of oral zinc gluconate on glucose effectiveness and insulin sensitivity in humans

Zinc improves both insulin secretion and insulin sensitivity, and exerts insulin-like effects. We investigated its acute effects on the parameters of glucose assimilation determined with the minimal model technique from frequent sampling intravenous glucose tolerance test (FSIVGTT) in seven healthy volunteers. FSIVGTTs (0.5 g/kg of glucose, followed by 2 U insulin iv injection at 19 min) were performed after the subjects had taken 20 mg zinc gluconate twice (the evening before and 30 min before the beginning of the test) or placebo pills (simple blind randomized protocol). Glucose assimilation was analyzed by calculating Kg (slope of the exponential decrease in glycemia), glucose effectiveness Sg (i.e., ability of glucose itself to increase its own disposal independent of insulin response), and SI (insulin sensitivity, i.e. the effect of increases in insulinemia on glucose disposal). The two latter parameters were calculated by fitting the experimental data with the two equations of Bergman’s “minimal model”. Zinc increased Kg (p<0.05) and Sg (p<0.05), whereas SI and insulin first-phase secretion did not significantly increase. This study suggests that zinc improves glucose assimilation, as evidenced by the increase in Kg, and that this improvement results mainly from an increase in glucose effectiveness (insulin-like effect), rather than an action on insulin response or insulin sensitivity.     

Cleavage of double-stranded DNA by 'metalloporphyrin-linker-oligonucleotide' molecules: influence of the linker.

Manganese porphyrin-linker-triple-helix-forming oligonucleotide molecules were prepared and their ability to cleave in vitro a double-stranded DNA target present in the HIV-1 genome was studied. The nature of the linker is a determining factor of the cleavage efficiency. Cleavage yields as high as 80% were observed when the linker was a spermine residue and in the absence of a large excess of free spermine known to stabilize triplex structures. The hydrophobic nature of aliphatic diamine linker modified the cleaver-DNA interactions and reduced the efficiency of DNA cleavage.