Common treatment of polycystic ovarian syndrome and major depressive disorder: case report and review.

We present the case of a young woman with treatment-resistant major depression, who presented to the Mood Disorders Clinic with a Hamilton Psychiatric Rating Scale for Depression (HAM-D-21) score of 28, after a year-long treatment with Effexor-XR. The patient also had untreated Polycystic Ovarian Syndrome (PCOS). The resolution of her depressive symptoms resulted from the treatment for PCOS with metformin and spironolactone. The patient remained euthymic 5 months after discontinuation of the antidepressant while continuing therapy for PCOS. We briefly overview of the pertinent literature of the pathophysiology of PCOS and affective disorders, highlighting an overlap in phenotypical presentations between these two disorders. Dysregulation of the hypothalamo-pituitary axis and various end organ systems are implicated in both PCOS and affective disorders. As such, several clinical and biochemical markers are common to both disorders, namely insulin resistance, obesity, and hyperandrogenism. In addition, these metabolic abnormalities are interrelated, causing women with PCOS or affective disorders to get caught in a "vicious cycle" of hormonal dysregulation. The case report presented here illustrates how treatment of symptoms such as insulin resistance and hyperandrogenism can lead to remission of major depressive disorder and PCOS. We suggest that through treatment of underlying metabolic defects, both the mood of the patient and the metabolic condition of PCOS can be assisted.

Distinct mRNAs that encode La autoantigen are differentially expressed and contain internal ribosome entry sites.

Analysis by reverse transcription-polymerase chain reaction has suggested the existence of at least two La autoantigen-encoding mRNAs that contain different 5' noncoding regions (NCRs) linked to the same La coding region (Troster, H., Metzger, T. E., Semsei, I., Schwemmle, M., Winterpacht, A., Zabel, B., and Bachmann, M. (1994) J. Exp. Med. 180, 2059-2067). La-encoding transcripts La1 and La1' contain 115- and 483-nucleotide 5' NCRs, respectively. To determine whether the various La transcripts are functional mRNAs, the expression and polysomal association of natural La1 and La1' RNAs were examined. Although La1 transcripts were ubiquitously expressed in human tissues, La1' transcripts were predominantly expressed in peripheral blood leukocytes, especially in B, T, and natural killer cells. Both La1 and La1' transcripts associated with polysomes in natural killer cells, suggesting that these transcripts were functional mRNAs. Upon activation of B cells with the mitogens phorbol 12-myristate 13-acetate and ionomycin, the amount of La1' mRNA, but not La1, declined. In contrast, after chemical activation of T cells, the amount of La 1 mRNA, but not La1', declined. The mechanism by which the La1 and La1' 5' NCRs initiate translation initiation was tested in cultured human HeLa cells and in two different in vitro translation systems. It was found that both 5' NCRs can mediate translation initiation by internal initiation. These findings indicate that the constitutive expression of La1 mRNA and the tissue-specific expression of La1' mRNA can both allow La protein synthesis under conditions when cap-dependent translation is compromised, such as inflammation, apoptosis, or certain viral infections.

Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray.

Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5' end- dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5' noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.

A splicing-dependent regulatory mechanism that detects translation signals.

Premature termination codons (PTCs) can cause the decay of mRNAs in the nuclear fraction of mammalian cells. This enigmatic nuclear response is of interest because it suggests that translation signals do not restrict their effect to the cytoplasm, where fully assembled ribosomes reside. Here we examined the molecular mechanism for this putative nuclear response by using the T-cell receptor-beta (TCR-beta) gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic ontogeny. We found that PTCs had little or no measurable effect on TCR-beta pre-mRNA levels, but they sharply depressed TCR-beta mature mRNA levels in the nuclear fraction of stably transfected cells. A PTC split by an intron was able to trigger the down-regulatory response, implying that PTC recognition occurs after an mRNA is at least partially spliced. However, intron deletion and addition studies demonstrated that a PTC must be followed by at least one functional (spliceable) intron to depress mRNA levels. One explanation for this downstream intron-dependence is that cytoplasmic ribosomes adjacent to nuclear pores scan mRNAs still undergoing splicing as they emerge from the nucleus. We found this explanation to be unlikely because PTCs only 8 or 10 nt upstream of a terminal intron down-regulated mRNA levels, even though this distance is too short to permit PTC recognition in the cytoplasm prior to the splicing of the downstream intron in the nucleus. Collectively, the results suggest that nonsense codon recognition may occur in the nucleus.

A regulatory mechanism that detects premature nonsense codons in T-cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro.

Gene rearrangement during the ontogeny of T- and B-cells generates an enormous repertoire of T-cell receptor (TCR) and immunoglobulin (Ig) genes. Because of the error-prone nature of this rearrangement process, two-thirds of rearranged TCR and Ig genes are expected to be out-of-frame and thus contain premature terminations codons (ptcs). We performed sequence analysis of reverse transcriptase-polymerase chain reaction products from fetal and adult thymus and found that newly transcribed TCR-beta pre-mRNAs (intron-bearing) are frequently derived from ptc-bearing genes but such transcripts rarely accumulate as mature (fully spliced) TCR-beta transcripts. Transfection studies in the SL12.4 T-cell line showed that the presence of a ptc in any of several TCR-beta exons triggered a decrease in mRNA levels. Ptc-bearing TCR-beta transcripts were selectively depressed in levels in a cell clone that contained both an in-frame and an out-of-frame gene, thus demonstrating the allelic specificity of this down-regulatory response. Protein synthesis inhibitors with different mechanism of action (anisomysin, cycloheximide, emetine, pactamycin, puromycin, and polio virus) all reversed the down-regulatory response. Ptc-bearing transcripts were induced within 0.5 h after cycloheximide treatment. The reversal by protein synthesis inhibitors was not restricted to lymphoid cells, as shown with TCR-beta and beta-globin constructs transfected in HeLa cells. Collectively, the data suggest that the ptc-mediated mRNA decay pathway requires an unstable protein, a ribosome, or a ribosome-like entity. Protein synthesis inhibitors may be useful tools toward elucidating the molecular mechanism of ptc-mediated mRNA decay, an enigmatic response that can occur in the nuclear fraction of mammalian cells.

Characterization of the dopa decarboxylase gene of Manduca sexta and its suppression by 20-hydroxyecdysone.

Manduca dopa decarboxylase (DDC) cDNA was isolated, sequenced, and found to be most closely related to Drosophila DDC (72% amino acid identity). Culture of Day 2 fourth instar larval epidermis with 20-hydroxyecdysone (20E) showed that 20E was necessary to determine the later expression of the gene, but its removal was required for this expression to occur. Experiments with the protein synthesis inhibitors, cycloheximide and anisomycin, and the mRNA synthesis inhibitor, alpha-amanitin, showed that 20E induced a protein(s) which suppressed transcription of the DDC gene. Gel mobility shift assays using epidermal extracts and various fragments of the first 1.1 kb of the 5' flanking region of the DDC gene showed only one DNA fragment 87 to 167 bp upstream of the 5' initiation site that bound a nuclear protein(s) with the expected developmental specificity. The protein was abundant at the time of high ecdysteroid titer when no DDC mRNA was present, but low both before the rise (no DDC mRNA) and after the decline of ecdysteroid titer (maximal DDC mRNA). Thus, this protein(s) is a candidate for an ecdysteroid-induced transcription factor which acts to suppress DDC transcription. DNase I footprinting assays confirmed by use of a specific oligonucleotide showed that this protein(s) bound to the sequence 5'-GGCTTATGCGCTGCA-3'.

T cell receptor-beta mRNA splicing during thymic maturation in vivo and in an inducible T cell clone in vitro.

The expression of TCR-beta mRNAs competent to encode functional V(D)JC beta proteins requires the activation of programmed DNA rearrangement events. It is not known whether other regulatory mechanisms control the steady-state levels of mature TCR-beta transcripts during thymic ontogeny. In this report, we demonstrate that TCR-beta pre-mRNAs accumulate in T cells, thus implicating RNA splicing as another potential level of regulation. Three methods were used to characterize the intron content of these pre-mRNA: Northern blot analysis, ribonuclease H mapping, and reverse transcription polymerase chain reaction analysis. Using these methods, we demonstrate that intron-containing TCR-beta transcripts derived from both the JC beta 1 and JC beta 2 loci accumulate in murine fetal and adult thymus. (VD)JC beta 1 pre-mRNAs that accumulate in the thymus possess unusually long poly(A) tails (> or = 300 nucleotides) and contain different combinations of four introns: the large intron between the J beta 1 and C beta 1 elements and the three introns within the C beta 1 element. The presence of an unusual transcript possessing IVS2C beta 1 at the 5' terminus suggests that cleavage of its splice acceptor is inefficient or negatively regulated. The profile of incompletely spliced TCR-beta transcripts present in the thymus in vivo is identical in intron content to those that we previously showed accumulate in the nucleus of the immature SL12.4 T lymphoma cell clone. An unstable negative regulatory protein may control TCR-beta expression in this cell clone because fully spliced TCR-beta transcripts are dramatically induced in the cytoplasm after treatment with any of five different protein synthesis inhibitors (cycloheximide, anisomyosin, emetine, puromycin, and pactamycin), all of which act by distinct mechanisms to inhibit protein synthesis.

Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma.

The rat preprotachykinin (PPT) gene encoding the neuropeptides substance P (SP), neurokinin A (NKA), neuropeptide K (NPK), and neuropeptide gamma was isolated from a lambda Charon 4A genomic library. Two overlapping clones contained all of the exons present in beta-PPT, including some 7 and 9 kb 5' and 3' flanking sequence, respectively. The presence of 1 major and 2 minor transcription initiation sites was determined from primer extension and nuclease protection experiments. Analysis of the nucleotide sequence homology between the rat and bovine revealed the presence of highly conserved regions throughout the entire coding region and within the 5' flanking sequences. Primer extension and nuclease protection experiments demonstrated that the primary transcript is differentially spliced primarily into gamma- and beta-PPT mRNA in all tissues examined in the adult rat where the gene is expressed. beta-PPT mRNA contains all of the exons, whereas gamma-PPT mRNA lacks exon 4, which encodes part of the N-terminus of NPK. The alpha-PPT mRNA, which lacks exon 6 (the sequence encoding NKA and processing sites), comprises about 1% of the total PPT mRNA. An RNA secondary structure model is proposed to account for these specific exon exclusion events in the RNA splicing process. These results are discussed with regard to the mechanisms regulating SP gene expression and the functional significance of differential RNA splicing in the rat.

Solution hybridization-nuclease protection assays for sensitive detection of differentially spliced substance P- and neurokinin A-encoding messenger ribonucleic acids.

In this chapter we discussed methods that can be used for the sensitive detection and quantitation of differentially or alternatively spliced mRNAs as well as mRNAs of low abundance. Although mechanisms responsible for splicing (and differential splicing in particular) have not been fully determined, many RNAs derived from a variety of genes have been observed to undergo the process. The impact of splicing with regard to the expanded potential of gene expression emphasizes the usefulness of the solution hybridization-nuclease digestion technique described here, compared to Northern blot analysis. The use of radiolabeled cRNA(s) provides for an assay of both high specificity and high sensitivity. While end-labeled cDNA probes can be used, they do not have the sensitivity inherent in the assay performed with uniformly radiolabeled cRNAs. If multiple mRNAs are derived from a single gene as a result of differential or alternative precursor RNA splicing, however, the results with a cRNA probe may initially appear to be quite complicated, and end-labeled cDNAs may yield more easily interpretable results. Nonetheless, both types of probes are useful in the context of gene expression analysis, and it is clear that for routine purposes of quantitation cRNA probes in solution hybridization-nuclease protection assays are clearly more desirable than RNA blot analyses due to their truly quantitative nature as well as ease of assay.

Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A.

Synthetic oligonucleotides were used to screen a rat striatal cDNA library for sequences corresponding to the tachykinin peptides substance P and neurokinin A. The cDNA library was constructed from RNA isolated from the rostral portion of the rat corpus striatum, the site of striatonigral cell bodies. Two types of cDNAs were isolated and defined by restriction enzyme analysis and DNA sequencing to encode both substance P and neurokinin A. The two predicted preprotachykinin protein precursors (130 and 115 amino acids in length) differ from each other by a pentadecapeptide sequence between the two tachykinin sequences, and both precursors possess appropriate processing signals for substance P and neurokinin A production. The presence of a third preprotachykinin mRNA of minor abundance in rat striatum was established by S1 nuclease protection experiments. This mRNA encodes a preprotachykinin of 112 amino acids containing substance P but not neurokinin A. These three mRNAs are derived from one rat gene as a result of differential RNA processing; thus, this RNA processing pattern further increases the diversity of products that can be generated from the preprotachykinin gene.

Qualitative and quantitative fluorescein fluorescence in determining intestinal viability.

Clinical evaluation and qualitative (visual) and quantitative (fluorometric) fluorescence for predicting intestinal viability were compared in an animal model of temporary arterial occlusion with early revascularization. Quantitative fluorescence was determined with a perfusion fluorometer after an intravenous bolus of fluorescein. Qualitative fluorescence was determined by examination under a Wood's lamp in a darkened room. The effectiveness of each diagnostic technique in determining nonviability was expressed in terms of sensitivity, specificity, and accuracy. All three methods had 100 percent specificity; only bowel deemed nonviable proved to be so. Quantitative fluorescence also had a 100 percent sensitivity, but clinical evaluation and qualitative fluorescence had only a 33 and 11 percent sensitivity, respectively (some segments of bowel that were ultimately nonviable were not correctly predicted to be so). The inaccuracy of qualitative fluorescence was due to the fact that ischemic intestine with a hyperfluorescent pattern often progressed to necrosis. Fluorometric quantitation identified those hyperfluorescent segments that were viable. This study suggests that visual fluorescence is not reliable in assessing intestinal viability after early revascularization after arterial occlusion, but quantitative fluorometric fluorescence is reliable in almost all instances.

Flexor profundus injuries treated with delayed two-staged tendon grafting.

Eleven patients with flexor profundus avulsions or severances recognized more than 6 weeks following injury were treated with delayed two-stage tendon grafting. Twelve profundus grafts were performed. All were young, well-motivated men. Total active motion improved from 166 to 244. Grip strength was significantly improved in eight patients. One graft ruptured requiring a secondary repair and two tenolyses were performed.