Leishmania donovani: Oral efficacy and toxicity of formycin B in the infected hamster

Formycin B, a structural analog of inosine, was evaluated as an orally administrable antileishmanial agent. Against Leishmania donovani in hamsters, it achieved an 85–92% reduction in numbers of parasites in livers of infected animals after oral administration at 13 mg/kg/day for 4 days. Its efficacy by oral administration was approximately four to eight times that by intramuscular administration and four times that of the positive control drug Glucantime by intramuscular administration. The levels of formycin B in serum after the final oral administration of 26 mg/kg/day were 1.4 μg/ml at 1 hr and 0.3 μg/ml at 2 hr. The concentration in liver was greater (9.0 μg/ml at 1 hr) and declined more slowly. With this latter dosage or with 104 mg/kg/day there was no acute toxicity of formycin B to bone marrow or formed elements of the blood. The only statistically significant toxicity to the liver was a doubling of serum total bilirubin levels. Comparison of the in vivo efficacy of formycin B against L. donovani to the mild acute toxicity of the drug suggests that formycin B has potential as an oral agent against visceral leishmaniasis.     

Phosphates of riboflavin and riboflavin analogs: A reinvestigation by high-performance liquid chromatography

Phosphoric acid esters of riboflavin can be easily separated by reverse-phase high-performance liquid chromatography using eluants of 0.1 M ammonium formate in aqueous methanol. Commercial FMN preparations contained seven different flavin phosphates; the content of riboflavin 5'-phosphate was 70-75% and is in agreement with previous studies. Millimole amounts of crude FMN can be processed by preparative HPLC. The method permits the preparation of greater than 99%-pure 5'-FMN. The following compounds were isolated in pure form and their structures determined: riboflavin 4'-phosphate, riboflavin 3'-phosphate, riboflavin 4',5'-diphosphate; riboflavin 3',4'-diphosphate, and riboflavin 3',5'-diphosphate. The latter compound binds tightly to apoflavodoxin from Megasphaera elsdenii (KD = 9.7 X 10(-9) M). The bound flavin has high catalytic activity, thus representing a novel type of FMN analog. A wide variety of structural analogs of FMN can be obtained in pure form by preparative HPLC.     

Thyrotropin releasing hormone: autonomic effects upon cardiorespiratory function in endotoxic shock

These studies define potential sites and mechanisms by which thyrotropin releasing hormone (TRH) stimulates cardiorespiratory function in normotensive rats as well as in rats subjected to endotoxic shock. Changes in mean arterial pressure, pulse pressure, heart rate, and respiratory rate were determined in conscious animals following injection of TRH into the lateral, third, or fourth ventricular spaces. Injections of TRH into the third ventricular space resulted in a greater increase in cardiorespiratory variables than did fourth ventricular injection. In endotoxin-treated rats, the cardiorespiratory effects of intracerebroventricular (icv) TRH and its analog MK 771 were assessed. TRH and MK 771 were shown to act within the brain to reverse endotoxic shock hypotension; at the doses used, the pressor effects of these two tripeptides were achieved through selectively different actions upon heart rate and pulse pressure. Adrenal demedullated and sham-operated control rats subjected to endotoxic shock were injected with icv and intravenous (iv) TRH in order to evaluate the potential involvement of sympatho-medullary function in cardiorespiratory responses. The cardiovascular effects of icv TRH were dependent upon adrenal medullary integrity; effects of iv TRH were not. Doses of iv TRH which effectively reverse shock neither altered nociceptive latencies nor interfered with analgesic responses to morphine. Collectively, these studies reinforce the potential therapeutic utility of TRH and its analogs in the treatment of shock and indicate potential sites and mechanisms which mediate these salutary effects.     

Analogs of thyrotropin-releasing hormone (TRH): Receptor affinities in brains,spinal cords,and pituitaries of different species

[³H](3-Me-His²) thyrotropin-releasing hormone ([³H]MeTRH) bound to TRH receptors in rodent, rabbit and dog brain and spinal cord (SC), and in rat, sheep, bovine and dog anterior pituitary (PIT) glands, with high affinity (dissociation constants, K_ds=5–9 nM; n=3–4) but to different densities of these sites (B _max range 6–145 fmol/mg protein) (rabbit SC>sheep PITG.pig brain>dog brain>rat brain>bovine and dog PIT). Various TRH analogs competitively inhibited [³H]MeTRH binding in these tissues with a similar rank order of potency: MeTRH>TRH> CG3703RX77368MK-771>TRH Glycinamide>Glu¹-TRHCG3509NVal²-TRH>>>TRH free acid>>>and cyclo-His-Pro, indicating a pharmacological similarity of CNS and pituitary TRH receptors. While most TRH analogs displaced [³H]MeTRH binding with a similar potency in the different species, TRH exhibited a 2-fold lower affinity in the rat and G.pig brain than in other tissues of other species. Similarly, CG3703 was 2.4–4.5 times more active in the rabbit brain than in the rodent and dog brain, and also more potent in the rabbit brain as compared to the sheep PIT. However, MK-771 and RX77368 had a similar affinity for the brain TRH receptors in the different species but RX77368 was 2-fold more active in the SC preparations and 3–4-fold less active in the sheep PIT when compared to the brain homogenates. RX 77368 exhibited the highest affinity for the dog PIT TRH receptor. In contrast, MK-771 showed a similar affinity for the brain, SC and PIT TRH receptor apart from in the rat PIT where it had the highest affinity. Similarly, TRH glycinamide was more active in the dog brain than rodent and rabbit brain. These data suggest that while the rank order of potency of TRH analogs is similar in the species examined, certain analogs appear to be more potent in certain tissues of some species than in others. In addition, the current results have shown that CG3703 is almost equipotent with RX77368 and MK-771 in most species but is substantially more active than its related analog, CG3509 in the brain, SC and PIT. Taken together, these observations may have some relevance to the future clinical applications of these metabolically stabilized TRH analogs.     

大鼠胃泌酸腺细胞膜和肝匀浆对五肽胃泌素及其类似物的降解

 本文用HPLC分离五肽胃泌素(Pentagastrin,Boc-β-Ala-Trp-Met-Asp-Phe·NH_2,PG_(1-5))及其N-端无保护基的TFA·五肽(β-Ala-Trp-Met-Asp-Pne·NH_2·TFA,TFA·PA_(1-5))被胃泌酸细胞膜和肝匀浆降解之产物,并对它们的氨基酸组成作了分析,又用薄层层析作了进一步验证,从而提出了酶促降解时可能的酶切位点。实验结果表明,N-端的Boc-基团能大幅度增加对酶降解的抵抗力,这从一个方面解释了为什么商品五肽胃泌素的泌酸活性大于其它胃泌素类似物的原因。     

Kinetin-induced stimulation of electrogenic pumping in soybean suspension cultures is unrelated to signal transduction

Primary modes of action of cytokinins have been thought to involve stimulation of the electrogenic H⁺ pump and-or opening of plasmamembrane Ca²⁺ channels. In order to test these hypotheses, rapid changes in membrane transport in response to cytokinin application were studied in heterotrophic suspension-cultured callus of soybean (Glycine max (L.) Merr.) using electrophysiological techniques. Kinetin (N⁶-furfurylaminopurine; 2 M) elicited membrane hyperpolarization of 13±1 mV. This effect occurred even at membrane poteintials more negative than the most negative ionic equilibrium potential, and therefore might have resulted either from stimulation of the electrogenic pump, or from closure of ionic channels. The former mechanism of action appears most likely because (i) kinetin-induced membrane hyperpolarization is not accompanied by a significant change in plasma-membrane resistivity and (ii) hyperpolarization is abolished by cyanide, which inhibits electrogenic pump activity by depletion of cellular ATP.Electrogenic pumping is also activated by two other cytokinins: N⁶-(benzyl)adenine and trans-zeatin. However, it is unlikely that the hormonal effect on electrogenesis is directly related to transduction of the cytokinin signal, for the following reasons: (i) hormonally inactive, but chemically related compounds (cis-zeatin, adenine) also elicited membrane hyperpolarization; (ii) hormonally active, N⁹-substituted cytokinins failed to stimulate electrogenesis; (iii) the chemically unrelated cytokinin N,N-diphenylurea also failed to stimulate electrogenesis.The results imply that the kinetin effect on electrogenic pumping is related to adenine, or its metabolism, and not hormonal action. Adenine was absorbed by soybean cells, but not in sufficient quantities to have a significant effect on adeninenucleotide pools. It appears likely that the control of electrogenesis requires either the presence of a purine free base (i.e. no substituents at the N⁹ position) or phosphoribosylation of the free base. No evidence was found for cytokinin-induced Ca²⁺-channel opening, though it is argued that such an event might be physiologically relevant, yet undetectable with the methods employed. It is essential that future studies on cytokinin signal transduction — especially as they relate to membrane transport — take into account the possibility that metabolic effects unrelated to hormone action are dominant.Abbreviations and symbols bzl⁶Ade N⁶ (benzyl)adenine - SRB Soybean Recording Buffer - V_m membrane potential     

Characteristics of [D-Trp]-somatostatin-sensitive B50 phosphorylation

Inhibition of the phosphorylation of the synaptic plasma membrane (SPM) protein B50 by [D-Trp⁸]-somatostatin in vitro is time-dependent. Increasing the time of incubation of hippocampal synaptic plasma membranes with the peptide from 15 sec to 30 min prior to addition of 7.5 μM [γ-³²Ps]ATP results in a complete reduction of B50 phosphorylation. Incubation of synaptic plasma membranes for 30 min in the absence of peptide does not alter basal B50 phosphorylation. Neither ACTH nor β-endorphin produces similar effects—inhibition of B50 phosphorylation by ACTH is maximal at 15 sec and β-endorphin produces only a small inhibition, even after 30 min. [D-Trp⁸]-somatostatin is not activating a membrane-bound protease, since maximal inhibition of B50 phosphorylation by the peptide is seen in the presence of leupeptin or bacitracin. Hippocampal synaptic plasma membranes contain protein phosphatase activity. Assays of B50 phosphorylation in synaptic plasma membranes done under conditions that favor either net phosphorylation or dephosphorylation are consistent with inhibition of protein phosphatase activity by [D-Trp⁸]-somatostatin. This evidence suggests that [D-Trp⁸]-somatostatin interacts with SPM binding sites in the hippocampus, which may alter the activity of an endogenous protein phosphatase to determine the degree of B50 phosphorylation.     

Effect of Nucleotides on the Orientation and Mobility of Myosin Subfragment-1 in Ghost Muscle Fiber

Using polarization fluorimetry, the orientation and mobility of 1,5-IAEDANS specifically bound to Cys707 of myosin subfragment-1 (S1) were studied in ghost muscle tropomyosin-containing fibers in the absence and in the presence of MgADP, MgAMP-PNP, MgATPgammaS, or MgATP. Modeling of various intermediate states was accompanied by discrete changes in actomyosin orientation and mobility of fluorescent dye dipoles. This suggests multistep changes in the structural state of the myosin head during the ATPase cycle. Maximal differences in the probe orientation by 4 degrees and its mobility by 30% were found between actomyosin states in the presence of MgADP and MgATP. It is suggested that interaction of S1 with F-actin induces nucleotide-dependent rotation of the whole motor domain of the myosin head or only the dye-binding site and also change in the head mobility.     

Helical order in tarantula thick filaments requires the "closed" conformation of the myosin head

Myosin heads are helically ordered on the thick filament surface in relaxed muscle. In mammalian and avian filaments this helical arrangement is dependent on temperature and it has been suggested that helical order is related to ATP hydrolysis by the heads. To test this hypothesis, we have used electron microscopy and image analysis to study the ability and temperature dependence of analogs of ATP and ADP.Pi to induce helical order in tarantula thick filaments. ATP or analogs were added to rigor myofibrils or purified thick filaments at 22 degrees C and 4 degrees C and the samples negatively stained. The ADP.Pi analogs ADP.AlF4 and ADP.Vi, and the ATP analogs ADP.BeFx, AMPPNP and ATPgammaNH2, all induced helical order in tarantula thick filaments, independent of temperature. In the absence of nucleotide, or in the presence of ADP or the ATP analog, ATPgammaS, there was no helical ordering. According to crystallographic and tryptophan fluorescence studies, all of these analogs, except ATPgammaS and ADP, induce the "closed" conformation of the myosin head (in which the gamma phosphate pocket is closed). We suggest that helical order requires the closed conformation of the myosin head but is not dependent on the hydrolysis of ATP.     

Determination of endogenous and supplied deuterated abscisic acid in plant tissues by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry with multiple reaction monitoring

A specific, sensitive, and accurate method for determination of abscisic acid (ABA) in plant tissues is described. The method employs reversed-phase high-performance liquid chromatography and electrospray ionization-tandem mass spectrometry for multiple reaction monitoring of underivatized ABA and deuterated ABA analogs. Specific analogs were used to study the mechanism of ABA fragmentation, to select appropriate standards, and to identify compounds suitable for metabolic studies involving the supply of differentially labeled ABA. Limits of detection and quantification of 1.9 and 4.7 pg, respectively, were obtained over a linear calibration range of 0-1.5 ng ABA (on-column injected) using 5.8', 8', 8'-d(4) ABA as the internal standard. Accuracy and precision were within 15% for routine quality control samples. The method of standard additions, as applied to Arabidopsis thaliana seed extracts, was also used to validate the method for analysis of plant tissue samples. The utility of the method was further demonstrated by determining levels of ABA in western white pine seeds and of ABA and supplied 8', 8', 8', 9', 9', 9'-d(6) ABA in Brassica napus tissues, using 5.8', 8', 8'-d(4) ABA or 8', 8', 8'-d(3) ABA as the internal standard. Limits of quantification as low as 0.89 ng/g were achieved by optimizing the extraction procedure for each type of plant tissue.