Cleavage of the cell-surface O-sialoglycoproteins CD34, CD43, CD44, and CD45 by a novel glycoprotease from Pasteurella haemolytica.

The study of structural/functional characteristics of the cell-surface glycoproteins of leukocytes has led to a better understanding of the differentiation and maturation of hematopoietic cells. We have assessed the ability of a unique metalloprotease that is secreted by the bovine fibrinous pneumonia pathogen Pasteurella haemolytica, to cleave cell-surface glycoproteins expressed on human leukocytes. Biochemical analysis shows that the O-glycosylated cell surface Ag CD34, CD43 (leukosialin), CD44 (hyaluronic acid receptor), and CD45 (leukocyte common Ag), are all cleaved by this protease. Although these enzyme-sensitive structures contain N-linked glycans, they are all extensively glycosylated with O-linked carbohydrates, which are especially abundant on CD34 and CD43. In contrast, the glycoproteins CD18/11a,b,c (leukocyte integrins), CD71 (transferrin receptor), HLA class I, and 8A3 Ag, which contain N-linked glycans but no O-sialo-glycans, were resistant to the action of the enzyme. Inasmuch as previous studies using glycophorin A had indicated that the substrate specificity of this enzyme may be uniquely restricted to the cleavage of O-sialoglycoproteins, we have designated this activity, P. haemolytica glycoprotease. Immunofluorescence analysis with a variety of antibodies to different epitopes of the P. haemolytica glycoprotease-sensitive structures indicate that this enzyme may have widespread applications in epitope-mapping studies, and represents a novel tool with which to study structure/function relationships for O-sialoglycosylated cell-surface proteins. However, most significantly these results suggest that the P. haemolytica glycoprotease may be of use in the affinity purification and recovery of clinically important leukocyte subsets, such as primitive hematopoietic progenitors that express CD34.


"Universal" T helper cell determinants enhance immunogenicity of a Plasmodium falciparum merozoite surface antigen peptide.

Synthetic peptide constructs containing a limited number of epitopes are being currently investigated as subunit vaccines against a variety of pathogens. However, because of widespread nonresponsiveness to most such constructs, possibly attributable to MHC restriction, the choice of appropriate carrier molecules to enhance immunogenicity of peptides constitutes an important and essential aspect of designing synthetic immunogens for human use. Widely used vaccines such as tetanus toxoid (TT) have not been uniformly effective as carrier proteins because of the phenomenon of epitope-specific suppression in which induction of an immune response against a synthetic peptide conjugated to TT is prevented by preexisting immunity to TT. Recently, T cell determinants that can be recognized in the context of several class II MHC molecules have been identified in tetanus toxin as well as in the circumsporozoite protein of a human malarial parasite, Plasmodium falciparum. Such determinants can be potentially used to circumvent the problem of epitope-specific suppression. In the present study we evaluated two such T cell determinants, viz., tt830-844 from tetanus toxin and CST3 from the malarial parasite, for their ability to help induce a boostable antibody response and to overcome genetic nonresponsiveness to a synthetic 20-residue construct containing a B cell and an overlapping T cell epitope from a major merozoite surface protein of P. falciparum. Our data provide support for the view that widely recognized T cell determinants may be used as universal carrier molecules for general vaccination.


Monoclonal autoantibodies to subnucleosomes from a MRL/Mp(-)+/+ mouse. Oligoclonality of the antibody response and recognition of a determinant composed of histones H2A, H2B, and DNA.

MRL/Mp(-)+/+ mice produce antinuclear antibodies and develop a spontaneous autoimmune syndrome with lupus-like nephritis. We obtained a panel of seven histone-reactive IgG mAb from a single MRL/Mp(-)+/+ mouse. These antibodies do not react significantly with DNA or individual histones, but bind strongly to the histone H2A-H2B dimer and even more strongly to the H2A-H2B-DNA complex. These antibodies also bind to whole nuclei when tested by immunofluorescence, indicating that they recognize an epitope accessible in chromatin. The V region sequences of these antibodies have been determined. The H chain third complementarity-determining regions of these antibodies are similar to those found in anti-DNA antibodies even though the antibodies in our panel do not react with DNA in the absence of histones, suggesting that DNA is part of the subnucleosome epitope. Several of these antibodies are clonally related, supporting the hypothesis that the activation of these clones is Ag-driven. Analysis of the sequences of these antibodies indicates that they derive from autoreactive B cells that were clonally expanded and whose V region genes have undergone numerous somatic mutations.


A new method for detachment of Dynabeads from positively selected B lymphocytes.

This paper describes a method for the detachment of immunomagnetic beads from positively selected human B lymphocytes. After rosetting of B cells using anti-CD19 coated magnetic beads (Dynabeads M-450 Pan B, Dynal), the Dynabeads were rapidly detached (efficiency 80%) from the cells using goat anti-mouse-Fab antiserum (DETACHaBEAD, Dynal) at ambient temperature. Isolated B cells did not show significant differences in the expression of a number of B cell antigens when compared to B cells stained in fresh whole blood. In contrast, positively selected B cells that had detached from the beads following overnight incubation, demonstrated a significantly reduced expression of certain of the antigens examined (CD19, CD20 and CD23). It was further demonstrated that neither anti-CD19 nor anti-Fab resided on the surface of the cells after detachment. The cells were still in G0 phase (greater than 90%) at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulated the vast majority of the cells to leave the G0 phase, and to progress through S phase in the presence of growth factors. The cells could also be stimulated to differentiate, further confirming the normal functional capacity of the isolated cells. The method described in this paper can also be used for the detachment of other positively selected cells, such as CD4+ T cells, CD8+ T cells and CD34+ stem cells.


Physical separation of ICAM-1 binding cells.

Studies of interactions between specific molecules involved in adhesion between cells are often complicated since cells possess many different types of surface molecules influencing adhesion. To be able to further study interactions between the adhesive cell surface molecule LFA-1 and its ligand ICAM-1, we have produced an ICAM-1 fusion protein in which the extracellular part of ICAM-1 has been fused to a part of an IgG heavy chain. The fusion protein was produced by CHO cells and was easily purified in large quantities from the cell culture supernatant. The protein was coupled to magnetic beads and was shown to have binding characteristics similar to native ICAM-1. The coupled beads were used for magnetic isolation of ICAM-1 binding cells thereby providing a method for detecting changes in avidity for ICAM-1 and also for the analysis of ICAM-1 binding cells.


Viral culture and p24 antigenemia of human immunodeficiency virus (HIV)-infected individuals correlated with antibody profiles determined with recombinant polypeptides of all HIV-1 open-reading frames.

The association between viral activity and antibody profiles was investigated in 202 individuals infected by the human immunodeficiency virus (HIV) grouped according to their Walter Reed clinical stage. Each study group was subdivided into subjects positive or negative for markers of active viral replication: presence of serum p24 antigen and viral culture. In Western blots using recombinant antigens, sera of HIV-positive individuals with positive viral markers had a significantly lower antibody reactivity to several viral proteins than did individuals without viral markers. Noticeably, proteins of the gag (p24, p17) and env (gp120, COOH-terminal part of gp41) open-reading frames revealed a decreased reactivity. The antibody response to the regulatory proteins revealed no or poor association with viral activity in the host. The results suggest that seroreactivity is mainly influenced by factors reflecting the viral activity of an HIV-infected individual, while the clinical stage of the patient is less important. Especially, reductions in antibodies against gp120 and p17 were useful markers associated with increased viral activity.


Inhibition of human T cell leukemia virus by the plant flavonoid baicalin (7-glucuronic acid, 5,6-dihydroxyflavone).

The ability of baicalin (7-glucuronic acid, 5,6-dihydroxyflavone), a flavonoid compound purified from the Chinese medicinal herb, Scutellaria baicalensis georgi, to inhibit human T cell leukemia virus type I (HTLV-I) was examined. Baicalin produced concentration-dependent inhibition of HTLV-I replication in productively infected T and B cells. Moreover, baicalin treatment selectively reduced the detectable levels of HTLV-I p19 gag protein in infected cells by greater than 70% at concentrations that produced insignificant effects on total cellular protein and DNA synthesis with no loss in cell viability. Resistance to HTLV-I infection and virus-mediated transformation was noted in uninfected peripheral blood lymphocytes pretreated with baicalin before cocultivation with lethally irradiated chronically infected cells. Baicalin inhibited reverse transcriptase activity in HTLV-I-infected cells as well as the activity of purified reverse transcriptase from Moloney murine leukemia virus and Rous-associated virus type 2. These results suggest that baicalin may be a potential therapeutic agent against HTLV-I-associated T cell diseases.


Inhibition of endotoxin-induced macrophage tumor necrosis factor expression by a prostacyclin analogue and its beneficial effect in experimental lipopolysaccharide intoxication.

Tumor necrosis factor (TNF), a protein produced in large quantities by endotoxin-activated macrophages, has been implicated as an important mediator of the lethal effect of endotoxin. A stable prostacyclin analogue (iloprost) was investigated for its ability to interfere with TNF secretion of lipopolysaccharide (LPS)-stimulated macrophages. It could be demonstrated by bioassays that LPS-induced TNF production was suppressed in a dose-dependent manner when macrophages were treated with iloprost at the time of LPS stimulation. Northern blot analysis revealed that iloprost inhibited TNF production at the transcription level. In vivo, endotoxin-induced mortality rates in galactosamine-sensitized mice could be significantly (P less than .05) reduced by iloprost administration. It is assumed that prostacyclin modulates endotoxin-induced and TNF-mediated inflammation in septic shock.


The topography of the surface of potato virus X: tritium planigraphy and immunological analysis.

Thermally activated tritium atoms were used to probe the surface topography of the coat protein of potato virus X (PVX) potexvirus. The accessibility profile of amino acid residues in the polypeptide chain was determined from data on the intramolecular distribution of tritium label in the PVX coat protein. Tryptic peptides T1 and T2, as well as parts of peptides T3 and T5, from the PVX particles were all located in the N-terminal region of the PVX coat protein and were accessible to tritium labelling, whereas the C-terminal region of the coat protein was practically inaccessible to it. Indirect ELISA and immunoblotting with two PVX-specific monoclonal antibodies confirmed that the N terminus of the coat protein (residues 1 to 56) was exposed on the virus surface, and furthermore that this region forms a highly immunogenic virus-specific antigenic region. The data obtained support the spatial model of PVX, in which the N-terminal amino acids of the coat protein are exposed at the particle surface, and the C-terminal region is buried in the particle. The spatial organization of the PVX coat proteins differs from the model proposed for other filamentous plant viruses such as potyviruses and tobamoviruses where both the N and C termini of the coat protein are located at the particles' surface.


Hexamethylene bisacetamide stimulates herpes simplex virus immediate early gene expression in the absence of trans-induction by Vmw65.

Hexamethylene bisacetamide (HMBA) and DMSO are known to induce differentiation of cultured erythroleukaemic cells and to enhance the reactivation of latent herpes simplex virus (HSV) after explantation of ganglia. We report that the presence of these compounds in cell culture medium overcomes the replication defect of in1814, an HSV-1 mutant with an insertion mutation that inactivates the virion trans-inducing factor, Vmw65 (VP16). The effect of HMBA was not cell type-specific and was attained even by a short exposure (1.5 to 5 h) to the agent early after infection. The presence of HMBA resulted in an increase in immediate early (IE) RNA accumulation after infection of cells in the presence of cycloheximide, such that RNA levels in in1814-infected cells approached the values observed in wild-type HSV-1-infected cells in the absence of HMBA. Transport of viral DNA to the cell nucleus was not affected by HMBA. The results suggest that HMBA- and DMSO-mediated enhancement of reactivation from latency is due to an increase in IE RNA production. In addition, these studies demonstrate a primary effect of HMBA on gene regulation which may be a paradigm for initial events during erythroleukaemic cell differentiation.


Canine parvovirus empty capsids produced by expression in a baculovirus vector: use in analysis of viral properties and immunization of dogs.

The VP-2 genes of canine parvovirus (CPV) and a recombinant consisting of CPV and feline panleukopenia virus (FPV) sequences were cloned into baculovirus expression vectors, fused to the baculovirus polyhedrin promoter. Recombinant baculoviruses were prepared and the properties of the parvovirus proteins expressed in insect cells examined. The proteins produced were the same size as the authentic CPV VP-2 protein, and were produced late after infection; the quantity of proteins recovered from the insect cell cultures was similar to those produced in CPV infections. Parvovirus particles formed had the haemagglutination (HA), sedimentation and buoyant density properties of authentic CPV capsids. Both the CPV capsids and the CPV-FPV recombinant capsids from the baculovirus system expressed the same epitopes as those seen in the viable parvoviruses when tested with a panel of anti-parvovirus monoclonal antibodies. Lysates of recombinant baculovirus-infected cells were inoculated into dogs, giving rise to serum neutralizing and HA-inhibiting antibodies, and the immunized dogs were protected from clinical disease upon challenge with a virulent isolate of the most recent antigenic type of CPV.


Protection of mice from lethal influenza by defective interfering virus: T cell responses.

The immune-mediated lethal influenza in C3H/He-mg (H-2k) mice infected with A/WSN influenza virus (H1N1) was investigated. A primary class I major histocompatibility complex-restricted, CD8+ cytotoxic T lymphocyte (CTL) response was found in the lungs with a peak activity at 5 days post-infection. Monoclonal antibody depletion in vivo showed that a lethal CD8+ cell response as well as a lethal CD4+ response was generated during infection. Mice survived infection only if both CD8+ and CD4+ cells were depleted. Mice infected with the same dose of virus, but treated with defective interfering (DI) A/WSN virus develop only a transient sub-lethal respiratory disease even though multiplication of virus in the lungs is undiminished, and we have shown here that this correlates with a reduction in the local CTL response. The mechanism by which DI virus beneficially modulates the immune response is discussed; it is proposed that there is classical, but cell type-specific DI virus interference in lymphocytes but not in the cells of the lung in which virus multiplies productively.


Human immunodeficiency virus type 1 envelope glycoprotein gp120-mediated killing of human haematopoietic progenitors (CD34+ cells).

The effects of human immunodeficiency virus type 1 (HIV-1) and recombinant envelope glycoprotein gp120 on the in vitro growth of enriched human haematopoietic progenitors (CD34+ cells) have been investigated. A 2 h exposure to HIV-1 resulted in a progressive and significant reduction of viable CD34+ cell number in liquid cultures and of granulocyte-macrophage, erythroid and megakaryocytic progenitors in semisolid cultures. In virus-treated CD34+ cells, no signs of active virus replication were observed and the possibility of latent infection was excluded by quantitative polymerase chain reaction. Recombinant HIV-1 envelope glycoprotein gp120 added to CD34+ cell cultures displayed a dose-dependent inhibitory activity on CD34+ cell viability. Neutralizing antibody against gp120 was able to block completely the inhibitory activity on CD34+ cells of either HIV-1 or recombinant gp120. These results demonstrate that HIV-1 envelope glycoprotein gp120 has a direct cytotoxic effect on CD34+ cells.


Epitope mapping of the human papillomavirus type 16 E4 protein by means of synthetic peptides.

Eight overlapping icosapeptides covering the entire sequence of the E4 protein of human papillomavirus type 16 (HPV-16), were prepared and tested for their reactivity with human sera in IgG-specific ELISA. The strongest reactivity of sera from HPV-16 DNA-positive invasive cervical carcinoma (INCA) patients was detected with the peptide denoted 16/E4-6, covering amino acids 51 to 70. Subsequently nearly 200 sera were tested for the presence of the 16/E4-6-specific antibody. Reactivity was more frequent in cervical intraepithelial neoplasia patients and INCA patients than in matched control subjects. Sera from INCA patients were also tested for antibody reactive with peptide 16/E7-2 covering the major type-specific reactive region of the HPV-16 E7 protein. Only four of 13 sera possessing the 16/E4-6-specific antibody were reactive with the 16/E7-2 peptide.


The direct effects of graded axonal compression on axoplasm and fast axoplasmic transport.

The direct effects of mechanical compression on axoplasm and fast axoplasmic transport were studied by video-enhanced differential interference microscopy. Single axons, isolated from the squid, were compressed with 0.5, 5, 20, or 100 gram (g) weights placed over a 1 millimeter (mm) length of axon. Brief compressions (10 seconds) at low pressures (0.5 g/mm) momentarily deformed the axon, but the axoplasm and axon returned to their normal shape and position after the pressure was removed, and no residual changes in axoplasmic structures, fast axoplasmic transport or membrane function were seen. Compressing the axon with 5-20 g/mm, however, broke the axoplasm at the site of the crush and squeezed the axoplasm out from under the compression site. Though the axoplasm usually returned to the crush site after the weight was removed and organelles continued to move in the axoplasm under the crush, the organelles failed to cross a dense line that marked the site of the rejoined axoplasm, instead they accumulated over time at the crush site. This results suggests that the blockage of fast transport at moderate compressions was due to a mechanical breakage of the axoplasm at the compression site. The plasma membrane was apparently not transected after moderate compressions (5-20 g/mm) since the resting membrane potential returned to nearly control levels after the weight was removed. Compressions with 100 g/mm, however, did break the plasma membrane as evidenced by the rapid and irreversible loss of the action potential and resting potential and the ion-dependent liquefaction of axoplasm and loss of all organelle transport at the 100 g/mm compression site. Thus, small mechanical pressure elastically deformed the axoplasm, moderate pressures mechanically broke the axoplasm, and high pressures broke the axoplasm and the plasma membrane.


Pathophysiology of pelvic adhesions. Modern trends in preventing infertility.

Infertility is secondary to pelvic adhesions in 15-20% of cases. Pelvic adhesions result from pelvic inflammatory disease, previous pelvic surgery, foreign bodies and previous appendicitis with pelvic abscess. As a result of the insult to the peritoneal surfaces of the pelvic organs, the concentrations of peritoneal fluid leukotriene, B4 and prostaglandin E2 are increased. Also, there is a decrease in plasminogen activity. The end result will be the formation of fibrin deposits, which will end in the formation of pelvic adhesions. The diagnosis of adhesions can be achieved by a high index of suspicion in patients with a history of pelvic infections or surgery. A pelvic examination with fixation of the uterus and/or adnexa is also highly suggestive. A hysterosalpingogram might lead to a suspicion of the presence of pelvic adhesions; however, there is some degree of false-positive and -negative results. The definitive diagnosis depends on laparoscopy. The use of an internationally accepted classification, such as that of the American Fertility Society, allows investigators to compare the results of treatment. Various adjuvants have been used following lysis of adhesions to prevent their recurrence; they yield various results. The most significant recommendation is to prevent the occurrence of adhesions by following the principles of microsurgical technique during every surgical procedure.


Membrane topology and quaternary structure of cardiac gap junction ion channels.

The membrane topology and quaternary structure of rat cardiac gap junction ion channels containing alpha 1 connexin (i.e. Cx43) have been examined using anti-peptide antibodies directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold electron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic "loop" domain of at least Mr = 5100 (residues (101 to 142) is readily accessible to peptide antibody labeling. The native Mr = 43,000 protein can be protease-cleaved on the cytoplasmic side of the membrane, resulting in an Mr approximately 30,000 membrane-bound fragment. Western immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252-271. Immunoelectron microscopy demonstrated that the Mr approximately 13,000 carboxy-terminal peptide(s) is released after protease cleavage and does not remain attached to the Mr approximately 30,000 membrane-bound fragment via non-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of the alpha 1 polypeptide. Therefore, the Mr approximately 13,000 carboxyterminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexins indicates conservation in the molecular design of the gap junction channels formed by alpha or beta connexins.


ME491 melanoma-associated glycoprotein family: antigenic identity of ME491, NKI/C-3, neuroglandular antigen (NGA), and CD63 proteins.

Numerous monoclonal antibodies (MAbs) have been produced to antigens found in human melanomas. Three of the best characterized melanoma antigens include the melanoma-associated glycoproteins (MAGs) defined by two reagent families--the ME491 family (including ME491, 8-1H, and 8-2A) and the NKI/C-3 family (including NKI/C-3 and NKI/black-13)--as well as the neuroglandular antigen (NGA) defined by MAbs LS59, LS62, and LS140. These three antigens have significant similarities in tissue distribution, biosynthesis, and structure. The ME491 MAG has been cloned, mapped, and sequenced. Numerous non-melanoma-associated proteins (Sm23, CO-029, R2, TAPA-1, CD9, CD37, CD53, and CD63) have recently been shown to have significant homology to this sequence. We conducted this study to investigate the similarity between the two MAG antigens and NGA. Several reagents defining the three different melanoma antigens were compared, using competition immunoprecipitation, immunoassay, and inhibition radioimmunoassay techniques. Immunoassay experiments show that MAbs defining the three melanoma antigens bind to affinity-purified ME491 antigen and inhibit each other from binding in an inhibition radioimmunoassay. Competition immunoprecipitation experiments demonstrate that the ME491 and NKI/C-3 antibodies bind to NGA. Rabbit anti-ME491 idiotype serum recognizes determinants shared by NKI/C-3 and the anti-NGA MAbs. A competition immunoprecipitation experiment also confirms the identity of CD63, as defined by MAb RUU-SP 2.28, with the three melanoma antigens. These data indicate that the MAGs defined by ME491 and NKI/C-3 as well as the anti-NGA antibodies are epitopes of the same molecule, which is identical to CD63 by both immunochemical and molecular genetic investigations. Our results indicate that the data obtained in studies of these three melanoma antigens may be pooled, and we propose that the molecule recognized by these reagents be classified as CD63.


Randomized double-blind study comparing the effectiveness of balloon dilation of the prostate and cystoscopy for the treatment of symptomatic benign prostatic hyperplasia.

We attempted to determine the effectiveness of balloon dilation of the prostate for the treatment of symptomatic benign prostatic hyperplasia (BPH). A total of 33 men with symptomatic BPH signed informed consent to participate in a randomized, double-blind study comparing balloon dilation of the prostate and cystoscopy. Balloon dilation of the prostate and cystoscopy were performed with the patient under intravenous sedation after intraurethral instillation of viscous lidocaine and infiltration of the autonomic neural innervation of the prostate with 1% lidocaine. Efficacy was based upon improvement in Boyarsky symptom scores, increases in peak urinary flow rates and patient global assessment of symptomatic improvement. Efficacy was assessed at 1 and 3 months after treatment. A study nurse blinded to the randomization scheme administered the symptom score questionnaires and supervised the uroflowmetry studies. The study was randomized, since the mean baseline prostate sizes, peak urine flow rates, obstructive and irritative symptoms scores, post-void residual volumes and patient ages were similar in the 2 groups. The patient perceptions of treatment rendered were similar for the 2 treatment groups, confirming that the study was double-blind. The patients undergoing cystoscopy and balloon dilation of the prostate experienced a statistically significant improvement in the mean total symptom scores at 1 and 3 months. The mean total symptom scores after balloon dilation of the prostate and cystoscopy at 1 and 3 months were not significantly different. The mean peak urinary flow rates 1 and 3 months after balloon dilation of the prostate and cystoscopy were not significantly different from the baseline mean peak urinary flow rates. We demonstrate that balloon dilation of the prostate and cystoscopy are equally effective for the treatment of BPH. Since cystoscopy is considered a diagnostic modality, our study suggests that the efficacy previously observed after balloon dilation of the prostate is primarily placebo-related. The study does not support the indication of balloon dilation of the prostate for the treatment of symptomatic BPH.


The intraprostatic spiral: clinical results in 150 consecutive patients.

The clinical results of treatment of infravesical prostatic obstruction with an intraurethral coil in 150 consecutive patients are reported. A total of 80 patients had urinary retention and 70 had severe prostatism. Median observation time was 8.2 months, with a range of 0 to 40 months. In 75 patients the spiral was removed after a median of 4 months (range 0 to 30 months) because of planned prostatectomy in 17, urinary retention in 16, incontinence in 10, local discomfort in 7, no symptomatic improvement in 13 and causes not related to the spiral (stroke and so forth) in 7. Migration occurred 55 times in 42 patients but this only led to coil removal in 5. A total of 23 patients died with the coil in situ. Voiding symptoms improved considerably in the majority of the patients. Approximately two-thirds of the patients had no or few symptoms, while a fourth had moderate symptoms, leaving only approximately 10% with severe prostatism. Chronic bacteriuria was noted in 52 patients but was not a clinical problem. Calcification on the top and inside of the coil was noted mainly after long-term treatment, and probably necessitated exchange of the coil after 2 to 3 years. We conclude that the prostatic spiral is a useful alternative to an indwelling catheter. However, life-long followup is necessary in most patients.


Nuclear localization of androgen receptor in heterogeneous samples of normal, hyperplastic and neoplastic human prostate.

To facilitate an understanding of how androgens participate in the genesis of human benign hyperplasia and carcinoma we assayed androgen receptor in the epithelium and stroma of human prostatic tissue from 57 patients. Immunohistochemical staining of human androgen receptor was performed on 106 sections of normal prostate, benign prostatic hyperplasia (BPH) and prostate cancer. To determine variability of androgen receptor staining sections taken from different portions of the gland were studied. Frozen tissue sections were incubated with monoclonal antiandrogen receptor antibodies and staining was completed by the indirect avidin-biotin peroxidase method. Antibody staining was found mainly in the nucleus of prostatic epithelial cells, although some stromal cells also showed positive staining. Unlike normal prostate, there was a heterogeneous distribution of androgen receptor in BPH and prostate cancer. The androgen receptor content in well differentiated adenocarcinoma epithelium was significantly higher compared to moderately (p less than 0.05) and poorly (p less than 0.05) differentiated adenocarcinoma. Regardless of the origin of stromal tissue, some staining was observed. In each specimen studied the androgen receptor staining was consistent qualitatively and quantitatively for each pathological component throughout the specimen. These data confirm that androgen receptor is a nuclear receptor protein. Furthermore, they show the ability of monoclonal antibodies to reveal cellular/subcellular distribution of androgen receptor, and demonstrate a correlation between the degree of tumor differentiation and androgen receptor content in epithelial but not in stromal cells. These observations may have important implications for understanding the variable tumor response to hormone therapy.


Effects of rectal examination, prostatic massage, ultrasonography and needle biopsy on serum prostate specific antigen levels.

Measurement of serum prostate specific antigen (PSA) is commonly used to evaluate the prostate gland in a variety of clinical settings. We examined the effects of prostatic manipulations, including digital rectal examination, prostate massage, transrectal ultrasonography and transrectal needle biopsy, on serum PSA levels in 199 men. We detected no clinically significant difference between serum PSA levels obtained immediately before and at 5 or 90 minutes after rectal examination in 43 men. We observed falsely increased PSA levels (to greater than 4 ng./ml., Tandem-R) in 1 of 17 men (6%) following prostatic massage and in 3 of 27 men (11%) following ultrasonography. Transrectal needle biopsy caused an immediate increase in serum PSA in 92 of 100 men. In 29 of these 92 men (32%) when followed weekly serum PSA levels did not return to baseline as expected according to the published serum PSA half-life of 2 to 3 days. Biopsies taking 3 or fewer cores (7 patients) resulted in a smaller increase in serum PSA (mean 1.63 +/- 1.12 times the baseline level versus 6.24 +/- 1.10 times baseline, p less than 0.03) and a proportionally shorter duration of PSA elevation (mean 1.43 +/- 0.48 weeks versus 2.13 +/- 0.14 weeks, p = 0.20) than those taking 4 or more cores (93 patients). Prostate size and the presence of cancer had no influence on the duration of PSA elevation following biopsy. We conclude that digital rectal examination, prostatic massage and ultrasonography have minimal effects on serum PSA levels in most patients. However, prostatic needle biopsy usually causes marked elevations of serum PSA levels with a persistent PSA leak into the blood stream lasting longer than expected from the serum half-life of PSA in approximately 25% of the patients.


Prostate specific antigen density: a means of distinguishing benign prostatic hypertrophy and prostate cancer.

Isolated prostate specific antigen (PSA) determinations in asymptomatic individuals have not demonstrated sufficient sensitivity and specificity to be useful in the routine evaluation of prostate disease. To enhance the accuracy of serum PSA we have used a quotient of serum PSA and prostate volume, which we refer to as prostate specific antigen density (PSAD). Prostate volume in this study was calculated from magnetic resonance imaging determinations of benign prostatic hypertrophy (BPH) or from the dimensions of the surgical specimen of cancer using the formula, length x width x depth x 0.5 = volume. A total of 61 patients with prostatic disease clinically confined to the prostate glands (41 with prostate cancer undergoing radical prostatectomy and 20 with BPH) was evaluated. The mean PSAD for prostate cancer was 0.581 while that for BPH was 0.044 (p less than 0.002). No patient with BPH had a PSAD of greater than 0.117 and only 1 patient had a density of 0.1 or greater. Of 34 patients with a PSAD of 0.1 or greater 33 had prostate cancer. Only 2 of the 41 prostate cancer patients and 14 of the BPH patients had a PSAD of 0.05 or less. There were 11 patients with a PSAD of greater than 0.05 and less than 0.1, including 6 with prostate cancer (1 with P0 disease) and 5 with BPH. Of the 6 prostate cancer patients 5 had a PSA of 4.0 or less and among the 5 patients with BPH 4 had a serum PSA of greater than 4.0 and 1 had a PSA of greater than 10. These results suggest that PSAD may be useful in distinguishing BPH and prostate cancer.


The use of prostate specific antigen density to enhance the predictive value of intermediate levels of serum prostate specific antigen.

Prostate specific antigen (PSA) is an extremely valuable tumor marker. However, its use in detection is limited by its low positive and negative predictive values. The ability of serum PSA to distinguish between benign and malignant prostatic conditions is particularly poor in the intermediate range of 4.1 and 10 ng./ml. by the Hybritech assay. We used transrectal ultrasound determined prostate volumes in a well characterized population of 533 men to form a serum PSA/prostate volume ratio called prostate specific antigen density (PSAD). The prevalence of cancer in the entire population was 18.4%. Discriminant analysis according to negative or positive outcome allowed for the construction of nomograms, which resulted in a PSAD defined cancer risk ranging from 3 to 100%. Predictive value nomograms created from PSAD may allow for a more individualized approach to evaluation of patients with intermediate levels of Hybritech serum PSA.


Prostate cancer in nonurological patients with normal prostates on digital rectal examination.

Random systematic ultrasonographically guided transrectal core biopsies of the prostate were performed in 73 patients with pulmonary malignancies to exclude prostate cancer as a primary malignant neoplasm. Of the patients 41 had normal prostates as judged by digital rectal examination, 27 had firm prostates and 5 had clinical stage B nodules. Hypoechoic areas were seen on transrectal ultrasonography in 14 of the 41 patients (34%) with normal prostates. Biopsy of the hypoechoic areas in this subgroup detected only 1 grade II prostate cancer. In another patient with normal transrectal ultrasound grade I cancer was detected by mapping of the prostate with 6 systematic ultrasonographically guided transrectal core biopsies. Of the remaining 39 patients with normal prostates transrectal ultrasound detected no hypoechoic defect in 26, a specificity for the detection of prostate cancer of 67%. Multiple core biopsies revealed prostate cancer in 15 of the 32 cases of palpably abnormal prostates, including 12 that were hypoechoic. Prostate cancer is a rare histopathological finding in men with normal prostates on rectal examination. Transrectal ultrasound detected only 1 of 2 low volume prostate cancers in our study group. Thus, ultrasound seems to have little use in patients with prostates that appear normal on digital examination, and its specificity is low.


The distribution of prostate specific antigen in men without clinical or pathological evidence of prostate cancer: relationship to gland volume and age.

We estimated the in vivo prostate gland volume in 408 men (320 without clinical evidence of prostate cancer, and 88 with an abnormal digital rectal examination and/or transrectal prostate ultrasound and negative biopsies) using sequential step-section ultrasound analysis and correlated it to the serum prostate specific antigen (PSA) value. Of the men 331 (81.1%) had a PSA value of 4 ng./ml. or less. The PSA value was greater than 4 but less than or equal to 10 in 64 men (15.7%) and greater than 10 in 13 (3.2%). The men were subclassified by prostate gland volume at arbitrary break points. A total of 139 men (34.1%) had a gland of 25 cm.3 or less, 2.2% of whom had a PSA value of greater than 4. Further analysis revealed that the incidence of a PSA value greater than 4 increased as the prostate volume increased (18.4% for greater than 25 but less than or equal to 50, and 65.4% for greater than 50) and as age increased. We found a statistically significant association between prostate gland volume and patient age (p less than 0.00005) to the serum PSA concentration. The finding of a PSA value of greater than 10 was uncommon regardless of the prostate gland volume. Clinical implications of these results are discussed, and a statistical model to estimate the serum PSA by gland volume and patient age was constructed.


Screening for prostatic carcinoma with prostate specific antigen.

Prostate specific antigen (PSA), neutral serine protease secreted exclusively by prostatic epithelial cells, has a number of applications in the management of men with prostatic carcinoma. While it is widely recognized that elevated PSA correlates with the presence of carcinoma, little data exist regarding the use of PSA as the initial test in the early detection of prostatic cancer. We measured serum PSA levels in men older than 50 years and performed digital rectal examination and ultrasound guided prostate biopsy of those who had a PSA level of greater than 4.0 ng./ml. A total of 1,249 men entered the protocol, of whom 187 (15.0%) had PSA levels above 4.0 ng./ml. Digital rectal examination and ultrasound guided biopsy were performed at our facility in 105 patients (56.2%). A total of 32 carcinomas (30.5%) was detected, including 23 in men with PSA between 4.1 and 10.0 ng./ml. and 9 in men with a PSA of greater than 10.0 ng./ml. Of the 32 carcinomas 12 (37.5%) occurred in men with normal prostates or glands demonstrating only asymmetry on digital rectal examination, and 3 men had carcinoma despite normal digital rectal examination and no hypoechoic peripheral zone lesion detected on ultrasound. Of the 32 patients 30 had clinically localized carcinoma but 7 of the 16 undergoing radical prostatectomy had pathological upstaging. We conclude that PSA represents an important adjunct to digital rectal examination for the early detection of prostatic carcinoma. The efficacy of this or any other early detection test to decrease prostate cancer mortality necessitates the results of prospectively randomized clinical trials.


Serum prostate specific antigen as pre-screening test for prostate cancer.

Prostate cancer has become the most common cancer and the second cause of death due to cancer in men in North America. Since curative therapies are limited to early stages of the disease, the availability of an efficient, easy to perform, widely acceptable and cost-effective method of early detection of prostate cancer is particularly important. Thus, digital rectal examination, transrectal ultrasonography of the prostate as well as measurements of serum prostate specific antigen (PSA) were performed independently in a series of 1,002 men between 45 and 80 years old randomly selected from the electoral rolls of Quebec City and its vicinity as part of a screening program for prostate cancer. Using this population of randomly chosen men, various cutoff serum PSA values were selected in an attempt to find the optimal decision threshold that would indicate a much greater risk of having prostatic cancer. At a threshold value of 3.0 micrograms./l. the sensitivity and specificity of the test are 80.7 and 89.6%, respectively, while the area under the receiver operating characteristic curve reflecting the accuracy of the test is 87.8 +/- 3.3% (plus or minus standard deviation). Moreover, the negative predictive value was estimated at 98.6%, thus leaving only a 1.4% chance of missing cancer when the serum PSA value was 3.0 micrograms./l. or less. Most importantly, such a threshold level of serum PSA retains only 19% of the whole cohort as candidates for transrectal ultrasonography and expensive diagnostic procedures, thus leading to the finding of 1 prostate cancer of 4 such examinations. The present data indicate that simple measurement of serum PSA can be used efficiently as a pre-screening test for prostate cancer in the general population to identify, at a low cost, the subpopulation of men at a much greater risk of having prostate cancer, and who should then be submitted to the more elaborate and expensive diagnostic procedures.


The predictive significance of substaging stage A prostate cancer (A1 versus A2) for volume and grade of total cancer in the prostate.

Morphometric analysis was performed on 44 radical prostatectomy specimens for clinical stages A1 and A2 carcinoma of the prostate. The majority of stage A cancers (86%) were located in the transition zone of the prostate, while only 14% arose in the peripheral zone. The subclassification into stages A1 and A2 based on the percentage of cancer in the transurethral resection chips did not reliably distinguish those cancers of high volume (transurethral resection plus residual). All 6 cases with Gleason grade 4 elements in the transurethral resection chips had relatively high volume cancer. In 32 of the 44 cases (73%) unsuspected cancers unrelated to the tumor detected at transurethral resection were found in the radical prostatectomy specimen. Of these cancers 87% were nontransition zone tumors. Eight unsuspected cancers were larger than the stage A cancer but only 2 of them were larger than 1 cc. Post-resection serum prostate specific antigen (PSA) levels were elevated with increasing total residual cancer volume in the radical specimen. In 19 of 20 cases with a PSA of greater than 2.5 ng./ml. the total residual cancer volume was more than 0.9 cc, while in 7 of 8 with a PSA of less than 1 ng./ml. total residual tumor volume was lower than 0.4 cc.


Transrectal ultrasound imaging and ultrasound guided prostate biopsies in the detection of residual carcinoma in clinical stage A carcinoma of the prostate.

Planning treatment for patients diagnosed with stage A prostate cancer by transurethral resection or open enucleation can be difficult. Inability to determine the presence or absence of significant residual disease can result in unnecessary treatment for some individuals and inadequate treatment in others. Transrectal ultrasound imaging and systematic prostate biopsies offer a potential means of evaluating these patients. To determine the rate of residual cancer detection 3 groups of stage A prostate cancer cases were evaluated. Group 1 consisted of 39 patients who underwent radical prostatectomy. Preoperative ultrasound imaging revealed residual cancer in only 24%. Transrectal ultrasound guided systematic biopsies were performed in group 2 (25 patients) and revealed cancer in only 28%. Based on prior morphometric studies of prostatectomy specimens from stage A cases, a modification of the systematic biopsy method, which included anteriorly directed biopsies, was developed and applied to group 3 (47 patients). Cancer was detected in 47% of the patients, and it was detected by additional anterior biopsies alone in 11%.


Luteinizing hormone-releasing hormone downstaging of clinical stage C prostate cancer.

A total of 7 patients with clinical stage C prostate cancer determined by digital rectal examination, transrectal ultrasonography, radionuclide bone scanning and serum prostatic acid phosphatase determination was treated with luteinizing hormone-releasing hormone analogues for 2 to 5 months in an attempt to downstage the disease. Although substantial decreases in prostate specific antigen level and prostatic volume occurred, only 2 patients experienced pathological downstaging of disease. We conclude that luteinizing hormone-releasing hormone therapy for downstaging of clinical stage C prostatic cancer is of limited value.


Estramustine and vinblastine: use of prostate specific antigen as a clinical trial end point for hormone refractory prostatic cancer.

The combination of estramustine phosphate and vinblastine sulfate, 2 agents with separate and unique antimicrotubular effects, has demonstrated additive cytotoxicity against the DU145 human prostate derived cell line in vitro. We evaluated this combination in 25 patients with progressive hormone refractory prostate cancer. Of 24 patients with an elevated prostate specific antigen (PSA) level at the start of treatment 13 (54%, 95% confidence limits 34 to 74%) had a greater than 50% decrease in PSA levels on at least 3 consecutive biweekly determinations. The median decrease in PSA in responding patients was 64% (mean 71.7%) and the median duration of response was 7 months. In 5 patients with bidimensionally measurable disease 2 partial responses were observed. Treatment was well tolerated, with mild and manageable toxicity. This is a well tolerated outpatient treatment regimen for patients with hormone-refractory prostatic cancer which deserves further investigation.


Prostate specific antigen levels after radical prostatectomy in patients with organ confined and locally extensive prostate cancer.

A total of 230 patients with localized prostate cancer underwent radical retropubic prostatectomy at UCLA (pathological stage T1-3 N0, M0). Classification into groups included 115 patients with organ confined disease (group 1), 82 with invasion into or through the capsule (group 2) and 33 with seminal vesicle involvement (group 3). Median followup was 48 months. The 10-year, cause-specific survival was 96%, 90% and 63%, and 5-year, clinical, disease-free survival was 91%, 79% and 58% for the 3 groups, respectively. Recent prostate specific antigen (PSA) levels were measured in most patients, even those operated upon many years ago. Of the patients 41 had detectable (0.4 ng./ml. or greater) PSA levels without any other clinical evidence of progression and 15 with clinical evidence of progression had PSA levels in the detectable range at the time of clinical progression. When isolated detectable PSA was also considered an indicator of progression the 5-year and 10-year, disease-free rates were 61% and 41%, respectively. These data show that radical prostatectomy performed in patients with even microscopic invasion into the capsule, positive margins and seminal vesicle involvement is associated with a higher clinical progression rate than organ confined disease. If isolated detectable PSA is also considered an indicator of recurrence the disease-free survival after radical prostatectomy might be less than indicated by previous studies. The relationship among survival, local tumor extension and PSA must be carefully examined.


Urinary prostate specific antigen levels: role in monitoring the response of prostate cancer to therapy.

We have shown that prostate specific antigen (PSA) levels can be as readily obtained from voided urine as from serum samples. This procedure was found to give stable and reproducible results. PSA analyses were performed on voided urine collected from 42 patients with benign prostatic hypertrophy (BPH), 27 with stage D2 prostate cancer and 57 after radical prostatectomy. The 42 BPH samples had a mean urinary PSA level of 216 ng./ml., which did not correlate with estimated prostate size. For 4 of 5 patients with stage D2 disease who presented before hormonal therapy urinary PSA levels were greater than 50 ng./ml. For 22 stage D2 patients seen after initiation of hormonal therapy the majority had low urinary PSA levels. After initiation of hormonal therapy in most cases low urinary PSA levels were found in conjunction with high serum PSA values. However, in other cases we found high urinary PSA with low serum PSA levels. Of 43 patients who underwent radical prostatectomy for stages A to C disease it was noteworthy that 77% had elevated urinary PSA levels, while only 33% had elevated serum levels. Therefore, close to 80% of these patients have prostate tissue remaining locally after this operation.


Detection of local recurrence after radical prostatectomy by prostate specific antigen and transrectal ultrasound.

Twenty patients with detectable levels of prostate specific antigen (PSA) after radical prostatectomy with no identifiable distant metastases were evaluated for local recurrence by digital rectal examination and transrectal ultrasound combined with biopsies. Of the patients 9 (45%) were found to have histological evidence of local recurrence at the initial assessment. All 4 patients with an abnormal digital rectal examination had recurrent disease. Transrectal ultrasound displayed abnormalities in 12 of the 20 patients, 7 of whom had positive biopsies. Random biopsies of the vesicourethral junction were performed in 8 patients who had negative ultrasound findings and an unremarkable digital rectal examination, of whom 2 had histological documentation of local recurrence. Complications occurred in 1 patient (5%) who presented with clot retention. We conclude that PSA is an excellent tool for identification of recurrent disease after radical prostatectomy, and transrectal ultrasound guided biopsy is a useful diagnostic approach in patients suspected of local failure, especially when the digital rectal examination is unremarkable.


The clinical usefulness of serum prostate specific antigen after hormonal therapy of metastatic prostate cancer.

We longitudinally followed serum prostate specific antigen (PSA) levels in 48 patients who were treated with either orchiectomy, monthly luteinizing hormone-releasing hormone injection or continuous diethylstilbestrol for stage D2 prostate adenocarcinoma and achieved an objective response. Of the patients 34 had clinical evidence of disease progression (median remission duration 19 months). Median length of followup for the 14 patients who remained in remission was 42 months. Pretreatment performance status, pretreatment extent of metastases as measured by a bone scan and post-treatment nadir PSA level were univariately correlated with remission duration. After adjustment for the 2 former pretreatment variables, a highly significant independent effect of the nadir PSA level on remission duration persisted. Patients whose post-treatment nadir PSA level decreased below 4 ng./ml. had a significantly longer remission duration than those whose nadir PSA remained elevated (median 42 versus 10 months, p less than 0.0001). No cases were observed to progress (as defined by our criteria independent of PSA level) while the serial post-treatment PSA levels continued to decrease or remained at a plateau after reaching the nadir. The time at which the PSA began to increase once the nadir was reached predated objective evidence of progression in all patients except 2 in whom the 2 events occurred simultaneously (mean lead time 7.3 +/- 5.0 months). We conclude that following serial PSA levels in patients treated with androgen ablation for metastatic prostate cancer can aid in distinguishing favorable from nonfavorable responders early in the course of therapy and greatly assist in monitoring for progression.


Beneficial antiarrhythmic effect of beta-blockade in patients with dilated cardiomyopathy.

Antiarrhythmic effects of beta-blockade (BB) in patients with dilated cardiomyopathy were compared with those of various antiarrhythmic agents using ambulatory Holter monitoring. The BB therapy effectively suppressed ventricular extrasystoles (VEs) in 85% of patients, as evidenced by improvement in Lown's grade or a reduction in the number of the highest grade VEs greater than 50%. In contrast, conventional antiarrhythmic agents, except flecainaid and amiodarone, were poorly effective in suppressing VEs. BB therapy gradually increased left ventricular fractional shortening (16 +/- 6% to 22 +/- 12%) and improved 12-month survival rates as compared with those receiving conventional therapy (93 vs 69%). This antiarrhythmic potency seemed to be an additional therapeutic efficacy of BB in the management of patients with dilated cardiomyopathy, which frequently associated with serious VEs.


Endothelial leukocyte adhesion molecule-1-dependent adhesion of colon carcinoma cells to vascular endothelium is inhibited by an antibody to Lewis fucosylated type I carbohydrate chain.

Endothelial leukocyte adhesion molecule-1 (ELAM-1) has been determined to be the mediator of adhesion of colon carcinoma cells to interleukin-1 (IL-1)-activated endothelial cells. To identify ELAM-1 ligand in colon carcinoma cells, we have screened a series of 11 monoclonal antibodies directed to these cells and found that only one MBr8 was able to inhibit the IL-1-induced increment in adhesion of HT29 and of SW948 colon carcinoma lines to endothelial cells. In contrast, MBr8 did not bind to polymorphonuclear cells, monocytes, and lymphocytes and did not inhibit polymorphonuclear adhesion to IL-1-activated endothelial cells. As expected, an ELAM-1 monoclonal antibody strongly inhibited IL-1 induced increment of adhesion of HT29, SW948, and polymorphonuclear cells. As negative control, MG63 osteosarcoma cells were used. These cells adhere more efficiently to IL-1 activated endothelial cells but MBr8 and ELAM-1 monoclonal antibodies did not affect their adhesion. The effect of MBr8 was also tested in an experimental system in vivo. As described previously, radiolabeled HT29 cell retention in the lung of nude mice was increased in animals given IL-1. MBr8 administration to nude mice or pretreatment of tumor cells with it inhibited this effect. These data suggest that cell adhesion to ELAM-1 might be mediated by different, cell type specific, sugar ligands.


Effect of FK506 on insulin secretion in normal dogs.

In this report, we describe the effect of FK506 on glucose-mediated insulin release in normal dogs. Dogs were placed into one of two groups, group 1 dogs received FK506 (1 mg/kg/d orally) for 2 weeks, and group 2 dogs received FK506 at the same dose, but for 4 weeks. Following the treatment period, both groups of dogs were allowed a recovery period during which time no FK506 was administered. Intravenous glucose tolerance tests (IVGTT) were performed (0.5 mg/kg IV) before FK506 treatment, after 2 or 4 weeks of treatment, and following the recovery periods. Complete blood cell counts (CBC) and serum chemistries were also obtained at these times. Following FK506 treatment for either 2 or 4 weeks, the dogs experienced a delay in glucose disappearance in response to IV glucose injection. Insulin secretion during IVGTT was unchanged in dogs treated for only 2 weeks, but was significantly decreased in dogs treated for 4 weeks. Following the recovery period, glucose disappearance during IVGTT returned to normal in dogs that were treated for 2 weeks, and was more rapid than normal in dogs that had been treated for 4 weeks. Insulin secretion after the recovery period remained unchanged in group 1 dogs, but continued to be significantly reduced in group 2 dogs that had received FK506 for 4 weeks. No significant change was detected in the CBCs or serum chemistries.


Interactions of galanin and arginine on growth hormone, prolactin, and insulin secretion in man.

Galanin (GAL), a 29 amino acid neuropeptide, is known to increase both basal and growth hormone-releasing hormone (GHRH)-induced growth hormone (GH) secretion while not significantly increasing prolactin (PRL) secretion in man. GAL is also endowed with an inhibiting effect on glucose-stimulated insulin release in animals, but not in man. We studied the effect of GAL (80 pmol/kg/min infused over 60 minutes) on the arginine- (ARG, 30 g infused over 30 minutes) stimulated GH, PRL, insulin, and C-peptide secretion in eight healthy volunteers (age, 20 to 30 years). GAL induced an increase of GH (GAL v saline, area under curve [AUC], mean +/- SEM: 316.5 +/- 73.9 v 93.2 +/- 20.9 micrograms/L/h, P less than .05), but failed to modify both PRL and insulin secretion. GAL enhanced the ARG-induced stimulation of both GH (1,634.1 +/- 293.1 v 566.9 +/- 144.0 micrograms/L/h, P less than .02) and PRL secretion (1,541.9 +/- 248.8 v 1,023.8 +/- 158.7 micrograms/L/h, P less than .02). On the contrary, GAL blunted the ARG-stimulated insulin (816.3 +/- 87.7 v 1,322.7 +/- 240.9 mU/L/h, P less than .05), as well as C-peptide secretion (105.1 +/- 9.8 v 132.8 +/- 17.3 micrograms/L/h, P less than .02). ARG administration induced a transient increase of glucose levels (P less than .01 v baseline) followed by a significant decrease (P less than .05 v baseline). This latter effect was prevented by the coadministration of GAL. In conclusion, these results show that in man GAL potentiates the GH response to ARG, suggesting that these drugs act at the hypothalamic level, at least in part, via different mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)


Drosophila melanogaster paramyosin: developmental pattern, mapping and properties deduced from its complete coding sequence.

Several cDNA clones encoding the complete Drosophila paramyosin sequence, including two potential polyadenylation sites, have been obtained. Southern analysis and in situ hybridization to polytene chromosomes indicate that in Drosophila the paramyosin gene is single copy, located on the left arm of the third chromosome at region 66D14. Northern analyses show predominantly two different RNAs which are the products of the choice between the two alternative polyadenylation sites. The two species begin to be synthesized around 10 h of development when embryonic muscles are formed, expression peaking at the end of embryogenesis. The protein is first expressed at germ band shortening in association with muscle precursor cells. A second maximum of paramyosin RNA expression occurs at late pupal stages when the higher molecular weight form becomes more abundant. In young adults this species becomes the main transcript detected. The 102 kDa polypeptide sequence is highly similar to that of Caenorhabditis elegans paramyosin. The protein has a central alpha-helical coiled-coil rod, organized in 29 groups of four typical seven-residue repeats and flanked by two short non-alpha-helical regions. Several leucine zippers are located on the hydrophobic face of the alpha-helix in paramyosin which, together with disulfide bonds between cysteines, are probably involved in the stabilization of the dimer. The structural and functional properties of Drosophila paramyosin deduced from the sequence are compared with those of known invertebrate myosins and paramyosins.


Structure and expression of AtS1, an Arabidopsis thaliana gene homologous to the S-locus related genes of Brassica.

Genetic and molecular analysis of the self-incompatibility locus (S-locus) of the crucifer Brassica has led to the characterization of a multigene family involved in pollen-stigma interactions. While the crucifer Arabidopsis thaliana does not have a self-incompatibility system, S-related sequence were detected in this species by cross-hybridization with Brassica DNA probes. In this paper, we show that an A. thaliana S-related sequence, designated AtS1, is expressed specifically in flower buds. Sequence analysis suggests that AtS1 encodes a secreted glycoprotein that is most similar to the Brassica S-locus related protein SLR1. As has been proposed for SLR1, this gene may be involved in determining some fundamental aspect of pollen-stigma interactions during pollination. The molecular and genetic advantages of the Arabidopsis system will provide many avenues for testing this hypothesis.


Accumulation of chloroplast psbB RNA requires a nuclear factor in Chlamydomonas reinhardtii.

We have isolated and characterized a nuclear mutant, 222E, in Chlamydomonas reinhardtii, which is defective in photosystem II (PSII). Polypeptide P5, the product of psbB, is not produced in this mutant, leading to a destabilization of other PSII components. The mutant specifically fails to accumulate psbB transcripts and displays an altered transcription pattern downstream of psbB. Pulse-labelling experiments suggest that mRNA stability and/or processing are affected by the alteration of a nuclear gene product in this mutant. We show that the C. reinhardtii psbB gene is co-transcribed with a small open reading frame that is highly conserved in location and amino acid sequence in land plants. The 5' and 3' termini of the psbB transcript have been mapped to 35 bases upstream of the initiation codon and approximately 600 bases downstream of the stop codon. The 3' flanking region contains two potential stem-loops, of which the larger (with an estimated free energy of -46 kcal) is near the 3' terminus of the transcript.


A comparison of leaf thionin sequences of barley cultivars and wild barley species.

Leaf thionins of several barley cultivars and wild barley species were analysed. We found large differences in the numbers of leaf thionin genes in different Hordeum species. While, for instance, cultivars of Hordeum vulgare (Section Hordeum) contain more than 50 copies of thionin genes per haploid genome, the numbers are much lower in Hordeum species belonging to the sections Critesion and Stenostachys. The apparent number of genes correlates with the concentration of leaf thionin and its mRNA, which differs more than 100-fold among various Hordeum species. Leaf thionins are synthesized as high molecular weight precursor proteins that contain a signal peptide domain, a thionin domain and an acidic polypeptide domain. Analysis of cDNA clones of leaf thionins revealed a family of related transcripts. When the predicted amino acid sequences of the precursor molecules of wild barley species were compared, differences in the sequence variability of the three domains became apparent. The frequency of amino acid exchanges is much higher within the thionin domain than in the signal peptide and acidic polypeptide domains. The amino acid exchanges within the thionin domain do not occur at random but are confined to variable regions that alternate with highly conserved areas. Conserved regions comprise mostly cysteine residues and adjacent amino acids and may be important for the correct formation of the specific disulphide configuration of thionins.


Chloride transport blockers prevent N-methyl-D-aspartate receptor-channel complex activation.

In cultured spinal cord neurons, we found that blockers of chloride transport (furosemide, a widely used loop diuretic, and the related compounds piretanide and bumetanide, as well as niflumic and flufenamic acids, used as antiinflamatory agents) prevented N-methyl-D-aspartate (NMDA) receptor activation in a dose-dependent manner and are specific for this class of glutamate receptor. Antagonism of NMDA-mediated currents by chloride transport blockers was voltage independent and showed fast on-ff kinetics. The action was noncompetitive with NMDA and did not arise from interaction with the Zn2+ inhibitory site, because blockade of NMDA-induced responses by furosemide and Zn2+ was additive. The inhibition was greater in a low concentration of glycine, but it could not be overcome by increasing the glycine concentration (up to 100 microM). In contrast, the inhibition was attenuated by the polyamine spermine. Because the presence of spermine was not required for inhibition to develop, we conclude that chloride transport blockers are noncompetitive antagonists of the NMDA receptor, likely acting as inverse agonists of the polyamine site. This action may explain the protective effect that has been shown for some of these drugs in neuronal degeneration; because they also prevent neuronal swelling, they may be good starting compounds for synthesis of appropriate therapeutic agents to ameliorate excitotoxicity.


Phosphorothioate oligonucleotides are inhibitors of human DNA polymerases and RNase H: implications for antisense technology.

Phosphorothioate oligodeoxycytidine (S-dCn) was used as a model compound to examine the impact of the number of phosphorothioate linkages and their position on the inhibition of human DNA polymerases and RNase H in vitro. S-dCn with a chain length longer than 15 could inhibit human DNA polymerases and RNase H activities, in a linkage number-dependent manner. Longer oligomers were more potent inhibitors than shorter ones. Kinetic studies indicated that S-dC28 was a competitive inhibitor of DNA polymerase alpha and beta with respect to the DNA template, whereas it was a noncompetitive inhibitor of polymerases gamma and delta. S-dC28 was also a competitive inhibitor of RNase H1 and H2 with respect to RNA-DNA duplex. Susceptibility of these enzymes to inhibition by S-dC28 was in the order of delta approximately gamma greater than alpha greater than beta and RNase H1 greater than RNase H2. Structural-activity relationships were explored with a group of S-dC28 analogs that have phosphorothioate internucleotide linkages at various positions. The inhibitory effect depended on the total number of thioate linkages, rather than the position of the linkages within the oligomer or the chain length itself. No sequence specificity was found. In the presence of the complementary RNA, antisense phosphorothioates (S-oligos) exerted a biphasic effect on RNase H activity. At low concentrations S-oligos could enhance the cleavage of the RNA portion of S-oligo-RNA duplex, whereas at high concentrations (in excess of the complementary RNA) S-oligos could inhibit RNase H and protect the complementary RNA from degradation. Together, these results suggest that the non-sequence-specific inhibitory effect of S-oligos should be taken into consideration in designing antisense inhibitors. This inhibitory activity could be avoided by decreasing the number of phosphorothioate linkages at the backbone, and S-oligos of 15-20 residues are preferable in antisense molecule design.


Quinoxaline derivatives: structure-activity relationships and physiological implications of inhibition of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated currents and synaptic potentials.

The inhibitory potencies at excitatory amino acid (EAA) receptors of 11 quinoxaline derivatives were evaluated in two-electrode voltage-clamp recordings of Xenopus oocytes injected with rat cortex mRNA. Currents activated by kainate or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) in Xenopus oocytes were inhibited competitively by all the quinoxaline derivatives, with apparent Ki values ranging from 0.27 to 300 microM against kainate and from 0.25 to 137 microM against AMPA. An excellent correlation was observed between inhibitory potencies of the quinoxaline derivatives against kainate and AMPA currents, in support of the contention that in this preparation these two agonists act at a single site. All 11 quinoxaline derivatives also inhibited current activated by the combination of glycine and N-methyl-D-aspartate (NMDA), apparently acting at the glycine site, and did so over a narrower range of apparent Ki values (0.37-8.1 microM). The correlation between the quinoxalines' kainate/AMPA potencies and their glycine/NMDA potencies was relatively weak. Thus, the quinoxaline derivatives were all good antagonists of glycine/NMDA currents and displayed a greater range of potencies against kainate and AMPA. The inhibitory effects of the six quinoxaline derivatives most potent in the Xenopus oocyte experiments were also tested against the excitatory postsynaptic field potential (EPSFP) recorded in the pyramidal cell dendritic field of the CA1 region of hippocampal slices after stimulation of the Schaffer collateral-commissural pathways. In slices superfused with "normal" medium (containing 1 mM Mg2+), in which the EPSFP is mediated primarily by non-NMDA receptors, IC50 values correlated closely with the Ki values against kainate/AMPA obtained in oocyte experiments but were approximately 8-fold higher. Similarly, in slices superfused with nominally Mg(2+)-free medium, in which the EPSFP is amplified due to a relief of the Mg2+ block of NMDA receptors, IC50 values correlated closely with the Ki values against glycine/NMDA obtained in oocyte experiments but were 60-fold higher. This comparison of results from the two experimental systems lends further support to the argument that hippocampal synaptic transmission is mediated postsynaptically by kainate/AMPA-type and NMDA/glycine-type EAA receptors that are pharmacologically indistinguishable from those expressed in mRNA-injected Xenopus oocytes. Furthermore, it suggests that EAA receptors in situ may be nearly saturated by high local concentrations of the endogenous ligands, a condition that would contribute substantially to the apparent non-NMDA receptor selectivity of certain quinoxaline derivatives.


Detection by fluorescence analysis of DNA unwinding and unscheduled DNA synthesis, of DNA damage and repair induced in vitro by direct-acting mutagens on human lymphocytes.

The sensitivity and reliability of UDS and FADU in detecting mutagenic effects were compared by measuring DNA damage and repair in PBL treated in vitro with UV light, MMS and BPDE. The results indicate that FADU is more sensitive than UDS, as it is able to detect DNA damage at doses 3-4-fold lower. We also determined the DNA damage and repair induced by the above agents on lymphocyte samples from different donors by FADU and UDS, confirming that the DNA repair process in humans is characterized by interindividually variable efficiency.


Detection of benzo[a]pyrene-diol-epoxide-DNA adducts in white blood cells of psoriatic patients treated with coal tar.

An enzyme-linked immunosorbent assay (ELISA) was used to detect BPDE-DNA adducts in white blood cells of 23 psoriatic patients undergoing clinical coal tar therapy. Ten of these patients were reanalyzed 2-5 months after the end of the coal tar treatments. The results show that the mean adduct level during the treatment period was 0.26 +/- 0.16 fmole BPDE/micrograms DNA (7.7 +/- 4.9 adducts/10(8) nucleotides), while 2-5 months later the mean adduct level had decreased significantly (P less than 0.005) to 0.11 +/- 0.08 fmole BPDE/micrograms DNA (3.3 +/- 2.4 adducts/10(8) nucleotides). No relationship could be ascertained between the level of exposure and the amount of BPDE-DNA adducts. In addition, no difference in the level of DNA adducts was found between smoking and non-smoking patients.


Unscheduled DNA synthesis induced by the antitumor drug vincristine in germ cells of male mice.

The cytotoxic and genotoxic effects of vincristine (VCR) on germ cells of male mice were investigated. Several parameters (the scale and the time course of unscheduled and scheduled DNA synthesis in spermatocytes and spermatids and the number of sperm present in caudal epididymides) were analyzed. Our results show that intraperitoneal administration of a single dose of VCR resulted in: (1) damage to DNA in spermatocytes and spermatids; (2) a reduction in the rate of germ-cell development; and (3) killing of the non-proliferating spermatid cells. Damage of DNA in germ cells indicates that VCR may have potential genetic hazards to patients who receive it in antitumor therapy.


Micronucleus induction in bone-marrow cells following consumption of cooked beef in mice. Preliminary investigations.

The bone-marrow micronucleus assay was used to investigate whether the consumption of cooked meat could induce chromosome damage. There was no difference in the micronucleus frequency of mice on normal diet (cereal-based, non-purified diet) and mice on normal diet supplemented with microwaved meat. However, supplementing the normal diet with well-done pan-fried meat or rare charcoal-barbecued meat or well-done charcoal-barbecued meat produced significant increments in the micronucleus frequency of polychromatic erythrocytes--the increments were of the order of 73% (p = 0.045), 90% (p = 0.047) and 136% (p = 0.001) respectively and they were observed after a 21-day feeding trial. These results suggest that ingestion of well-done pan-fried or barbecued meat may increase genetic damage, however, the accompanying decreased intake of vegetable constituents may have also contributed to the observed changes.


Cytogenetic study of persons occupationally exposed to ethylene oxide.

Ten persons occupationally exposed to ethylene oxide (EO), used in the sterilization of medical instruments, were studied at a hospital. The estimated concentration to which they were exposed was 60-69 ppm, TWA. Peripheral blood samples from 10 workers and 10 controls of the same age and sex were taken to determine the frequency of sister-chromatid exchanges (SCE) and chromosomal aberrations (CA). The mean frequencies of SCE/cell (X = S) were 13.27 for the exposed workers and 6.05 for controls. Chromosome aberration frequencies in exposed individuals were significantly increased compared with controls. A significant relationship between the frequencies of SCE and CA and EO exposure was demonstrated. Blood chemistry parameters such as urea, creatinine, uric acid, lactic dehydrogenase, glutamic oxaloacetic and pyruvic transaminases, luteinizing gonadotropin and follicle stimulating gonadotropin and thyrotropin were also measured and found to be within the normal range.


The detection of promutagen activation by extracts of cells expressing cytochrome P450IA2 cDNA: preincubation dramatically increases revertant yield in the Ames test.

Two slightly different protocols, the plate incorporation method and the preincubation method, are used in the Ames Salmonella mutagen test. Using a preincubation method, we recently demonstrated efficient activation of a number of food-derived promutagens by extracts of mammalian cells expressing cDNAs of rat-liver cytochrome P450IA2 and of a P450IA2-IA1 hybrid. We report here that, for 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 1-aminoanthracene and several other promutagens, preincubation dramatically increased the number of revertant colonies in the Ames test when extracts of cytochrome P450IA2-containing transfected cells or low concentrations of rat-liver extracts were used as the source of activating enzymes. At higher concentrations of rat-liver extract protein, the effect of preincubation was less pronounced. The effect of preincubation was not due to the low protein concentrations in the assays since increasing the total protein concentration did not abolish the requirement for preincubation for the detection of MeIQ activation at low concentrations of rat-liver extract. In experiments where P450IA2 synthesized in transfected cells in culture is used to study promutagen activation, the plate incorporation protocol may seriously underestimate the capacity of cell extracts to activate promutagens. Thus, interlaboratory comparisons become difficult and unnecessarily large quantities of cell extract protein may be needed to detect promutagen activation. Whenever Ames test assays are carried out under conditions where P450 concentration limits revertant yield, it would be prudent to examine both the preincubation and plate incorporation protocol.


Plant extracts induce chromosome aberrations and sister-chromatid exchanges in Chinese hamster ovary cells and human lymphocytes.

Effects of extracts from Vicia faba were compared with those of Zea mays for the induction of sister-chromatid exchanges (SCEs) and of chromosome aberrations (CAs) in Chinese hamster ovary (CHO) cells. CA induction by the maize extract was also tested in human lymphocytes. The extracts from roots and leaves of Vicia faba induced CAs and SCEs in CHO cells. The extracts from maize leaves also induced SCEs and CAs in CHO cells, and CAs in human lymphocytes. Maize extracts were more potent in inducing SCEs than Vicia extracts and the SCE- and CA-inducing capacity of maize extracts decreased during preincubation before addition to cells.


Genotoxicity and cell proliferative activity of a nitrosated Oroxylum indicum Vent fraction in the pyloric mucosa of rat stomach.

In vivo genotoxic activity and cell proliferative activity were examined in the stomach mucosa of male F344 rats by in vivo short-term methods after oral administration of a nitrosated Oroxylum indicum Vent (OiV) fraction, which had been found to be mutagenic without S9 mix to Salmonella typhimurium TA98 and TA100. Administration of the nitrosated OiV fraction at doses of 1 and 2 g/kg body weight induced dose-dependent DNA single-strand scission (p less than 0.02), determined by the alkaline elution method, in the stomach pyloric mucosa 2 h after its administration: a dose of 2 g/kg body weight induced an 18-fold increase in the DNA elution rate constant. Administration of the nitrosated OiV fraction at doses of 0.7-2.8 g/kg body weight also induced dose-dependent increases, up to 11-fold (p less than 0.05), in replicative DNA synthesis in the stomach pyloric mucosa 16 h after its administration. Moreover administration of the nitrosated OiV fraction at doses of 0.25-2.0 g/kg body weight induced dose-dependent increases, up to 100-fold, in ornithine decarboxylase activity in the stomach pyloric mucosa with a maximum 4 h after its administration. These results demonstrate that the nitrosated OiV fraction has genotoxic and cell proliferative activity in the pyloric mucosa of rat stomach in vivo.


Inducible stable DNA replication in Escherichia coli uvr+ and uvr- cells, treated with genotoxic chemicals.

Inducible stable DNA replication (iSDR) provoked by a damaging treatment with MMS, MNU, MNNG, NFAA, NFN, 4NQO, NAL or MMC, was followed in both repair-competent E. coli PQ35 and its uvrA derivative E. coli PQ37. In contrast to SOS-inducible mutagenesis, which is more pronounced in excision-deficient cells, iSDR was more obvious in repair-competent cells. This may be due to special features of iSDR and need not indicate involvement of the uvrA gene product in it.


Bioassay-directed fractionation of mutagenic PAH atmospheric photooxidation products and ambient particulate extracts.

Simulated atmospheric gas-phase reactions of naphthalene, fluorene and phenanthrene have been carried out in an environmental chamber with bioassay-directed chemical analysis of the reaction products. Nitro-PAH were found to be the most significant mutagens formed from the reactions of naphthalene and fluorene. The mutagram (bar graph of mutagenic activity versus HPLC fraction) of the phenanthrene reaction products closely resembled that of an ambient air particulate extract with the most mutagenic activity being in a fraction more polar than that in which the nitro-PAH elute. Nitrophenanthrene lactones (nitro-6H-dibenzo[b,d]pyran-6-ones) were found to account for the observed activity of this polar fraction of the phenanthrene reaction products. It has been shown that the utilization of an environmental chamber with a known PAH-starting material and the ability to produce sufficient product for isomer-specific identifications of mutagens is a promising complement to bioassay-directed fractionation of ambient air particulate extracts.


Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein.

Expression of P-glycoprotein, the product of the MDR1 gene, confers multidrug resistance on cell lines and human tumours (reviewed in refs 1,2). P-glycoprotein (relative molecular mass 170,000) is an ATP-dependent, active transporter which pumps hydrophobic drugs out of cells, but its normal physiological role is unknown. It is a member of the ABC (ATP-binding cassette) superfamily of transporters, which includes many bacterial transport systems, the putative peptide transporter from the major histocompatibility locus, and the product of the cystic fibrosis gene (the cystic fibrosis transmembrane regulator, CFTR). CFTR is located in the apical membranes of many secretory epithelia and is associated with a cyclic AMP-regulated chloride channel. At least two other chloride channels are present in epithelial cells, regulated by cell volume and by intracellular Ca2+, respectively. Because of the structural and sequence similarities between P-glycoprotein and CFTR, and because P-glycoprotein is abundant in many secretory epithelia, we examined whether P-glycoprotein might be associated with one or other of these channels. We report here that expression of P-glycoprotein generates volume-regulated, ATP-dependent, chloride-selective channels, with properties similar to channels characterized previously in epithelial cells.


The cocaine-insensitive component of non-exocytotic efflux of noradrenaline from adrenergic axons in the isolated rat tail artery.

The working hypothesis was that the cocaine-insensitive component of non-exocytotic efflux of noradrenaline represents diffusion of the unprotonated amine across the axonal membrane. It was tested by examination of the effect of changing axoplasmic pH--and thus the fraction of extravesicular noradrenaline in the unprotonated form--on the overflows of endogenous noradrenaline and 3,4-dihydroxyphenylethylene glycol from rat tail arteries. The catechols were assayed by liquid chromatography with amperometric detection. To dissipate the H+ gradient across the axonal membrane, the tissues were incubated in media of different pH, in which Na+ was completely replaced with K+ and which were HCO3(-)-(and Ca(2+)-)free. Exposure of the tissues to these media produced substantial, but reversible increases in the overflow of noradrenaline. Subsequently, the overflows of both noradrenaline and the glycol kept rising, but their ratio did not change. Cocaine (0.1 mmol/l) lowered the (noradrenaline overflow: glycol overflow) ratio significantly. The ratio observed in its presence increased steeply with decreasing external and, presumably, axoplasmic pH. Addition of valinomycin and carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (1 mumol/l each) to the cocaine-containing media more than doubled the overflows without altering significantly the ratio. Under identical conditions, the overflow of noradrenaline from preparations with inactive neuronal monoamine oxidase did not decrease with decreasing pH. Since, in the presence of cocaine, the overflow ratio increased--rather than decreased--with decreasing pH, and because the overflow or noradrenaline from preparations with inactive monoamine oxidase did not decline with pH, the cocaine-insensitive component of noradrenaline efflux does not seem proportional to the axoplasmic concentration of the unprotonated amine.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular aspects of glutamate receptors and sodium-calcium exchange carriers in mammalian brain: implications for neuronal development and degeneration.

N-Methyl-D-aspartate (NMDA) and L-glutamate activate membrane receptors that produce substantial permeation of Na+, K+ and Ca2+ through the neuronal membrane. These ionic fluxes are intimately linked to processes that regulate neuronal survival, growth and differentiation. Intracellular free Ca2+ concentrations are thought to be particularly important determinants of the vulnerability of neurons to excessive excitatory stimulation produced through activation of NMDA receptors. In order to understand the molecular events involved in both NMDA receptor activation and regulation of intracellular Ca2+ levels, we have purified and reconstituted the protein complexes that form the NMDA/glutamate receptors in rat brain synaptic membranes and those that constitute the Na(+)-Ca2+ antiporters in bovine brain synaptic membrane. The molecular properties of these protein complexes are described, and information from the most recent studies of exploration of the molecular structures of these receptors and transport carriers is summarized.


Possible role of cGMP in excitatory amino acid induced cytotoxicity in cultured cerebral cortical neurons.

Using cultured cerebral cortical neurons at mature stages (9 days in culture, d.i.c.) it was demonstrated that glutamate, NMDA (N-methyl-D-aspartate) and to a lesser extent KA (kainate) increase the intracellular cGMP concentration ([cGMP]i) whereas no such effect was observed after exposure of the cells to QA (quisqualate) and AMPA (2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate). No effect of glutamate, NMDA and KA was observed in immature neurons (2 d.i.c.). The pharmacology of these cGMP responses was investigated using the glutamate antagonists APV (2-amino-5-phosphonovalerate) with selectivity for NMDA receptors, CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione) with selectivity for non-NMDA receptors and the novel KA selective antagonists AMOA (2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionate) and AMNH (2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)methyl-5-methyl-3- oxoisoxazolin-4-yl]propionate). In addition, the cytotoxicity of glutamate, NMDA and KA was studied and found to be enhanced by addition of the non-metabolizable cGMP analogue 8-Br-cGMP. On the contrary, the toxicity of QA and AMPA was not affected by 8-Br-cGMP. Pertussis toxin augmented the toxicity elicited by glutamate, NMDA, KA and QA but not that induced by AMPA. On the other hand, only glutamate and KA induced toxicity was potentiated by cholera toxin, which also enhanced the stimulatory effect of glutamate and NMDA but not that of KA on the cellular cGMP content. The toxicity as well as the effects on intracellular cGMP levels could be antagonized by the specific excitatory amino acid (EAA) antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)


Presynaptic modulation of amino acid release from synaptosomes.

Using synaptosomes prepared from whole rat brain, the spontaneous, calcium-independent, and calcium-dependent release of glutamate and GABA was assessed. Time intervals of 1-30 seconds were studied. Spontaneous release of glutamate (but not GABA) was elevated by 10 microM NMDA or AMPA by thirty seconds. This stimulation was partially calcium-dependent. Calcium-dependent release induced by 30 mM KCl was biphasic, confirming previous findings. This release was stimulated at all time periods by the presence of 10 microM NMDA or AMPA in an antagonist-sensitive manner. These data suggest that glutamate and GABA are released from vesicular stores in rat synaptosomes and that some of this release is modulated by presynaptic glutamate receptors.


Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used convention strategies in our search for a suitable composition of a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization - homogeneity of tertiary structure--is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.


Zinc-induced molt: evidence for a direct inhibitory effect on granulosa cell steroidogenesis.

Results from previous studies indicate that the use of dietary zinc may provide an effective means to initiate an induced molt in laying hens. Although much evidence indicates that high concentrations of zinc (10,000 to 20,000 ppm) cause the cessation of lay primarily by depressing feed intake, recent data suggest that lower concentrations (2,800 ppm) in a calcium-deficient diet may act via a direct action on the ovary. Therefore, a series of in vitro studies was conducted to evaluate whether zinc can affect granulosa cell progesterone production. Incubation of granulosa cells from the largest preovulatory (F1) follicle with zinc as zinc sulfate (.1 to 10 microM) had no effect on basal progesterone production. By contrast, ovine luteinizing hormone-stimulated progesterone production was inhibited (P less than .05) in a dose-related fashion by zinc in both the sulfate and acetate forms (1 to 10 microM). Furthermore, zinc attenuated oLH- and forskolin-induced cyclic adenosine monophosphate (cAMP) formation, and inhibited 8-bromo-cAMP- and calcium ionophore (A23187)-induced progesterone production. Such results indicate both pre- and post-cAMP sites of action for zinc's inhibitory actions on progesterone production in F1 granulosa cells. Finally, ovine follicle-stimulating hormone-stimulated cAMP accumulation and progesterone production in granulosa cells collected from 9- to 12-mm follicles (a stage of development representing the early, rapid growth phase) were suppressed (P less than .05) by co-incubation of cells with zinc.(ABSTRACT TRUNCATED AT 250 WORDS)


Continuous endotoxin infusion suppresses rat spleen cell production of cytokines.

Endotoxin, i.e., lipopolysaccharides, was continuously infused into rats at a nonlethal dose by means of an implanted osmotic pump for up to 2 weeks. The pump was connected to the jugular vein by a polyethylene catheter. Administration of endotoxin via the pump compromised the ability of spleen cells to produce the lymphokines interleukin 1 and tumor necrosis factor after stimulation in vitro with endotoxin. In addition, the ability of the spleen cells to produce alpha/beta-interferon in response to endotoxin in vitro was also examined, as was the capability of the spleen cells to produce gamma-interferon following stimulation with concanavalin A. Suppression of the expected interleukin 1 and tumor necrosis factor production by spleen cells from rats continuously infused with endotoxin was observed. There was also a moderate effect on interferon production, but this was much less. These results provide further findings indicating the unresponsiveness of spleen cells to lipopolysaccharides, as well as to a nonspecific plant mitogen, following continuous infusion of endotoxin into rats via an implanted osmotic pump. Additional studies are needed to determine the mechanisms involved in such suppression.


Correlations between catecholamine levels and sexual behavior in male zebra finches.

In zebra finches, the combined actions of estrogens and androgens activate male courtship, including singing, and also strongly modulate norepinephrine (NE) levels and turnover in brain areas known to be involved in controlling courtship behavior. To determine whether changes in NE levels mediate changes in courtship, we administered DSP-4 to males and measured its effects on monoamine levels and reproductive behavior. DSP-4 treatment did not affect serotonin (5-HT), had small, variable effects on dopamine (DA), and caused moderate, nonsignificant reductions in NE. However, in DSP-4-treated males, NE levels in specific vocal-control nuclei showed high positive correlations with courtship singing. There were no significant correlations between NE levels in hypothalamic nuclei and any behavior or DA or 5-HT levels in any nuclei and any behavior. DSP-4-treated males took longer to begin singing and performed fewer song bouts and courtship displays, but their songs could not be differentiated from those of control males. This suggests that their behavioral deficits resulted from deficits in attention rather than an inability to sing.


Effects of DM-9384, a pyrrolidone derivative, on ischemia-induced changes in the central monoamine systems.

Alterations in brain tissue levels of monoamines and monoamine metabolites were studied in gerbils 60 min after cerebral ischemia induced by 10 min carotid ligation after pretreatment with the antiischemic drug DM-9384 (1, 3, 10, 30 mg/kg, PO). The DA levels decreased in striatum after the ischemia, while cortical and hippocampal DA levels increased. The DOPAC levels increased in cortex, but were essentially unaffected in other regions. The HVA levels increased in all forebrain regions studied. NA levels decreased in hippocampus and superior colliculus, while a general increase in MHPG levels was seen. Decreases in 5-HT levels were seen in all forebrain regions except cortex. The 10 mg/kg and 30 mg/kg doses of DM-9384 counteracted the decrease in striatal 5-HT and hypothalamic MHPG/NA ratio, respectively. Thus pretreatment with DM-9384 exerted minor protective effects on the alterations induced in monoamine systems by transient forebrain ischemia.


The substance P fragment SP(1-7) stimulates motor behavior and nigral dopamine release.

Earlier studies have shown that the undecapeptide substance P (SP) alters motor behavior and dopamine metabolism following injection into the substantia nigra (SN) in rat, even though the SN appears largely devoid of SP-specific (NK-1) receptors. In this report, intra-nigral injections of the amino-terminal SP fragment SP(1-7) enhanced rearing, sniffing and locomotor activity, and increased the nigral DOPAC-to-DA ratio. In addition, SP(1-7) increased 3H-DA release from the SN in vitro. These findings suggest that some of the effects of nigral SP on motor behavior and dopamine release are mediated by amino-terminal fragments of SP.


Galanin is more common than NPY in vascular sympathetic neurons of the brush-tailed possum.

The distribution of galanin (Gal) in sympathetic vascular neurons of adult and juvenile brush-tailed possums (Trichosurus vulpecula), was examined using double-labelling immunohistochemistry. This was compared with the distribution of neuropeptide Y (NPY) in the same tissues. Immunoreactivity (IR) to galanin was present in the majority (64-99%) of nerve cell bodies in paravertebral sympathetic ganglia, where it mostly co-existed with IR to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). Gal-IR also was present in most, if not all, TH-IR perivascular axons supplying systemic arteries and veins. NPY-IR was less common than Gal-IR in all sympathetic ganglia and perivascular axons examined. Some sympathetic, TH-IR axons supplying the abdominal aorta and renal artery contained both Gal-IR and NPY-IR, while TH-IR axons supplying cephalic and thoracic vessels contained Gal-IR but not NPY-IR. Limited observations on sympathetic neurons in two species of wallabies indicated that Gal-IR also was more common than NPY-IR in other marsupial species, but the incidence of NPY-IR was higher in these wallabies than in the brush-tailed possum. Together with previous studies, this work suggests that the coexistence of galanin and NPY may be the primitive condition for sympathetic neurons in tetrapods. The differential expression of these peptides in specific populations of sympathetic neurons may have important functional consequences in the autonomic control of the circulation.