Bicistronic expression of ecdysone-inducible receptors in mammalian cells.

The recent emergence of inducible expression systems for mammalian cells has greatly facilitated the in vivo analysis of gene function. The ecdysone-inducible expression system is particularly attractive because of (i) extremely low basal expression and high-level induced expression, (ii) the lack of pleiotropic effects caused by the inducer or activator, and (iii) the rapid penetrance and clearance of the inducer. Here, we describe an improved receptor expression vector. The required ecdysone receptor proteins (VgEcR and RXR) are co-expressed from a bicistronic cytomegalovirus (CMV) expression cassette in the vector pERV3. The CMV promoter in this vector can be readily replaced with a cell type-specific promoter of interest. Using the ecdysone analogs, muristerone A or ponasterone A, induction ratios of up to three orders of magnitude were attained in the transient transfection assays and in a cell line stably transformed with both pERV3 and an ecdysone-inducible reporter vector. Fine control of luciferase expression was achieved bv varying both the induction time and inducer concentration. Here, we describe a set of cell lines stably transformed with the vector pERV3, in which the ecdysone receptors are expressed at optimal levels for the high-level induction of gene expression.

Personality factors and disorders in chronic pain.

It has long been recognized that there is a relationship between certain personality types and personality disorders (PD) and chronic nonmalignant pain (CP). The relationship, however, is far from understood and the physiological and psychological mechanisms that underlie it are unclear. Those who treat chronic pain face many challenges when dealing with individuals who have personality disorders and they often become frustrated when interacting with these patients. Patients with certain traits and personality disorders may continue to worry and ruminate about their symptoms long after the tissue pathology has resolved. Other individuals may overly rely on the clinician and assume a passive role in their treatment, thereby decreasing the likelihood for a positive outcome. Moreover, patients with personality disorders may be demanding (eg, borderline), self-absorbed (eg, narcissistic), or substance seeking (eg, antisocial, borderline). In an attempt to improve management of such patients, pain specialists have attempted to better understand the complex relationship between personality and chronic pain. In this article, we will review the predominant historical and current theories of pain and personality, discuss aspects of the gate-control theory of pain that may relate to personality, and discuss the diathesis-stress model of personality disorders in pain. Last, we will review studies of personality and personality disorders in chronic pain and their treatment implications. We conclude that, based on the underlying neurochemistry, there may be a direct or indirect link between PD and CP, but further prospective research, both on the biological and psychological relationship, should be conducted.

Acupuncture: does it work and, if so, how?

This article is intended to assist physicians in giving advice to their patients about acupuncture. Despite 20 years of research, the efficacy of acupuncture in general is not established. One of the difficulties with drawing conclusions from the existing literature is that the term acupuncture is used to describe a variety of treatments that differ in many important aspects, both theoretical and technical. This article critically reviews the existing evidence supporting the various effects that have been proposed to result from acupuncture treatments. The evidence is classified according to level of effect (eg, local, segmental, generalized) and type of acupuncture treatment (eg, manual v electrical acupuncture).

Painful peripheral neuropathies.

This article reviews the current pathophysiology of painful peripheral neuropathies, their differential diagnosis, and management.

An Escherichia coli expression vector that allows recovery of proteins with native N-termini from purified calmodulin-binding peptide fusions.

We describe a T7-based Escherichia coli expression vector in which protein coding sequence is seamlessly fused to the N-terminal calmodulin-binding peptide (CBP) purification tag. We combined the use of the site-specific protease enterokinase (EK) and the type IIs restriction enzyme Eam1104 I, which cleave outside their respective (amino acid and nucleotide) target sequences, such that any amino acid sequence may be fused directly C-terminal to the EK cleavage site without codon constraints conferred by the cloning method. PCR products are cloned using ligation-dependent or ligation-independent methods with high cloning efficiencies (>10(6) cfu/microg vector), allowing production of insert quantities sufficient for several cloning experiments with a limited number of PCR cycles, resulting in a significant time-savings and reduced likelihood of accumulating PCR-derived mutations. CBP fusion proteins are expressed to high levels when the CBP peptide is positioned at the N-terminus. CBP binds to calmodulin with nanomolar affinity, and fusion proteins are purified to near homogeneity from crude extracts with one pass through calmodulin affinity resin using gentle binding and elution conditions. We show high efficiency seamless cloning of three inserts into the pCAL-n-EK vector, including one encoding the protein c-Jun N-terminal kinase (JNK). CBP-EK-JNK fusion protein was synthesized to 10-20 mg/liter culture and purified to near homogeneity in one step with calmodulin affinity resin. The fusion tag was efficiently removed with EK to yield active JNK with native N-terminal amino acid sequence.

Differential effects of rat pregnancy on uterine and lung atrial natriuretic factor receptors.

We investigated if the refractoriness to the tocolytic effects of atrial natriuretic factor (ANF) during rat pregnancy is due to a downregulation of one or both guanylyl cyclase (GC)-coupled GC-A and GC-B ANF receptors; lungs were used as controls. Uteri and lungs of virgin, pregnant (days 7, 16, and 21), and day 2 postpartum rats expressed mRNAs for GC-A and GC-B as well as GC-uncoupled ANF-C receptors. GC-B receptor protein was more abundant than GC-A in uteri; the reverse was the case in lungs. Pregnancy decreased uterine mRNAs and proteins for GC-A and GC-B receptors as well as the effects of ANF and C-type natriuretic peptide (CNP) on uterine GC activity; lung ANF receptors and effects of ANF and CNP on lung GC activity were not modulated by pregnancy. It is concluded that pregnancy induces organ-specific modulation of ANF receptors and a downregulation of ANF-GC receptors would minimize interference with uterine motility during pregnancy.

A new expression vector for high level protein production, one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins.

Calmodulin-binding peptide (CBP), a peptide of 26 amino acids derived from muscle myosin light chain kinase (MLCK), binds to calmodulin with nanomolar affinity. Proteins fused in frame with CBP can be purified from crude E. coli lysates in a single step using calmodulin affinity chromatography (Stofko-Hahn et al., 1992). Because the binding between CBP and calmodulin is calcium-dependent, the fusion protein can be eluted from the resin with virtually any buffer containing EGTA (2 mM) and used directly for many applications. To take full advantage of this affinity purification system, we constructed the versatile CBP fusion protein expression vector pCAL-n. The CBP coding sequence was positioned for fusion at the N-terminus, an advantage that ensures consistent high level synthesis of fusion proteins due to the efficient translation of the CBP in E. coli. The production of fusion proteins from pCAL-n is controlled by the tightly regulated T7(lac)O promoter. A versatile multiple cloning site (MCS) was included to facilitate the cloning of genes of interest. The protein coding sequence for the enzyme c-Jun N-terminal kinase (JNK) was inserted into the MCS of pCAL-n, and the resulting fusion protein CBP-JNK synthesized in E. coli cells at 15-20 mg/1 culture. CBP-JNK was purified to near homogeneity in one step with calmodulin affinity resin. Purified CBP-JNK is fully active, and the CBP peptide tag can be removed by cleavage with thrombin. We also show that CBP can be efficiently phosphorylated by cAMP-dependent protein kinase. Hence, the purified fusion proteins can be labeled directly with [gamma-32P]ATP and used to probe protein-protein or protein-nucleic acid interactions.

Downregulation of renal atrial natriuretic factor receptors and receptor mRNAs during rat pregnancy.

Using renal glomeruli and papillae from virgin, pregnant (15- to 17-day), and postpartum (day 2) rats, we investigated whether the decrease in the renal effects of atrial natriuretic factor (ANF) during pregnancy is caused by a downregulation of ANF receptors. Pregnancy decreased the maximal binding of 125I-labeled ANF to guanylyl cyclase (GC)-linked ANF-GC receptors in glomeruli and papillae and increased the binding to clearance receptors (ANF-C) in glomeruli; ANF-C receptors were not detected in the papillae Ribonuclease protection assay detected mRNAs for all the three receptors in the papillae; pregnancy decreased GC-A and ANF-C but not GC-B-receptor mRNAs. Western blots revealed a decrease in GC-A receptors in the papillae of pregnant rats; GC-B-receptor protein was barely detectable. Effects of ANF on guanosine 3', 5'-cyclic monophosphate (cGMP) production by the glomeruli and papillae were decreased during pregnancy and returned to virgin levels during postpartum. It is concluded that a decrease in the renal effects of ANF during pregnancy is caused by a downregulation of renal ANF GC-A receptors and receptor-coupled cGMP production.

Downregulation of adrenal atrial natriuretic peptide receptor mRNAs and proteins by pregnancy in rats.

The main objective of this study was to find out if the reported changes in the aldosterone-suppressant activity of atrial natriuretic peptide (ANP) during different hormonal states in rats are due to a modulation of ANP receptors. In zona glomerulosa cells, ribonuclease protection assay detected mRNAs for guanylate cyclase (GC)-coupled ANP GC-A and GC-B receptors, and for ANP C receptors, which are not coupled to GC. Western analysis using polyclonal anti-GC-A and anti-GC-B receptor antibodies revealed the presence of GC-A but not GC-B receptor proteins in zona glomerulosa cells. Pregnancy (days 7, 16 and 21), oestradiol-17 beta and progesterone decreased mRNAs for all the three ANP receptors in zona glomerulosa cells. Pregnancy decreased GC-A receptor proteins in zona glomerulosa cells, but these recovered to virgin values on day 2 postpartum. ANP receptor mRNAs in zona glomerulosa cells increased by postpartum day 2, but did not reach the values found in virgin rats. Zona fasciculata mainly contained GC-A receptor mRNA. It is concluded that ANP receptors in rat adrenal zona glomerulosa are modulated by pregnancy, oestrogen and progesterone; a decrease in ANP GC-A receptors during pregnancy might explain the accompanying decrease in the aldosterone-suppressant effects of ANP.

Hormonal modulation of atrial natriuretic factor receptors in adrenal fasciculata cells from female rats.

We recently reported that the hormonal status of female rats modified atrial natriuretic factor (ANF) receptors and the aldosterone-suppressant activity of ANF in adrenal glomerulosa cells; here we investigated if this was also true for adrenal fasciculata cells. Adrenal fasciculata cells from animals in different hormonal states contained guanylate cyclase linked ANF-R1 receptors but not ANF-R2 (clearance) receptors. The concentration of ANF-R1 receptors in cells from intact virgin rats was insignificantly higher than in cells from 13- to 15-day pregnant rats and significantly higher than in cells from ovariectomized (OVX), OVX beta-estradiol-treated, and OVX progesterone-treated rats. Under none of the hormonal states did ANF suppress adrenocorticotropic hormone (ACTH) stimulated corticosterone secretion. Data suggest that the interactions between ANF and ACTH on mineralocorticoid and glucocorticoid synthesis markedly differ.

Hormonal modulation of atrial natriuretic factor receptors and effects on adrenal glomerulosa cells of female rats.

This study was done to determine if a decrease in the aldosterone-suppressant effect of atrial natriuretic factor (ANF) by progesterone and an increase by estrogen was caused by modulation of adrenal zona glomerulosa ANF receptors. Freshly dispersed glomerulosa cells from virgin, 13-15 day pregnant, ovariectomized (OVX) estradiol-17 beta-treated and OVX progesterone-treated rats were used. Competitive displacement of specifically bound [125I]ANF1-28 with unlabelled ANF1-28 yielded concentrations of guanylate cyclase-linked ANF-R1 plus ANF-R2 (clearance) receptors and the displacement with unlabelled ANF4-23 yielded ANF-R2 receptors; the difference between the two was treated as the concentration of ANF-R1 receptors. Pregnancy and progesterone decreased and estrogen increased the number of glomerulosa ANF-R1 receptors. ANF produced a significantly greater suppression of potassium-induced aldosterone secretion in cells from OVX estradiol-treated rats than in cells from OVX progesterone-treated animals. These data suggest that the inhibition of the aldosterone-suppressant activity of ANF by progesterone is the result of a downregulation of ANF-R1 receptors.

A constitutive enhancer in the bovine papillomavirus upstream regulatory region shares genetic elements with the viral P1 promoter.

The bovine papillomavirus upstream regulatory region represents a common element in the regulation of transcription from the five early viral promoters. We have determined the sequences required for transcription from the viral P1 promoter, which is located at the 5' end of the upstream regulatory region. In vitro transcription from P1 requires a 123-bp fragment (nucleotides 7153 to 7275; -33 to +90) consisting of an upstream TATA-like sequence as well as an unidentified protein which binds to sequences immediately downstream of the initiation site. In vivo, this promoter requires additional downstream sequences (to position +160; nucleotide 7345) for maximal activity but does not require any additional DNA sequence upstream of a putative TATA box. Four regions within the downstream sequence from +9 to +160 are protected from DNase I digestion by proteins present in a HeLa cell extract. The presence of these sites correlates with the level of P1 activity. A constitutive enhancer maps to this same region, and mutations in this enhancer have been shown to affect downstream promoters. Deletion analysis indicates that the same sequences are required by both the P1 promoter and the constitutive enhancer, suggesting that the same proteins function in both activities.

The E2 transactivator of bovine papillomavirus type 1 is expressed from multiple promoters.

The E2 proteins of bovine papillomavirus type 1 (BPV-1) are a family of site-specific DNA-binding proteins which regulate viral transcription by repression and activation. Repressors E2-TR and E8/E2 are expressed from promoters P5 (P3080) and P3 (P890), respectively. Previous reports have provided evidence that the transcript for the 48-kilodalton transactivator is initiated from a promoter proximal to the open reading frame encoding this protein (P2440 or P4). Our studies extend these findings and show that the E2 transactivation gene is expressed from multiple promoters. We have described the isolation of a cDNA (N15-2) which represents an RNA species expressed from the P3 promoter. The major exon of this species was produced by splicing to an acceptor located at nucleotide 2558 and contained the complete E2 open reading frame. The acceptor is probably utilized by yet another more abundant mRNA expressed from the P2 promoter (A. Stenlund, J. Zabielski, H. Ahola, J. Moreno-Lopez, and U. Pettersson, J. Mol. Biol. 182:541-554, 1985). Linked to a surrogate promoter, the N15-2 cDNA can transactivate an E2-responsive reporter gene. BPV-1 plasmids containing mutations either in the 2558 splice acceptor or in the P4 promoter showed significantly reduced transforming ability and reduced ability to transactivate an E2-responsive reporter, while a double mutant was inactive in both assays. The transformation defect was complemented by an E2 expression vector, and the BPV genome absolutely required the E2 protein to transactivate in the second assay. Thus, these genetic experiments show that alternate modes of E2 expression contribute to the E2 mRNA pool. Direct analysis of cytoplasmic RNA from transformed cultured cells proves that transcripts containing the 2558 acceptor exon are approximately as abundant as the P4 type E2 mRNAs. Furthermore, analysis of the E2 proteins present in various cell lines harboring specific BPV-1 mutants, including the 2558 acceptor mutant, proves that alternate modes of E2 expression exist. The ability of the E2 activator and repressors to each be independently expressed from multiple E2-responsive promoters probably adds to the resiliency of the latent virus as a plasmid and may be important for its homeostasis within the cell in different environmental or developmental situations.

Bovine papillomavirus type 1 encodes two forms of a transcriptional repressor: structural and functional analysis of new viral cDNAs.

Genetic and biochemical evidence has established that the E2 open reading frame (ORF) of bovine papillomavirus type 1 encodes at least two different site-specific DNA-binding proteins, one which activates and the other which represses expression from a viral promoter (P. F. Lambert, B. A. Spalholz, and P. M. Howley, Cell 50:69-78, 1987). We have obtained data which show that a second form of the repressor gene is expressed in transformed cells harboring stable viral plasmids. The structural details of this gene have been discerned by cDNA cloning, by RNase protection, and by primer extension analysis of in vivo RNA. Moreover, data from in vitro transcription experiments support the notion that this form of the E2 repressor is expressed from a novel viral promoter and that a small exon from another ORF is linked to an active repressor domain in E2. Thus, two different forms of the repressor are expressed from different promoters and might be independently regulated either in the cell cycle or in different tissue types. We show by functional in vivo assays utilizing a cDNA vector encoding this gene that the trans-acting factor has in vivo activities similar to those of the known repressor. Our screen of a cDNA library for cDNA clones representing bovine papillomavirus transcripts has also revealed a number of other novel structures defining new donor and acceptor RNA-processing sites. Notably, clones which conceptually can be translated to yield an E7 protein, the viral M gene, and the entire E2 ORF have been characterized. Finally, truncated versions of putative E8 cDNAs were also obtained.

[Quasi-normal cerebrospinal fluid in patients with acquired immunodeficiency syndrome and cryptococcal meningitis]. / Liquide céphalo-rachidien quasi-normal chez des patients atteints du syndrome d'immunodéficience acquise et d'une méningite à cryptocoque.

We report eight patients with cryptococcal meningitis and a cerebrospinal fluid characterized by few or no white blood cells and chemistries that may be near normal. In four of these patients, only testing for cryptococcal antigen allowed the initial diagnosis. Seven of the patients had a certain diagnosis of AIDS. Six have died. Autopsies performed in two cases indicated a poor meningeal inflammatory response. Contrary to the findings in most immunodeficient patients, in AIDS cryptococcal meningitis may present with few cellular or biochemical abnormalities in the cerebrospinal fluid. In AIDS patients presenting with headache and fever or change in mental status, examination of the cerebrospinal fluid should not be limited to routine studies.

Identification of an antigen-specific immunoglobulin M antibody associated with acute Toxoplasma infection.

We used the protein gel transfer technique (Western blotting) to analyze the immune response to individual electrophoretically resolved Toxoplasma antigens. Toxoplasma antigens were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The array of resolved proteins was electrophoretically transferred to CNBr-activated filter paper and incubated with serum samples from patients with either acute or chronic Toxoplasma infections or with serum samples from seronegative controls. Serum immunoglobulin G and immunoglobulin M bound to individual Toxoplasma antigens were detected by radioiodinated staphylococcal protein A and a goat anti-human immunoglobulin M antibody, respectively. A complex array of Toxoplasma antigens was revealed by this procedure. Immunoglobulin M directed against a low-molecular-weight antigen was detected in sera from all acutely infected adults (19 sera tested) and was absent from all sera from individuals with chronic infections (9 sera tested) and from sera from seronegative controls (9 sera tested).