Localization and secretion of inhibins in the equine fetal ovaries

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.     

Three new ursene carboxylic acids from Uncaria thwaitesii

Three new ursene carboxylic acids, uncaric acid, diketouncaric acid and diacetyluncaric acid were isolated from the woody part of Uncaria thwaitesii, interrelated and their structures established.     

Microbial metabolism. Part 13. Metabolites of hesperetin

The fungal culture, Mucor ramannianus (ATCC 2628) transformed hesperitin (1) to four metabolites: 4'-methoxy-5,7,8,3'-tetrahydroxyflavanone (8-hydroxyhesperetin) (2), 5,7,3',4'-tetrahydroxyflavanone (eriodictyol) (3), 4'-methoxy-5,3'-dihydroxyflavanone 7-sulfate (hesperetin 7-sulfate) (4) and 5,7,3'-trihydroxyflavanone 4'-O-α-quinovopyranoside (eriodictyol 4'-O-α-quinovopyranoside) (5). The structures were established by spectroscopic methods.     

Experimental evidence for sequestering C with biochar by avoidance of CO2 emissions from original feedstock and protection of native soil organic matter

There is a need for further studies to compare the decomposition of biochar to that of the original feedstock and determine how these amendments affect the cycling of native organic matter (NOM) of different soils to improve our understanding of the resulting net C sequestration potential. A 510‐days incubation experiment was conducted (i) to investigate the evolution of CO₂ from soils amended with either fresh corn stover (CS) or with biochars produced from fresh CS at either 350 (CS‐350) or 550 °C (CS‐550), and (ii) to evaluate the priming effect of these amendments on NOM decomposition. Two soil types were studied: an Alfisol and an Andisol, with organic C contents of 4% and 10%, respectively. Except for the controls (with no C addition), all treatments received 7.18 t C ha^?1. We measured C efflux in short‐term intervals and its isotopic signature to distinguish between C evolved from C₄ amendments and C₃‐dominated NOM. Emission rates were then integrated for the whole time period to cover total emissions. Total CO₂‐C evolved from the original C in fresh CS, CS‐350 and CS‐550 was greater in the Andisol (78%, 13% and 14%) than in the Alfisol (66%, 8% and 7%). For both soils, (i) no significant differences (P > 0.05) were observed in the rate of CO₂ evolution between controls and biochar treatments; and (ii) total accumulated CO₂ evolved from the uncharred amendment was significantly higher (P < 0.05) than that from the other treatments. In the Alfisol, a significant (P < 0.05) net positive priming effect on NOM decomposition was observed when amended with fresh CS, while the opposite was detected in biochar treatments. In the Andisol, no significant (P > 0.05) net priming effect was observed. A C balance indicated that the C lost from both biochar production and decomposition ‘broke even’ with that lost from fresh residue decomposition after <35 weeks. The ‘break‐even’ point was reached earlier in the Andisol, in which the fresh CS mineralizes faster. These results provided experimental evidence for the potential of biochar to sequester C and avoid CO₂ emissions from original feedstock while protecting native soil organic matter.     

Laserspray Ionization, a New Atmospheric Pressure MALDI Method for Producing Highly Charged Gas-phase Ions of Peptides and Proteins Directly from Solid Solutions

The first example of a matrix-assisted laser desorption/ionization (MALDI) process producing multiply charged mass spectra nearly identical to those observed with electrospray ionization (ESI) is presented. MALDI is noted for its ability to produce singly charged ions, but in the experiments described here multiply charged ions are produced by laser ablation of analyte incorporated into a common MALDI matrix, 2,5-dihydroxybenzoic acid, using standard solvent-based sample preparation protocols. Laser ablation is known to produce matrix clusters in MALDI provided a threshold energy is achieved. We propose that these clusters (liquid droplets) are highly charged, and under conditions that produce sufficient matrix evaporation, ions are field-evaporated from the droplets similarly to ESI. Because of the multiple charging, advanced mass spectrometers with limited mass-to-charge range can be used for protein characterization. Thus, using an Orbitrap mass spectrometer, low femtomole quantities of proteins produce full-range mass spectra at 100,000 mass resolution with <5-ppm mass accuracy and with 1-s acquisition. Furthermore, the first example of protein fragmentation using electron transfer dissociation with MALDI is presented.Two primary differences between ESI and MALDI methods are the sample environment (solution versus solid) and the observable charge state(s) (multiply versus singly charged). The multiply charged ions observed in ESI mass spectrometry (MS) enhance the yields of fragment ions, a key benefit in structure characterization, and allow analysis of high molecular weight compounds on mass spectrometers with a limited mass-to-charge (m/z) range. In contrast, MALDI MS is ideal for the analysis of heterogeneous samples because it often requires less sample, and spectra of singly charged ions are easier to interpret. We report here the astonishing observation of highly charged molecular ions by laser ablation of a solid matrix/analyte mixture typically used in MALDI MS analyses. The distribution and abundances of the observed ions are similar to those obtained by ESI. Importantly, the MALDI mechanism that produces singly charged ions can be “turned on” at the operator''s will by changing only the matrix or matrix preparation conditions; this capability is not available with any other ionization method. These findings show for the first time that singly charged ions as well as multiply charged ions are available in MALDI. Besides having important mechanistic implications relating to MALDI and ESI, our findings have enormous practical analytical utility.ESI and MALDI combined with MS revolutionized the study of biological materials and earned the Nobel Prize in Chemistry for their ability to ionize proteins for analysis using MS. However, after two decades of extensive studies, the mechanism for ion formation in MALDI remains controversial (1–8). At the heart of these debates lies the predominance of singly charged ions in MALDI mass spectra; the exception being very high mass compounds. A mechanism for the formation of multiply charged ions in MALDI has previously been proposed (1) based on molecular modeling studies (9, 10) and glimpses of multiply charged ions have been observed in lower molecular weight compounds (11–14). The formation of these multiply charged ions has been attributed to sample preparation, high laser fluence, a metal-free sample stage, use of an IR laser, and atmospheric pressure (AP)1 conditions. Multiply charged ions were also recently observed by laser ablation of a liquid surface in the presence of a high electric field (15). The inability in that experiment to observe ions from a solid MALDI matrix/analyte sample or in the absence of an electric field suggests an ionization process involving liquid droplets in a high field similar to ESI (16) or other liquid based, field-induced ionization methods (17, 18).Here, we show analytically useful ESI-like MALDI mass spectra obtained using standard MALDI conditions but using a nontraditional source (19) mounted in place of the standard atmospheric pressure ionization source on a mass spectrometer most commonly used with ESI. The utility of this MALDI MS method for extending the mass range of mass spectrometers as well as the capability of peptide/protein sequencing using electron transfer dissociation (ETD) (20) is demonstrated. Because highly charged ions have not previously been observed with any MALDI ion source configuration, we briefly discuss the fundamental concepts that lead to their production. Key aspects of laserspray ionization (LSI) are laser ablation using a UV laser aligned in transmission geometry (TG) (21–23), field-free (FF) at AP (24), using a heated AP to vacuum ion transfer capillary. In order to emphasize the MALDI sample preparation but distinguish laserspray from conventional AP-MALDI, the new ionization method will hereafter be referred to as FF-TG AP-MALDI.     

Exposure of neonatal female rats to p-tert-octylphenol disrupts afternoon surges of luteinizing hormone, follicle-stimulating hormone and prolactin secretion, and interferes with sexual receptive behavior in adulthood

The present study investigated the effects of exposure of neonatal female rats to p-tert-octylphenol (OP) on estrogen-induced afternoon surges of LH, FSH, and prolactin (PRL) secretion, and on sexual behavior in adulthood. After birth, one group of female Wistar rat pups received s.c. injections of OP (100 mg/kg body weight [BW]; OP group) dissolved in DMSO, while the control group received DMSO only (DMSO group). In order to make a qualitative comparison, a third group was injected with estradiol-17beta (500 microg/kg BW; estradiol group) dissolved in DMSO. Injections were given on Days 1, 3, 5, 7, 9, 11, 13, and 15 of age. The rats from the OP and estradiol groups that were used for subsequent experiments were in persistent vaginal estrus. Spontaneous LH surge measured at Postnatal Days (PND) 78-81 was observed only in the DMSO group on the afternoon of the day of proestrus. At PND 115, randomly selected rats from each of three treatment groups were bilaterally ovariectomized (ovx), and 8 days later, Silastic capsules containing estradiol-17beta were implanted under the skin. Estrogen implants stimulated afternoon surges of LH, FSH, and PRL for two consecutive days in the DMSO group, but not in the OP and estradiol groups. Rats from the OP and DMSO groups underwent ovx at PND 186, and 6 days later they were treated with a combination of estradiol benzoate s.c. (15 microg/kg BW) and progesterone s.c. (2 mg/kg BW) to test the lordosis reflex. In response to this hormone treatment and mounting stimulus delivered by the stud male rats, the OP-treated rats were less receptive compared with control DMSO-treated rats, and thus the lordosis quotient and lordosis rating were significantly (P < 0.05) reduced in the OP group compared with the DMSO group. Analysis of the area of the sexually dimorphic nucleus of the preoptic area of the brain revealed that the area of this nucleus was larger in the OP group than it was in control DMSO rats. We conclude that the exposure of neonatal female rats to higher doses of OP disrupts the cyclic release of LH, FSH, and PRL, and interferes with the display of sexual receptive behavior in adulthood.     

Evidence for a secretory pathway Ca2+-ATPase in sea urchin spermatozoa

Plasma membrane, sarco-endoplasmic reticulum and secretory pathway Ca2+-ATPases (designated PMCA, SERCA and SPCA) regulate intracellular Ca2+ in animal cells. The presence of PMCA, and the absence of SERCA, in sea urchin sperm is known. By using inhibitors of Ca2+-ATPases, we now show the presence of SPCA and Ca2+ store in sea urchin sperm, which refills by SPCA-type pumps. Immunofluorescence shows SPCA localizes to the mitochondrion. Ca2+ measurements reveal that approximately 75% of Ca2+ extrusion is by Ca2+ ATPases and 25% by Na+ dependent Ca2+ exchanger/s. Bisphenol, a Ca2+ ATPase inhibitor, completely blocks the acrosome reaction, indicating the importance of Ca2+-ATPases in fertilization.     

Regulation of follicle-stimulating hormone secretion by estradiol and dimeric inhibins in the infantile female rat.

Plasma and ovarian levels of the dimeric forms of inhibin and plasma estradiol-17beta were investigated and compared with changes in plasma gonadotropins from Postnatal Day (PND) 5 to PND 30 in the female rat. The inhibin subunit proteins were localized in follicular granulosa cells of the ovary. Plasma immunoreactive inhibin levels were low until PND 15 and increased thereafter. Plasma levels of inhibin B (alpha and beta(B) subunits) remained very low until PND 15 and then increased by approximately 24-fold. In contrast, plasma levels of inhibin A (alpha and beta(A) subunits) were relatively low and steady until PND 20, then increased by approximately 3-fold at PND 25. Changes in ovarian inhibin A and B levels closely resembled those in plasma levels. Plasma FSH levels were low at PND 10 but started to peak from PND 15 and remained high until PND 20, followed by a remarkable reduction at PNDs 25 and 30. This dramatic fall in FSH coincided with the rise of inhibin A. A significant inverse correlation was observed between plasma FSH and plasma inhibin A (r = -0.67, P < 0.0002), ovarian inhibin A (r = -0.48, P < 0.01), plasma inhibin B (r = -0.48, P < 0.05), and ovarian inhibin B (r = -0.54, P < 0.01). Plasma estradiol-17beta levels were elevated from PND 5 through PND 15, then fell sharply through PND 30. Plasma estradiol-17beta was significantly and positively (r = 0.75, P < 0.0002) correlated with plasma FSH. Plasma LH rose to higher levels at PND 15 and tended to be lower thereafter. The inhibin alpha, beta(A), and beta(B) subunits were localized to primary, secondary, and antral and large antral follicles, but the types of these immunopositive follicles varied with age. It appeared that, at PND 25 and afterward, all three subunits were mainly confined to large antral follicles in the ovary. We conclude that estradiol-17beta likely is the major candidate in stimulation of FSH secretion in the infantile female rat. We also conclude that inhibin regulation of pituitary FSH secretion through its negative feedback in the infantile female rat begins to operate after PND 20. We suggest that this negative feedback is achieved by increases in plasma levels of the two dimeric forms, and that inhibin A appears to be the major physiological regulator of FSH secretion at the initiation of this mechanism. We also conclude that large antral follicles in the ovary are the primary source of these bioactive inhibins that are secreted in large amounts into the circulation after PND 20.     

Synthesis and solution conformation studies of 3-substituted uridine and pseudouridine derivatives

A series of 3-substituted uridine and pseudouridine derivatives, based on the naturally occurring 3-(3-amino-3-carboxypropyl) modification, were synthesized. Their aqueous solution conformations were determined by using circular dichroism and NMR spectroscopy. Functional group composition and chain length were shown to have only a subtle influence on the distribution of syn/anti conformations of the modified nucleosides. The dominating factor appears to be the glycosidic linkage (C- vs. N-glycoside) in determining the nucleoside conformation.