Feedback effects of ovarian steroids on the hypothalamic-hypophyseal axis in the rabbit

The feedback effects of two ovarian steroids, estradiol-17 beta (E2) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha OH), were examined in both intact (INT) and ovariectomized (OVEX) does. We measured steroid-induced alterations in endogenous gonadotropin-releasing hormone (GnRH) from sequential 10-min samples of hypothalamic perfusates, simultaneous changes in peripheral plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the modification of pituitary responsiveness, i.e., increments in plasma LH (delta LH) and plasma FSH (delta FSH), after 50 ng, 250 ng, and 1 microgram of exogenous GnRH in individual does of 6 treatment groups. The groups were: INT does, OVEX does, OVEX does receiving either one (1 E2) or two (2 E2) E2-filled Silastic capsules, OVEX does receiving a 20 alpha OH-filled capsule (20 alpha OH), and OVEX does receiving both capsules of E2 and 20 alpha OH (1 E2 + 20 alpha OH). Ovariectomy enhanced the pulsatile release of hypothalamic GnRH and pituitary LH and FSH, and increased the LH response (delta LH) to exogenous GnRH (OVEX vs. INT, p less than 0.05). Replacement of E2 at the time of ovariectomy prevented the increased GnRH and gonadotropin secretion as well as the enhanced delta LH that were observed in untreated OVEX does. The release of hypothalamic GnRH in the 20 alpha OH group was lower (p less than 0.05) than that in the OVEX group and not different from that in the INT group. The release of pituitary LH and FSH and the delta LH in the 20 alpha OH group was not different from that in the OVEX group, but these parameters were greater (p less than 0.05) than those in the INT group. The hypothalamic GnRH pulse frequency in the 1 E2 + 20 alpha OH group was lower (p less than 0.05) than that in either the 1 E2 or the 20 alpha OH group, but the delta LH in the 1 E2 + 20 alpha OH group was not different from that in either the 1 E2 or the 20 alpha OH group. The highest dose (1 microgram) of exogenous GnRH stimulated a modest increase in FSH in the OVEX, 20 alpha OH, 1 E2 + 20 alpha OH, and 1 E2 groups; but a steroid effect on delta FSH among these 4 groups was not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changing frequency of pulsatile luteinizing hormone and progesterone secretion during the luteal phase of the menstrual cycle of rhesus monkeys

Experiments were conducted to examine the pulsatile nature of biologically active luteinizing hormone (LH) and progesterone secretion during the luteal phase of the menstrual cycle in rhesus monkeys. As the luteal phase progressed, the pulse frequency of LH release decreased dramatically from a high of one pulse every 90 min during the early luteal phase to a low of one pulse every 7-8 h during the late luteal phase. As the pulse frequency decreased, there was a corresponding increase in pulse amplitude. During the early luteal phase, progesterone secretion was not episodic and there were increments in LH that were not associated with elevations in progesterone. However, during the mid-late luteal phase, progesterone was secreted in a pulsatile fashion. During the midluteal phase (Days 6-7 post-LH surge), 67% of the LH pulses were associated with progesterone pulses, and by the late luteal phase (Days 10-11 post-LH surge), every LH pulse was accompanied by a dramatic and sustained release of progesterone. During the late luteal phase, when the LH profile was characterized by low-frequency, high-amplitude pulses, progesterone levels often rose from less than 1 ng/ml to greater than 9 ng/ml and returned to baseline within a 3-h period. Thus, a single daily progesterone determination is unlikely to be an accurate indicator of luteal function. These results suggest that the changing pattern of mean LH concentrations during the luteal phase occurs as a result of changes in frequency and amplitude of LH release. These changes in the pulsatile pattern of LH secretion appear to have profound effects on secretion of progesterone by the corpus luteum, especially during the mid-late luteal phase when the patterns of LH concentrations are correlated with those of progesterone.     

A simple approach to improve the sensitivity of enzyme-linked immunosorbent assay: Using the IMAPlate 5RC96 for result readout

In this article, we describe a new enzyme-linked immunosorbent assay (ELISA) setup to improve the sensitivity of commercial or homemade ELISAs. In the new ELISA setup, an IMAPlate 5RC96, a disposable multi-utility lab device developed by NCL New Concept Lab is used as a self-uptaking microcuvette array to read out the result of the ELISA that is performed in the normal 96-well plate with reduced substrate solution and stop solution. A commercial interleukin-6 (IL-6) ELISA reagent kit was used for the evaluation. Compared with the conventional ELISA setup, the new ELISA setup could easily increase the absorbance values by up to more than 10-fold. Therefore, the sensitivity (change in absorbance/change in concentration [ΔAbs/ΔConc]) is increased accordingly. In addition, methods to extend the upper detection limit of plate readers for the IMAPlate 5RC96 are described. This new ELISA setup may be more notable for the approach employed than for the specific analyte. It should generally be applicable to any conventional ELISA and should serve as an example of a simple solution that increases the detection sensitivity and/or detection range of other assays as well. We expect the approach to have a substantial practical impact on analytical methods and to accelerate discovery, research, and application of analytes at low concentration in life sciences and diagnostics.     

Preparation of Agarose with Low Net Negative Charge Density Using an Inexpensive Anion Exchanger

A method of preparing agarose of low net negative charge density by the use of Chromium tanned gelatin is described. Using commercial agarose (agarose c) as standard of comparison the product has a sulphate content approximately 0.1 that of agarose c and its pyruvate content was reduced to zero as shown by NMR in DMSO. The product was still able to adsorb and desorb serum glycoproteins similar to agarose c indicating that charge on the agarose c gel particles played a minor role if any on the binding of the serum glycoproteins.     

Alternative exon usage and processing of the major histocompatibility complex-encoded proteasome subunits.

The finding that two subunits of the proteasome, LMP2 and LMP7, are encoded in the major histocompatibility complex (MHC) has linked the proteasome which represents a major extralysosomal proteolytic system to the processing of intracellular antigens. Here we describe a second form of the human LMP7 cDNA, LMP7-E2, which has been identified during the characterization of novel genes in the MHC. The analysis of the genome organization of LMP7 revealed that LMP7-E1 and LMP7-E2 arise by alternative exon usage. Using specific antibodies against LMP2 and LMP7, we show that they are co-expressed with class I MHC molecules as well as a putative peptide transporter. The polypeptides encoded by LMP7 and LMP2 undergo proteolytic processing when incorporated into proteasomes, and the LMP7 precursor is derived mainly from LMP7-E2. Furthermore, our data suggest that LMP7 and LMP2 are mutually dependent for their incorporation into the proteasomal complex.     

DNA-based identification of Alaska skates (Amblyraja, Bathyraja and Raja: Rajidae) using cytochrome c oxidase subunit I (coI) variation

Variation at the mitochondrial cytochrome c oxidase subunit I (mt-COI) gene was examined in 15 species of North Pacific skates. Thirteen species had unique sequences, indicating that a DNA-based barcoding approach may be useful for species identification.     

Twelve microsatellite loci for lake trout (Salvelinus namaycush)

We describe 12 microsatellite loci isolated from lake trout (Salvelinus namaycush). The number of alleles at these loci ranged from two to 11 with an average of 5.3 alleles per locus. The expected heterozygosity ranged from 0.29 to 0.76, with an average of 0.68. Accidental (or illegal) introductions of lake trout into watersheds are decimating native trout populations in the northern Rocky Mountains, and these loci will be useful for identifying the source of these introductions and for estimating the number of founding individuals.     

A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase

RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. Several of its activities are regulated by the DNA sequence chi (5'-GCTGGTGG-3'), which is recognized in cis by the translocating enzyme. When RecBCD enzyme encounters chi, the intensity and polarity of its nuclease activity are changed, and the enzyme gains the ability to load RecA protein onto the chi-containing, unwound single-stranded DNA. Here, we show that interaction with chi also affects translocation by RecBCD enzyme. By observing translocation of individual enzymes along single molecules of DNA, we could see RecBCD enzyme pause precisely at chi. Furthermore, and more unexpectedly, after pausing at chi, the enzyme continues translocating but at approximately one-half the initial rate. We propose that interaction with chi results in an enzyme in which one of the two motor subunits, likely the RecD motor, is uncoupled from the holoenzyme to produce the slower translocase.