Acute and chronic suppression of leukotriene B4 synthesis ex vivo in neutrophils from patients with rheumatoid arthritis beginning treatment with methotrexate. To compare the cumulative effects of oral methotrexate (MTX) therapy (after 6-8 weeks) with the acute effects (24 hours after a dose) on arachidonic acid metabolism by the 5-lipoxygenase (5-LO) pathway in neutrophils from patients with active rheumatoid arthritis (RA) who were beginning therapy with MTX. Neutrophils and monocytes were isolated from whole blood from 7 patients with RA, immediately before and 24 hours after their first weekly dose of 7.5 mg of MTX, and again after their dose at 6-8 weeks. Total immunoreactive leukotriene B4 (LTB4) formation in neutrophils activated ex vivo with calcium ionophore A23187 was significantly suppressed (by 33%) before the 6-8-week dose, compared with the level before the first dose (mean +/- SEM 8.29 +/- 1.24 ng/10(6) cells at predose 6-8 weeks versus 12.29 +/- 2.13 ng/10(6) cells at predose 1; P = 0.03). Reductions were also observed after the first dose (27%; P = 0.07) and after the 6-8-week dose (43%; P = 0.05) compared with the respective predose levels. MTX treatment produced significant reductions in the total generation of 5-LO pathway products (5-hydroxyeicosatetraenoic acid + 6-trans-LTB4 + LTB4 + omega-oxidation products of LTB4) by calcium ionophore-activated neutrophils, as quantitated by integrated optical density after resolution on reverse-phase high-performance liquid chromatography. Decreases were observed after the first dose (26%; P = 0.025), immediately before the 6-8-week dose (23%; P = 0.05), and after the 6-8-week dose (47%; P = 0.0033) compared with levels before the first dose, and after the 6-8-week dose compared with the level before it (32%; P = 0.04). The generation of LTB4 by calcium ionophore-activated monocytes was not significantly affected by MTX therapy. The significant decreases in the formation of omega-oxidation products of LTB4 and in the total generation of neutrophil 5-LO pathway products in the absence of a significant change in the release of 3H-arachidonic acid or the generation of platelet-activating factor suggest that the activity of the 5-LO enzyme in neutrophils is inhibited. We conclude that weekly oral MTX therapy in patients with active RA inhibits neutrophil 5-LO pathway product generation in a pattern consistent with inhibition of the activity of the 5-LO enzyme; an effect is observed after the first dose. The inhibition of 5-LO is cell-selective and cumulative, with a superimposed incremental inhibition observed after the weekly MTX dose.


Neuroendocrine peptide receptors on cells of the immune system. The functional role of neuropeptide receptors in the immune system seems to be sensory and regulatory. Signaling molecules (i.e. neuropeptides/neurotransmitters) secreted by immune or neuroendocrine cells can interact with specificity to immunocyte membrane bound neuroendocrine peptide receptors resulting in changes in immune homeostasis. At the onset of this review, three criteria were established in order to formally define the existence of a receptor including the pharmacologic, biochemical, and biological profiles. Applying these criteria, at least ten neuroendocrine peptide/peptide neurotransmitter receptors have met the requirements (table 1). Perhaps more striking, many of these receptors have characteristics which are nearly identical to those receptors found on neuroendocrine tissue. This observation implies irregularities in the physiology of a receptor system in one tissue compartment (neuroendocrine or immune) may be mirrored by the receptors found in the other compartment (neuroendocrine or immune) as well. This rather hypothetical concept is, in fact, supported by data studying ACTH, CRH, and TRH receptors on immune cells relative to the expression or function of these receptors in neuroendocrine tissue taken from patients with neuroendocrine disorders. It is likely that future work will use this relationship to further study the dynamic interaction between the immune and neuroendocrine systems in defining neurologic, neuroendocrine, and autoimmune disorders.


Renal transplantation.

Renal transplantation. This article provides an overview of the renal transplantation process from the evaluation phase through hospitalization and follow-up care. Nursing care of the postoperative transplant patient in the intensive care unit is detailed in this article, using nursing various diagnoses as a guideline.


Muscarinic and gamma-aminobutyric acid-ergic receptor changes during vestibular compensation. A quantitative autoradiographic study of the vestibular nuclei complex in the rat. Involvement of the neurotransmitters acetyl choline and gamma-aminobutyric acid (GABA) in vestibular compensation has been suggested by electrophysiological and pharmacological experiments. In this investigation we used quantitative autoradiography to study the modification of muscarinic and benzodiazepine receptors in each nucleus of the rat's vestibular nucleus complex. Tissues were examined 3, 14, 23 h and 3, 12, 37 and 90 days after unilateral surgical labyrinthectomies. The present results demonstrated a muscarinic receptor supersensitivity in the deafferented side in the superior vestibular nucleus 90 days after surgery. This increase was not large enough to support the cholinergic receptor supersensitivity hypothesis for vestibular compensation. The changes in the benzodiazepine receptors observed for a short time following surgery were reversed after a few days. These findings support a transient involvement of GABAergic pathways in vestibular compensation.


gamma-Aminobutyric acid receptor activation of outer hair cells in the guinea pig cochlea. Gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter of the central nervous system, may also be released from olivocochlear efferent nerves reaching the outer hair cells of the cochlea. In the present study the cell potential of isolated outer hair cells of the third and fourth turns of the guinea pig cochlea was measured with patch-clamp electrodes. GABA-receptor agonists and antagonists were applied extracellularly. The cell membrane hyperpolarized in a reversible manner with increasing concentrations of extracellular GABA. Half-maximal hyperpolarization (2 mV) was achieved with approximately 10(-7) M GABA. Desensitization was not observed. The hyperpolarizing effect of GABA was potentiated by the benzodiazepine clorazepate and was blocked by picrotoxin.


Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene. We have isolated a number of Drosophila cDNAs on the basis of their encoding calmodulin-binding proteins. A full-length cDNA clone corresponding to one of these genes has been cloned and sequenced. Conservation of amino acid sequence and tissue-specific expression are observed between this gene and the transient receptor potential (trp) gene. We propose the name transient receptor potential-like (trpl) to describe this newly isolated gene. The trpl protein contains two possible calmodulin-binding sites, six transmembrane regions, and a sequence homologous to an ankyrin-like repeat. Structurally, the trpl and trp proteins resemble cation channel proteins, particularly the brain isoform of the voltage-sensitive Ca2+ channel. The identification of a protein similar to the trp gene product, yet also able to bind Ca2+/calmodulin, allows for a reinterpretation of the phenotype of the trp mutations and suggests that both genes may encode light-sensitive ion channels.


The trp gene is essential for a light-activated Ca2+ channel in Drosophila photoreceptors. Invertebrate phototransduction is an important model system for studying the ubiquitous inositol-lipid signaling system. In the transient receptor potential (trp) mutant, one of the most intensively studied transduction mutants of Drosophila, the light response quickly declines to baseline during prolonged intense light. Using whole-cell recordings from Drosophila photoreceptors, we show that the wild-type response is mediated by at least two functionally distinct classes of light-sensitive channels and that both the trp mutation and a Ca2+ channel blocker (La3+) selectively abolish one class of channel with high Ca2+ permeability. Evidence is also presented that Ca2+ is necessary for excitation and that Ca2+ depletion mimics the trp phenotype. We conclude that the recently sequenced trp protein represents a class of light-sensitive channel required for inositide-mediated Ca2+ entry and suggest that this process is necessary for maintained excitation during intense illumination in fly photoreceptors.


Nitric oxide-induced blockade of NMDA receptors. We studied the effects of nitric oxide (NO)-producing agents on N-methyl-D-aspartate (NMDA) receptor activation in cultured neurons. 3-Morpholino-sydnonimine (SIN-1) blocked both NMDA-induced currents and the associated increase in intracellular Ca2+. The actions of SIN-1 were reversible and suppressed by hemoglobin. A degraded SIN-1 solution that did not release NO was unable to block NMDA receptors. This showed that the SIN-1 effects were due to NO and not to another breakdown product. Similar results were obtained with 1-nitrosopyrrolidine (an NO-containing drug) and with NO released from NaNO2. Pretreatment with hemoglobin potentiated NMDA-induced effects, demonstrating that endogenous NO modulates NMDA receptors. Since NMDA receptor activation induces NO synthesis, these results suggest a feedback inhibition of NMDA receptors by NO under physiological condition.


muI Na+ channels expressed transiently in human embryonic kidney cells: biochemical and biophysical properties. We describe the transient expression of the rat skeletal muscle muI Na+ channel in human embryonic kidney (HEK 293) cells. Functional channels appear at a density of approximately 30 in a 10 microns 2 patch, comparable to those of native excitable cells. Unlike muI currents in oocytes, inactivation gating is predominantly (approximately 97%) fast, although clear evidence is provided for noninactivating gating modes, which have been linked to anomalous behavior in the inherited disorder hyperkalemic periodic paralysis. Sequence-specific antibodies detect a approximately 230 kd glycopeptide. The majority of molecules acquire only neutral oligosaccharides and are retained within the cell. Electrophoretic mobility on SDS gels suggests the molecules may acquire covalently attached lipid. The channel is readily phosphorylated by activation of the protein kinase A and protein kinase C second messenger pathways.


cDNA that encodes active agrin.

cDNA that encodes active agrin. Agrin is thought to mediate the motor neuron-induced aggregation of AChRs and AChE on the surface of muscle fibers at neuromuscular junctions. We have isolated a cDNA from a chick brain library that, based on sequence homology and expression experiments, codes for active agrin. Examination of the sequence reveals considerable similarity to homologous cDNAs previously isolated from ray and rat libraries. A conspicuous difference is an insertion of 33 bp in chick agrin cDNA, which endows the encoded protein with AChR/AChE aggregating activity. Homologous transcripts having the 33 bp insertion were detected in the ray CNS, which indicates that an insertion of similar size is conserved in agrin in many, if not all, vertebrate species. Results of in situ hybridization studies and PCR experiments on mRNA isolated from motor neuron-enriched fractions of the spinal cord indicate that, consistent with the agrin hypothesis, motor neurons contain transcripts that code for active agrin.


The agrin gene codes for a family of basal lamina proteins that differ in function and distribution. We isolated two cDNAs that encode isoforms of agrin, the basal lamina protein that mediates the motor neuron-induced aggregation of acetylcholine receptors on muscle fibers at the neuromuscular junction. Both proteins are the result of alternative splicing of the product of the agrin gene, but unlike agrin, they are inactive in standard acetylcholine receptor aggregation assays. They lack one (agrin-related protein 1) or two (agrin-related protein 2) regions in agrin that are required for its activity. Expression studies provide evidence that both proteins are present in the nervous system and muscle and that, in muscle, myofibers and Schwann cells synthesize the agrin-related proteins while the axon terminals of motor neurons are the sole source of agrin.


Slower spontaneous excitatory postsynaptic currents in spiny versus aspiny hilar neurons. In the hilar region of the rat hippocampus, large spontaneous excitatory postsynaptic currents (sEPSCs) mediated by non-NMDA glutamate receptors are present in both excitatory spiny mossy cells and inhibitory aspiny hilar interneurons, making these neurons ideal candidates for a comparative study using the tight seal whole-cell recording technique. Although sEPSCs have similar amplitude distributions, the rise and decay times are significantly slower in spiny versus aspiny neurons. Similar kinetic differences are observed in synaptic currents evoked by mossy fiber stimulation. These results demonstrate a physiological difference between the excitatory drive to excitatory and inhibitory neurons in the hilus that certainly contributes to differences in synaptic strength and that may be applicable to other brain regions. Furthermore, since the development or modification of individual spines or groups of spines may affect synaptic strength, these results may be pivotal in establishing a role for spines in modulating synaptic activity.


Signal transduction and pharmacological characteristics of a metabotropic glutamate receptor, mGluR1, in transfected CHO cells. The signal transduction and pharmacological properties of a metabotropic glutamate receptor, mGluR1, were studied in CHO cells permanently expressing the cloned receptor. mGluR1 stimulated phosphatidylinositol (PI) hydrolysis in the potency rank order of quisqualate greater than L-glutamate greater than or equal to ibotenate greater than L-homocysteine sulfinate greater than or equal to trans-ACPD. This receptor also evoked the stimulation of cAMP formation and arachidonic acid release with comparable agonist potencies. DL-AP3 and L-AP4, the effective antagonists reported for glutamate-stimulated PI hydrolysis in brain slices, showed no appreciable effects on mGluR1, suggesting the existence of an additional subtype of this receptor family. Pertussis toxin and phorbol ester produced distinct effects on the three transduction cascades, implying that mGluR1 independently links to the multiple transduction pathways probably through different G proteins.


Transplantation and functional integration of an identified respiratory interneuron in Lymnaea stagnalis. The possibility that damaged neural circuitries can be repaired through grafting has raised questions regarding the cellular mechanisms required for functional integration of transplanted neurons. Invertebrate models offer the potential to examine such mechanisms at the resolution of single identified neurons within well-characterized neural networks. Here it is reported that a specific deficit in the respiratory behavior of a pulmonate mollusc, caused by the ablation of a solitary interneuron, can be restored by grafting an identical donor interneuron. The transplanted interneuron not only survives and extends neurites within the host nervous system, but under specific conditions forms synapses with appropriate target neurons and is physiologically integrated into the host's circuitry, thereby restoring normal behavior.


Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide. Vasoactive intestinal polypeptide (VIP), a 28 amino acid peptide hormone, plays many physiological roles in the peripheral and central nerve systems. A functional cDNA clone of the VIP receptor was isolated from a rat lung cDNA library by cross-hybridization with the secretin receptor cDNA. VIP bound the cloned VIP receptor expressed in mouse COP cells and stimulated adenylate cyclase through the cloned receptor. The rat VIP receptor consists of 459 amino acids with a calculated Mr of 52,054 and contains seven transmembrane segments. It is structurally related to the secretin, calcitonin, and parathyroid hormone receptors, suggesting that they constitute a new subfamily of the Gs protein-coupled receptors. VIP receptor mRNA was detected in various rat tissues including liver, lung, intestines, and brain. In situ hybridization revealed that VIP receptor mRNA is widely distributed in neuronal cells of the adult rat brain, with a relatively high expression in the cerebral cortex and hippocampus.


Infection risk in patients with small cell lung cancer receiving intensive chemotherapy and recombinant human granulocyte-macrophage colony-stimulating factor. The effect of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was assessed in 17 patients with small cell lung cancer. GM-CSF was initially given alone by subcutaneous injection for 10 days at 50-500 micrograms/m2 per day. There was a significant rise in neutrophils and eosinophils and to a lesser extent in monocytes at all dose levels. During the next phase, patients received chemotherapy (etoposide, ifosfamide and doxorubicin), and GM-CSF was given on alternate cycles, the patients acting as their own controls, so that the amelioration of chemotherapy could be assessed. Despite partial abrogation of the neutropenia associated with chemotherapy (P = 0.04), GM-CSF failed to reduce the frequency of febrile episodes in association with neutropenia, with six episodes occurring on GM-CSF and seven while patients were not receiving GM-CSF after a total of 66 cycles of chemotherapy. After GM-CSF, there was a reduction in polymorph phagocytic ability and chemotaxis in 6/12 and 9/11 patients, respectively. Timed blood counts after GM-CSF administration showed that peak leucocytosis occurred at 8-12 h and fell to two-thirds of this level at 24 h. Toxicity consisting of lethargy, myalgia and bone pain occurred at all dose levels but was manageable. 2 patients had thromboembolism. This study failed to demonstrate a reduction in the infection risk associated with moderately intensive chemotherapy for small cell lung cancer despite the partial abrogation of neutropenia.


Multistage carcinogenesis in paediatric and adult cancers. Retinoblastoma (RB) and the familial adenomatous polyposis/colorectal cancer (FAP/CRC) complex provide well-characterised examples of multistage carcinogenesis and inheritance of a predisposition to cancer. Retinoblastoma appears to conform to the simple two-step model first proposed by Knudson. The gene responsible for RB, now called Rb1, has been located in chromosome region 13q14. The Rb1 gene has been cloned and subjected to extensive analysis. It is probable that the Rb1 gene product has a role in the regulation of transcription. The familial form of RB occurs as the result of a germline mutation of one of the copies of the Rb1 gene. Colorectal cancer, in contrast, appears to be the result of four or five steps involving both activation of oncogenes and inactivation of antioncogenes. The FAP gene has been located in chromosome region 5q21 by genetic linkage, and a candidate gene, MCC (mutated in colon cancer), has been cloned. Other mutations in previously-identified genes that have been identified as important in the genesis of CRC include the activation of p53 and of Ki-ras. A gene lying in chromosome region 18q which is deleted in colorectal cancer, and hence named DCC has been cloned. Its protein product has sequence homology to neural cell adhesion molecules and other related cell-surface glycoproteins. Delineation of the genes involved in the development of tumours such as RB and CRC provides insight into the mechanisms by which sequential mutations result in carcinogenesis.


Potentiation of etoposide cytotoxicity against a human ovarian cancer cell line by pretreatment with non-toxic concentrations of methotrexate or aphidicolin. Exposure of human ovarian cancer SW626 cell line to 0.08 mumol/l methotrexate or 25 mumol/l aphidicolin for 24 h caused no cytotoxicity but enhanced etoposide cytotoxicity. Methotrexate or aphidicolin treatment induced a reversible blockade at the beginning of S phase which was reversed upon drug removal with a consequent wave of synchronisation. The enhancement of etoposide cytotoxicity was not due to higher etoposide intracellular uptake in the methotrexate or aphidicolin-pretreated cells. The topoisomerase II content in methotrexate or aphidicolin pretreated SW626 cells was higher than in control cells assessed by western blotting or flow cytometry. The higher etoposide cytotoxicity observed after synchronization with methotrexate or aphidicolin was apparently unrelated to the number of drug-induced DNA-topoisomerase II complexes evaluated as DNA double strand breaks or DNA-protein crosslinks. These data support the view that etoposide-induced DNA-topoisomerase II complexes are more cytotoxic in cells which are in S-phase.


Comparison of two carboplatin-containing regimens with standard chemotherapy for small cell lung cancer in a randomised phase II study. The EORTC Lung Cancer Cooperative group. The EORTC Lung Cancer Cooperative group performed a randomised phase II study in patients with small cell lung cancer comparing the standard cyclophosphamide/doxorubicin/etoposide (CDE) regimen with two regimens containing the new and active cisplatin derivative, carboplatin, 400 mg/m2 in combination with ifosfamide, a drug without important myelotoxicity, at a dose of 5 g/m2 (IMP) or the non-myelotoxic drug vincristine twice 2 mg (VP). Of 178 evaluable patients, 63 received CDE [30 limited disease (LD), 33 extensive disease (ED)], 55 received IMP (22 LD, 33 ED) and 60 (26 LD, 34 ED) were treated with VP. The response duration was not statistically different: CDE 31 weeks, IMP 29 weeks and VP 21 weeks. The time to progression after CEE was 28 weeks, IMP 24 weeks and VP 17 weeks. This was significantly shorter after VP than after CDE (P = 0.017). The 60% response rate of the VP combination was low compared with CDE (83%) and IMP (77%). Toxicity of all three regimens was acceptable, and dose reduction for myelosuppression was necessary in only a minority of the patients. We conclude from this study that the combination of carboplatin, at the maximally tolerated dose of 400 mg/m2, in combination with ifosfamide 5 g/m2, is an active regimen with efficacy comparable with the standard CDE regimen.


[Prevalence and significance of the C virus antibody in chronic hepatopathy not related to B virus in alcoholics]. The presence of antibody to the hepatitis C virus was determined in 254 alcoholic patients with non-B chronic hepatitis and a titre of antinuclear antibodies of 1/40 or lower. Alcoholic hepatitis was present in 12 patients, steatohepatitis in 20, active chronic hepatitis in 22, cirrhosis in 181, and hepatocarcinoma in 19. Twenty patients had previously received blood transfusion alone or during surgery, 49 had undergone previous surgery without transfusion, a clinical episode of hepatitis could be traced in 14, 4 patients were drug addicts, 41 had received blood transfusion after the diagnosis was made, and 128 presented with alcoholism alone. Anti-hepatitis C antibody was found in 20 out of 2,000 blood donors (1%) in our hospital. Anti-hepatitis C antibody was found in 87 patients (34.2%) in our series, a figure unaltered by past medical history. Patients with anti-HC antibody had higher levels of AST, ALT, total proteins, gamma-globulin, and IgG. The incidence of active chronic hepatitis was higher among patients with anti-HC antibody, whereas the incidence of steatohepatitis was higher among patients without anti-HC. Regarding findings on liver biopsy, the incidence of anti-HC was significantly higher (p less than 0.001) among patients with active chronic hepatitis (72.7%) than in any other group; no significant differences were found between patients with cirrhosis (33.3%), hepatocarcinoma (31.5%), steatohepatitis (15%), or alcoholic hepatitis (16.7%). Among HBsAg-negative patients, the incidence of anti-HC was similar between those with (39.7%) and without other serum markers of HB (32.9%).(ABSTRACT TRUNCATED AT 250 WORDS)


[Prevalence of hepatitis C virus antibody in chronic HBsAg-negative non alcoholic hepatopathy]. The presence of antibody to hepatitis C virus was determined in 316 HBsAg-negative patients with non-alcoholic chronic hepatitis who did not receive any blood transfusion once the diagnosis was made. A titre of antinuclear antibodies of 1/40 or lower was found in 18 patients. Persistent chronic hepatitis was present in 21 patients, active chronic hepatitis in 145, hepatic cirrhosis in 128, and hepatocarcinoma in 22 patients. One hundred and three patients had previously received blood transfusion, 76 had undergone previous surgery without transfusion, a clinical episode of hepatitis could be traced in 14, 13 patients were drug addicts (all of them HIV negative), 1 patient had received multiples injections, another had been treated with acupuncture, and 108 patients were free of any of the above. Anti-HCV was present in 76.6% of patients; a significantly higher proportion (87.4%) was found among patients who had received blood transfusion than in patients with previous surgery (72.4%) (p = 0.012), clinical hepatitis (57.1%), or without previous hepatic disease (70.3%) (p = 0.003). The incidence of anti-HCV was lower among cirrhotics (70.3%) than in patients with active chronic hepatitis (84.1%) (p = 0.006); in contrast, previous blood transfusion was significantly higher (p = 0.001) among the latter (40.7%) than in cirrhotics (21.9%). The incidence of anti-HCV was similar among patients with (78.6%) and without (75.8%) type B infection. Our results suggest that infection with virus C may account for a high proportion of non-alcoholic non-B chronic hepatitis.


[Neoplasms during the 1st year of life: a study of 116 cases treated during the last 10 years]. 116 infants under one year of age (14 per 100) with malignancies including 35 neuroblastomas, 23 retinoblastomas, 14 Wilm's tumours, 10 hepatoblastomas, 10 brain tumours, 9 germ cell tumours, 8 histiocytic and 7 soft tissue sarcomas, were treated in the last ten years. Hepatoblastoma is the highest relative incidence tumor in the first year of life and with brain tumours has the worst prognosis (50 and 40 per 100, respectively). The disease-free survival rate is most than 80 per 100 in neuro and nephroblastoma with medical and surgical treatment. Familiar incidence in Wilm's tumor is 42 per 100 and chemotherapy side effects are 23 per 100. Familiar incidence in neuroblastoma is 20 per 100 and chemotherapy side effects 21 per 100. In conclusion, in children with malignancies under one year of age have good prognosis (75 per 100 survival at five years), with lower surgical complications rate. Early diagnosis and response to chemotherapy, the side effects of which are considerable and should be strictly controlled, are key factors in the better prognosis and increased life expectancy in this group of patients.


The receptor for human sex steroid binding protein (SBP) is expressed on membranes of neoplastic endometrium. Sex steroid binding protein (SBP) receptor was detected on cell membranes obtained from human endometrium adenocarcinoma. The binding of SBP was proved to be highly specific, saturable, and at high affinity. It was, additionally, shown to occur at two sites at different affinities, as previously described for other human tissues. SBP was, therefore, demonstrated to recognized a specific receptor on endometrium adenocarcinoma membranes. The effect of steroid hormones on SBP-receptor interaction was also evaluated. Both dihydrotestosterone and estradiol were shown to inhibit the binding of SBP to its specific receptor on neoplastic membranes. Testosterone at a dose of 10(-9) M was shown not to interfere to a significant extent with SBP-receptor binding. The sensitivity for estradiol we had previously observed in normal premenopausal endometrium was completely lost in postmenopausal neoplastic tissue. These observations suggest that the SBP-membrane recognition system is still present in neoplastic postmenopausal endometrium, but it has been modified either by the postmenopausal endogenous milieu or by the neoplastic transformation.


Plasma levels of C19 steroid glucuronides in pre-menopausal women with non-classical congenital adrenal hyperplasia. Recent reports have thrown doubt on the role of measurements of plasma 5 alpha-androstane-3 alpha,17 beta-diol glucuronide (3 alpha-diolG) as a marker of peripheral androgen metabolism in women with polycystic ovarian syndrome and idiopathic hirsutism. It has been suggested that a plasma profile of C19 steroid glucuronides may be more informative. While preliminary data indicates that both 3 alpha-diolG and androsterone G (ADTG) may arise from adrenal steroid precursors, there have been no reports of C19 steroid glucuronides in women with non-classical, or late-onset congenital adrenal hyperplasia (NC-CAH), who constitute a significant proportion of the hirsute female population. We therefore measured plasma levels of 3 alpha-diolG, ADTG and dihydrotestosterone G (DHTG) before and following a standard Cortrosyn test in 15 symptomatic and 3 asymptomatic NC-CAH patients, 5 heterozygote carriers for 21-hydroxylase deficiency (NCHETS) and 18 normal women. The effects of chronic glucocorticoid (GCR) therapy (greater than 3 months) on the C19 steroid glucuronide profile in the symptomatic patients was also investigated. Baseline plasma levels of all 3 glucuronides were significantly (P less than 0.001) higher in symptomatic patients compared with either normals or NCHETS. However, the order of discrimination was ADTG greater than 3 alpha-diolG greater than DHTG. There were no significant differences between steroid glucuronide levels for NCHET and normal women and the C19 steroid glucuronide concentrations for the asymptomatic NC-CAH patients were greater than 2 SD above the normal means. Moderate clinical improvement was observed in all patients receiving oral GCR therapy and was accompanied by approx. 80% suppression of the plasma levels of all 3 C19 steroid glucuronides. This contrasts with a mean suppression of androstenedione of only 50%. However, plasma levels of the C19 steroid glucuronides were not significantly increased in response to a short ACTH stimulation test. This may be explained by the fact that the androgen glucuronides are thought to be peripherally formed metabolites derived from unconjugated glandular secreted androgen precursors and thus their synthesis at 60 min following adrenal stimulation may lag substantially behind that of their respective precursors. There were significant linear correlations between the levels of all 3 glucuronides, but neither correlated with Ferriman-Gallway scores, body mass index or 17-hydroxyprogesterone levels.(ABSTRACT TRUNCATED AT 400 WORDS)


Heteroconjugate antibodies enhance cell-mediated anti-herpes simplex virus immunity in vivo. Heteroconjugate antibodies are generated by covalently linking two mAbs with different specificities. When anti-CD3 mAb, capable of activating effector T cells, is coupled to anti-herpes simplex virus (HSV) mAb, which binds HSV antigens on virally-infected target cells, the resulting heteroconjugate antibody can be used in vitro to enhance anti-HSV immunity. Specifically, these heteroconjugate antibodies can augment anti-HSV immunity among lymphocytes previously lacking cytotoxicity against HSV-infected target cells. However, the efficacy of these specialized reagents in enhancing anti-HSV immunity in vivo has not been determined. We report here that anti-HSV heteroconjugates used in an adoptive transfer murine model of HSV-1 infection inhibited HSV replication in vivo and improved long-term survival. These results demonstrate that heteroconjugate antibodies have a potential therapeutic role in enhancement of anti-HSV immunity.


Reaction of dioxygen with cytochrome c oxidase reduced to different degrees: indications of a transient dioxygen complex with copper-B. The reactions of the fully reduced, three-electron-reduced, and mixed-valence cytochrome oxidase with molecular oxygen have been followed in flow-flash experiments, starting from the CO complexes, at 445 and 830 nm at pH 7.4 and 25 degrees C. With the fully reduced and the three-electron-reduced enzyme, four kinetic phases with rate constants in the range from 1 x 10(5) to 10(3) s-1 can be observed. The initial fast phase is associated with an absorbance increase at 830 nm. This is followed by an absorbance decrease (2.8 x 10(4) s-1), the amplitude of which increases with the degree of reduction of the oxidase. The third phase (6 x 10(3) s-1) displays the largest absorbance change at both wavelengths in the fully reduced enzyme and is not seen in the mixed-valence oxidase at 830 nm; a change with opposite sign but with a similar rate constant is found at 445 nm in this enzyme form. The slowest phase (10(3) s-1) is also largest in the fully reduced oxidase and not seen in the mixed-valence enzyme. It is suggested that O2 initially binds to reduced CuB and is then transferred to cytochrome a3 before electron transfer from cytochrome a/CuA takes place. The fast oxidation of cytochrome a seen with the fully reduced enzyme is suggested not to occur during natural turnover. A reaction cycle for the complete turnover of the enzyme is presented. In this cycle, the oxidase oscillates between electron input and output states of the proton pump, characterized by cytochrome a having a high and a low reduction potential, respectively.


Use of 1H NMR ROESY for structural determination of O-glycosylated amino acids from a serine-containing glycopeptidolipid antigen. A serine-containing glycopeptidolipid antigen isolated from Mycobacterium xenopi typified a new class of mycobacterial glycopeptidolipid antigens devoid of the C-mycoside core structure [Rivière, M., & Puzo, G. (1991) J. Biol. Chem. 266, 9057-9063]. The lipopeptide core assigned to C12-Ser-Ser-Phe-alloThr-OCH3 exhibits three potential sites of glycosylation. The carbohydrate parts are composed of 3-O-methyl-6-deoxy-alpha-L-talopyranosyl and 2,3,4-tri-O-methyl-L- rhamnopyranosyl(alpha 1----3)-2-O-lauroyl-L-rhamnopyranosyl(alpha 1----3)-L- rhamnopyranosyl(alpha 1----3)-2,4-di-O-(acetyl, lauroyl)-6-deoxy-alpha-L-glucopyranosyl appendages. In the present work, the carbohydrate attachment sites were successfully determined by ROESY experiments on the native glycopeptidolipid using chloroform as solvent. From the NOE contacts, we unambiguously established that the acylated serine is glycosylated by the 3-O-methyl-6-deoxy-alpha-L-talopyranosyl appendage while the 2,3,4-tri-O-methyl-L-rhamnopyranosyl(alpha 1----3)-2-O- lauroyl-L-rhamnopyranosyl(alpha 1----3)-L-rhamnopyranosyl(alpha 1----3)-2,4-di- O-(acetyl, lauroyl)-6-deoxy-alpha-L-glucopyranosyl appendage is bound to the C-terminal alloThr-OCH3. From these data, the acetyl and lauroyl residues on the C-2 and C-4 of the basal monosaccharide unit were successfully localized. Furthermore, the "L" absolute configuration for the serines and the phenylalanine residues and the "D" configuration for the allothreonine were established. The primary structure of this novel type of mycobacterial antigen, a serine-containing glycopeptidolipid, has now been fully established.


Sequence-specific 1H NMR assignments and secondary structure of the streptococcal protein G B2-domain. Two-dimensional NMR spectroscopy has been used to obtain sequence-specific 1H NMR assignments for the IgG-binding B2-domain of streptococcal protein G. Secondary structure elements were identified from analysis of characteristic backbone-backbone NOE patterns and amide proton exchange data. The B2-domain contains a four-stranded beta-sheet region in which the two inner strands form a parallel beta-sheet with each other and antiparallel beta-sheets with the outer strands. The outer strands are connected via a 16-residue alpha-helix and short loops on both ends of the helix. The alpha-helix and beta-sheet structures contain well-defined polar and apolar sides, and numerous long-range NOEs from the apolar helix to apolar sheet regions were used to derive a model for the global fold of the B2-domain. While the overall fold is similar to that obtained for B1-type domains, differences in amide proton exchange rates and hydrophobic packing are observed.


Fast internal main-chain dynamics of human ubiquitin. The fast internal dynamics of human ubiquitin have been studied by the analysis of 15N relaxation of backbone amide nitrogens. The amide 15N resonances have been assigned by use of heteronuclear multiple-quantum spectroscopy. Spin lattice relaxation times at 60.8 and 30.4 MHz and the steady-state nuclear Overhauser effect at 60.8 MHz have been determined for 67 amide 15N sites in the protein using two-dimensional spectroscopy. These data have been analyzed in terms of the model free treatment of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The global motion of the protein is shown to be isotropic and is characterized by a correlation time of 4.1 ns rad-1. The generalized order parameters (S2) of backbone amide N-H vectors in the globular region of the protein range from 0.5 to 0.95. No apparent correlation between secondary structure and generalized order parameters is observed. There is, however, a strong correlation between the magnitude of the generalized order parameters of a given N-H vector and the presence of hydrogen bonding of the amide hydrogen or its peptide bond associated carbonyl. Using a chemical shift tensor breadth of 160 ppm, the N-H vectors of peptide linkages participating in one or more hydrogen bonds to the main chain show an average generalized order parameter of 0.80 (SD 0.06), while those amide NH of peptide linkages free of hydrogen-bonding interactions with the main chain show an average order parameter of 0.69 (SD 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)


Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast ferricytochromes c. Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 90% of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 ferricytochrome c further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.


Intersubunit communications in Escherichia coli cyclic AMP receptor protein: studies of the ligand binding domain. Escherichia coli cAMP receptor protein (CRP) is a homodimer in which each subunit is composed of two domains. The C-terminal domain is responsible for DNA recognition, whereas the larger N-terminal domain is involved in cAMP binding. Biochemical and genetic evidence suggests that both intersubunit and interdomain interactions play important roles in the regulatory mechanism of this protein. Essentially all intersubunit contacts occur via a long C-helix which is a part of the N-terminal domain. In this work, intersubunit interactions in CRP were studied with the use of two proteolytic fragments of the protein. Subtilisin digestion produces a fragment (S-CRP) which includes residues 1-117 and in which about 85% of the C-helix is removed, whereas chymotrypsin digestion produces a fragment (CH-CRP) consisting of residues 1-136, in which the whole C-helix is preserved. Both fragments were purified and subjected to functional tests which included cAMP binding, subunit assembly, and hydrodynamic properties in the presence and absence of cAMP. S-CRP binds cAMP with a similar affinity to that of native CRP but with reduced cooperativity. CH-CRP exhibits about 1 order of magnitude tighter binding of cAMP than S-CRP or CRP and the highest degree of negative cooperativity. Both fragments are dimeric with dimerization constants around 10(8) M-1. Ligand binding promotes dimerization and induces a small contraction of both S-CRP and CH-CRP. There is no apparent correlation between dimer stability and cooperativity of ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)


Reaction of O6-alkylguanine-DNA alkyltransferase with O6-methylguanine analogues: evidence that the oxygen of O6-methylguanine is protonated by the protein to effect methyl transfer. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) repairs the promutagenic O6-methylguanine lesion by transferring the methyl group to a cysteine residue on the protein. A mechanism in which AGT activates the guanyl moiety as a leaving group by protonation of a heteroatom on guanine was probed by reacting AGT with analogues of O6-methylguanine in which the heteroatoms were changed. The initial rates of reaction were measured at various substrate concentrations in 50 mM Hepes, 1 mM EDTA, 1 mM DTT, and 10% glycerol, pH 7.8 at 37 degrees C. The kinact (h-1) and Kin (mM) were determined for O6-methylguanine (1.66 +/- 0.19, 1.51 +/- 0.32), 6-methoxypurine (1.07 +/- 0.25, 10.6 +/- 4.2), S6-methyl-6-thioguanine (0.63 +/- 0.04, 1.17 +/- 0.18), 6-methylthiopurine (no reaction), Se6-methyl-6-selenoguanine (1.76 +/- 0.28, 10.6 +/- 5.0), 6-methylselenopurine (2.51 +/- 0.62, 15.7 +/- 6.3), O6-methyl-1-deazaguanine (1.71 +/- 0.34, 14.8 +/- 4.4), O6-methyl-3-deazaguanine (1.90 +/- 0.24, 2.54 +/- 0.59), and O6-methyl-7-deazaguanine (1.97 +/- 0.26, 2.56 +/- 0.72). These results indicate that replacement of the nitrogens does not affect the kinact parameter but the Kin is increased upon removal of the exocyclic amino group and the nitrogen at the 1-position. Replacement of the oxygen with sulfur decreases the kinact, and replacement with selenium increases the Kin. The results are consistent with a mechanism in which O6-methylguanine binds to the active site of AGT with hydrogen bonds to the oxygen, the exocyclic amino group, and the nitrogen at the 1-position of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of enzyme and ligand protonation on the binding of folates to recombinant human dihydrofolate reductase: implications for the evolution of eukaryotic enzyme efficiency. There is marked pH dependence of the rate constant (koff) for tetrahydrofolate (H4folate) dissociation from its ternary complex with human dihydrofolate reductase (hDHFR) and NADPH. Similar pH dependence of H4folate dissociation from the ternary complex of a variant of hDHFR with the substitution Phe31----Leu (F31L hDHFR) causes this dissociation to become rate limiting in the enzyme mechanism at pH approximately 5, and this accounts for the marked decrease in kcat for this variant as the pH is decreased from 7 to 5. This decreased kcat at low pH is not seen for most DHFRs. koff for dissociation of folate, dihydrofolate (H2folate), and H4folate from their binary complexes with hDHFR is similarly pH dependent. For all the complexes examined, the pH dependence of koff in the range pH 5-7 is well described by a pKa of about 6.2 and must be due to ionization of a group on the enzyme. In the higher pH range (7-10), koff increases further as the pH is raised, and this relation is governed by a second pKa which is close to the pKa for ionization of the amide group (HN3-C4O) of the respective ligands. Thus, ionization of the ligand amide group also increases koff. Evidence is presented that the dependence of pH on koff for hDHFR accounts for the shape of the kcat versus pH curve for both hDHFR as well as its F31L variant and contributes to the higher efficiency of hDHFR compared with bacterial DHFR.


Identification of the phosphoribulokinase sugar phosphate binding domain. A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (form I; NADH dependent) has been expressed in and purified to homogeneity from Escherichia coli that harbor the prkA gene in the plasmid pKP1565b. Restriction digestion of the phosphoribulokinase-encoding plasmid produces a tractable 450 bp fragment that encodes amino acid residues 28-179, which include a region (residues 42-54) highly conserved among phosphoribulokinase proteins. Using overlap extension polymerase chain reaction methodology, directed mutagenesis was performed to produce mutant proteins in which basic residues in this conserved region were replaced by neutral amino acids. Lysine-53, implicated by affinity labeling studies, has been replaced by methionine; little effect on substrate binding or catalysis is apparent. In contrast, when histidine-45 is replaced by asparagine, a 40-fold increase in the Km for ribulose 5-phosphate results; a 200-fold increase results when arginine-49 is replaced by glutamine. Implication of this region as part of the sugar phosphate binding site is compatible with previous results that indicate targeting by an ATP analogue containing a reactive functionality esterified to the gamma-phosphoryl group. The phosphoribulokinase reaction involves a single in-line phosphoryl transfer, requiring that the gamma-phosphoryl of ATP be closely juxtaposed to the bound cosubstrate. It follows that any reactive group attached to the gamma-phosphoryl in a nucleotide analogue that is bound to PRK in the absence of the cosubstrate will be favorably positioned to modify the sugar phosphate binding site.


Conformational similarities between one-chain and two-chain tissue plasminogen activator (t-PA): implications to the activation mechanism on one-chain t-PA. Tissue plasminogen activator (t-PA) is an exceptional serine protease, because unlike most other serine protease zymogens single-chain tissue plasminogen activator (sct-PA) possesses a substantial amount of proteolytic activity. The unusual reaction of sct-PA afforded the opportunity to directly compare the active site environment of sct-PA and two-chain tissue plasminogen activator (tct-PA) in solution through the application of a series of nitroxide spin labels and fluorophores. These labels, which have been previously shown to covalently label the catalytic serine of other serine proteases, inactivated both sct-PA and tct-PA. The labels can be divided into two classes: those which form tetrahedral complexes (sulfonates) and those which form trigonal complexes (anthranilates). Those which formed tetrahedral complexes were found to be insensitive to structural differences between sct-PA and tct-PA at the active site. In contrast, those which formed trigonal complexes could differentiate and monitor the sct-PA to tct-PA conversion by fluorescence spectroscopy. Models of the structure of sct-PA and tct-PA were constructed on the basis of the known X-ray structures of other serine protease zymogen and active enzyme forms. One of the nitroxide spin labels was modeled into the sct-PA and tct-PA structures in two possible orientations, both of which could be sensitive to structural differences between sct-PA and tct-PA. These models formed the structural rationale used to explain the results obtained with the "tetrahedral" and "trigonal" probes, as well as to offer a possible explanation for the unique reactivity of sct-PA.


The antitumor agent CC-1065 inhibits helicase-catalyzed unwinding of duplex DNA. The antitumor drug CC-1065 is thought to exert its effects by covalent bonding to N3 of adenine in DNA and interfering with some aspect of DNA metabolism. Therefore, it is of interest to determine what effect this drug has on enzymes involved in various aspects of DNA metabolism. In this report, we examine the ability of two DNA helicases, the dda protein of phage T4 and helicase II of Escherichia coli, to unwind CC-1065-adducted, tailed, oligonucleotides. It is shown that the presence of the drug on DNA strongly inhibits unwinding catalyzed by the T4 and E. coli proteins. A significant difference between the results obtained with the two helicases is that DNAs containing drug on either the tailed or the completely duplex strands are poor substrates for helicase II but dda protein-mediated unwinding is inhibited only when the drug is on the tailed strand. The drug-modified, helicase-released, strands migrate abnormally through a native gel, suggesting that the drug traps an unusual secondary structure generated in the course of protein-mediated unwinding. A kinetic analysis of the drug-inhibited reactions reveals that the helicases are trapped by the DNA-drug complex. This is evidenced by a decrease in the rate of helicase exchange between drug-bound substrate and drug-free duplex. The implications of these results with respect to the mechanism of action of CC-1065 in vivo are discussed.


Replication inhibition and translesion synthesis on templates containing site-specifically placed cis-diamminedichloroplatinum(II) DNA adducts. A series of site-specifically plantinated, covalently closed circular M13 genomes (7250 bp) was constructed in order to evaluate the consequences of DNA template damage induced by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP). Here are reported the synthesis and characterization of genomes containing the intrastrand cross-linked adducts cis-[Pt(NH3)2[d(ApG)-N7(1),-N7(2)]], cis-[Pt-(NH3)2[d(GpCpG)-N7(1),-N7(3)]], and trans-[Pt(NH3)2[d(CpGpCpG)-N3(1),-N7(4)]]. These constructs, as well as the previously reported M13 genome containing a site-specifically placed cis-[Pt(NH3)2[d-(GpG)-N7(1),-N7(2)]] adduct, were used to study replication in vitro. DNA synthesis was initiated from a position approximately 177 nucleotides 3' to the individual adducts, and was terminated either by the adducts or by the end of the template, located approximately 25 nucleotides on the 5' side of the adducts. Analysis of the products of these reactions by gel electrophoresis revealed that, on average, bypass of the cis-DDP adducts occurred approximately 10% of the time and that the cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] intrastrand cross-link is the most inhibitory lesion. The cis-[Pt(NH3)2[(GpCpG)-N7(1),-N7(3)]] adduct allowed a higher frequency of such translesion synthesis (ca. 25%) for two of the polymerases studied, modified bacteriophage T7 polymerase and Escherichia coli DNA polymerase I (Klenow fragment). These enzymes have either low (Klenow) or no (T7) associated 3' to 5' exonuclease activity. Bacteriophage T4 DNA polymerase, which has a very active 3' to 5' exonuclease, was the most strongly inhibited by all three types of cis-DDP adducts, permitting only 2% translesion synthesis. This enzyme is therefore recommended for replication mapping studies to detect the location of cis-DDP-DNA adducts in a heterologous population. The major replicative enzyme of E. coli, the DNA polymerase III holoenzyme, allowed less than 10% adduct bypass. Postreplication restriction enzyme cleavage studies established that the templates upon which translesion synthesis was observed contained platinum adducts, ruling out the possibility that the observed products were due to a small amount of contamination with unplatinated DNA. The effects on in vitro replication of a recently characterized adduct of trans-DDP [Comess, K. M., Costello, C. E., & Lippard, S. J. (1990) Biochemistry 29, 2102-2110] were also evaluated. This adduct provided a poor block both to DNA polymerases and to restriction enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)


Comparative NMR study of A(n)-bulge loops in DNA duplexes: intrahelical stacking of A, A-A, and A-A-A bulge loops. We have prepared a series of deoxyoligonucleotide duplexes of the sequence d(G-C-A-T-C-G-X-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-G-C), in which X represents either one (A), two (A-A), or three (A-A-A) unpaired adenine basis. Using two-dimensional proton and phosphorus NMR spectroscopy, we have characterized conformational features of these bulge-loop duplexes in solution. We find that Watson-Crick hydrogen bonding is intact for all 12 base pairs, including the GC bases that flank the bulge loop. Observation of NOE connectivities in both H2O and D2O allows us to unambiguously localize all of the bulged adenine residues to intrahelical positions within the duplex. This is in contrast to an earlier model for multiple-base bulge loops in DNA [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840], in which all but the most 5' bulged base are looped out into solution. We find that insertion of two or three bases into the duplex results in the disruption of specific sequential NOEs for the base step across from the bulge loop site on the opposite strand. This disruption is characterized by a partial shearing apart of these bases, such that certain sequential NOEs for this base step are preserved. We observe a downfield-shifted phosphorus resonance, which we assign in the A-A-A bulge duplex to the 3' side of the last bulged adenine residue. Proton and phosphorus chemical shift trends within the An-bulge duplex series indicate that there is an additive effect on the structural perturbations caused by additional unpaired bases within the bulge loop. This finding parallels previous observations [Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840; Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4833-4837] on the magnitude of the induced bending of DNA duplexes by multiple-base bulge loops.


Solution structure of a trinucleotide A-T-A bulge loop within a DNA duplex. We have synthesized an oligodeoxynucleotide duplex, d(G-C-A-T-C-G-A-T-A-G-C-T-A-C-G).d(C-G-T-A-G-C-C-G-A-T-C-G), with a three-base bulge loop (A-T-A) at a central site in the first strand. Nuclear Overhauser experiments (NOESY) in H2O indicate that the GC base pairs flanking the bulge loop are intact between 0 and 25 degrees C. Nuclear Overhauser effects in both H2O and D2O indicate that all bases within the bulge loop are stacked into the helix. These unpaired bases retain an anti conformation about their glycosidic bonds as they stack within the duplex. The absence of normal sequential connectivities between the two cytosine residues flanking the bulge site on the opposite strand indicates a disruption in the geometry of this base step upon insertion of the bulged bases into the helix. This conformational perturbation is more akin to a shearing apart of the bases, which laterally separates the two halves of the molecule, rather than the "wedge" model often invoked for single-base bulges. Using molecular dynamics calculations, with both NOE-derived proton-proton distances and relaxation matrix-calculated NOESY cross peak volumes as restraints, we have determined the solution structure of an A-T-A bulge loop within a DNA duplex. The bulged bases are stacked among themselves and with the guanine bases on either side of the loop. All three of the bulged bases are displaced by 2-3 A into the major groove, increasing the solvent accessibility of these residues. The ATA-bulge duplex is significantly kinked at the site of the lesion, in agreement with previously reported electron microscopy and gel retardation studies on bulge-containing duplexes [Hsieh, C.-H., & Griffith, J. D. (1989) Proc. Natl. Acad. Sci. U.S.A 86, 4833-4837; Bhattacharyya, A., & Lilley, D. M. J. (1989) Nucleic Acids Res. 17, 6821-6840]. Bending occurs in a direction away from the bulge-containing strand, and we find a significant twist difference of 84 degrees between the two base pairs flanking the bulge loop site. This value represents 58% of the twist difference for base pairs four steps apart in B-DNA. These results suggest a structural mechanism for the bending of DNA induced by unpaired bases, as well as accounting for the effect bulge loops may have on the secondary and tertiary structures of nucleic acids.


Synthesis of fully active biotinylated analogues of parathyroid hormone and parathyroid hormone-related protein as tools for the characterization of parathyroid hormone receptors. The synthesis, purification, and characterization of biotinylated analogues of parathyroid hormone (PTH) and PTH-related protein (PTHrP) are described. A novel methodology was developed which allowed the selective biotinylation during solid-phase synthesis of either the Lys13 or Lys26 residue in PTH/PTHrP sequences. Incorporation of orthogonally protected N alpha-Boc-Lys(N epsilon-Fmoc) at a selected position in the sequence, followed by selective side-chain deprotection and biotinylation of the epsilon-amino group, permitted modification of the specific lysine only. Biotinylated analogues of [Nle8,18,Tyr34]bPTH(1-34)NH2 (analogue 1a) were prepared by modification of Lys13 with a biotinyl group (analogue 1) or a biotinyl-epsilon-aminohexanoyl group (analogue 2) or at Lys26 with a biotinyl-epsilon-aminohexanoyl group (analogue 3). A biotinylated PTHrP antagonist [Leu11,D-Trp12,Lys13(N epsilon-(biotinyl-beta-Ala))]PTHrP(7-34)NH2 (analogue 5), was also prepared. In a different synthetic approach, selective modification of the thiol group of [Cys35]PTHrP(1-35)NH2, in solution, with N-biotinyl-N'-(6-maleimidohexanoyl)hydrazide, resulted in analogue 4. The high affinities of the biotinylated analogues for PTH receptors present in human osteosarcoma B-10 cells or in porcine renal cortical membranes (PRCM), were comparable to those of the underivatized parent peptides. The analogues were also highly potent in stimulation of cAMP formation (analogues 1-4) or inhibition of PTH-stimulated adenylyl cyclase (analogue 5) in B-10 cells. The most potent analogue (analogue 1) had potencies in B-10 cells (Kb = 1.5 nM, Km = 0.35 nM) and in porcine renal membranes (Kb = 0.70 nM) identical or similar to those of its parent peptide, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of the cellular actions of nerve growth factor by staurosporine and K252A results from the attenuation of the activity of the trk tyrosine kinase. The protein kinase inhibitors staurosporine and K252A inhibit some of the cellular actions of nerve growth factor (NGF). To explore the molecular mechanisms involved, we test the ability of these agents to block one of the earliest cellular responses to NGF, protein tyrosine phosphorylation. Concentrations of 10-100 nM staurosporine and K252A inhibit NGF-dependent tyrosine phosphorylation in PC12 cells and inhibit trk oncogene-dependent tyrosine phosphorylation in trk-transformed NIH3T3 (trk-3T3 cells). In contrast, these compounds are without effect on epidermal growth factor (EGF)-stimulated tyrosine phosphorylation in PC12 cells. NGF-stimulated tyrosine phosphorylation of the pp140c-trk NGF receptor and tyrosine phosphorylation of pp70trk are also inhibited by similar concentrations of staurosporine and K252A, whereas tyrosine phosphorylation of the EGF receptor, insulin receptor, and v-src is not affected. Both staurosporine and K252A inhibit the autophosphorylation of pp70trk on tyrosine residues in an in vitro immune complex kinase reaction. Incubation of trk-3T3 cells with 10 nM staurosporine causes rounded transformed cells to revert to a normal flattened phenotype, whereas src-transformed cells are unaffected by this agent. These data suggest that staurosporine and K252A specifically inhibit the trk tyrosine kinase activity through a direct mechanism, probably accounting for the attenuation by these agents of the cellular actions of NGF.


Structural studies of the acidic transactivation domain of the Vmw65 protein of herpes simplex virus using 1H NMR. We have overproduced and purified the carboxy-terminal transactivation domain of Vmw65 (VP16) of herpes simplex virus, and studied potential folding of the domain by 1H NMR. Two species of the acidic domain were obtained from the bacterial expression system, and we demonstrate that one of these represents read-through of the natural amber termination codon of the Vmw65 reading frame producing a larger polypeptide. Additional residues in the read-through product were identified by total amino acid analysis and by NMR. Study of the correctly terminated product by 1D NMR gave resonances which were clustered into groups around their random-coil chemical shift positions, and 2D NMR demonstrated that, even in mixed solvents containing up to 80% MeOH, there was very little evidence of secondary structure. Together these results indicate that the isolated acid domain has little if any alpha-helical content of any stable nature. We discuss these results with reference to the demonstrated activity of the acidic domain in a wide variety of polypeptide contexts.


Albumins activate peptide hydrolysis by the bifunctional enzyme LTA4 hydrolase/aminopeptidase. Albumins from several species activated the bifunctional, Zn2+ metalloenzyme amino-peptidase/leukotriene A4 hydrolase (EC 3.3.2.6). Bovine serum albumin, 1 mg/mL, increased hydrolysis of L-proline-p-nitroanilide and leucine-enkephalin by 12-fold and 7-fold, respectively. The apparent Km for L-proline-p-nitroanilide was inversely proportional to the albumin concentration from 0 to 1 mg/mL, declining from 9.4 to 0.7 mM without an appreciable change in apparent Vmax. These data imply a random activation process in which the enzyme-activator complex is catalytically dominant. Hill plots indicated a 1:1 stoichiometric relationship between albumin and enzyme. Secondary plots of slope versus the reciprocal of albumin concentration indicated that it binds to the enzyme with an affinity constant of 0.9 microM. The pH optimum of the nonactivated enzyme occurred at pH 8; the albumin-activated enzyme had an optimum near pH 7. Neither ultrafiltration nor dialysis of albumin altered its activating effect, but boiling abolished it. Albumin did not affect other cytosolic or microsomal leucine aminopeptidases, or gamma-glutamyltransferase. Albumin functions as a nonessential activator, since enzymatic activity was always detectable in its absence. Chloride ions, which activate other Zn2+ metalloenzymes, also activated leukotriene A4 hydrolase/aminopeptidase with an EC50 = 50 mM, increasing its initial velocity 2.2-fold in the absence of albumin. Zn2+ activated the enzyme, increasing its apparent Vmax but not its apparent Km, suggesting it replaced Zn2+ lost from the active site, especially at acidic pH. At concentrations greater than 30-50 microM, Zn2+ was inhibitory. Albumin mitigated the effect of chloride, but not the effect of Zn2+ or that of the competitive inhibitor, captopril.(ABSTRACT TRUNCATED AT 250 WORDS)


An attempt to discriminate catalytic and regulatory proton binding sites in membrane-bound, thiol-reduced chloroplast ATPase. The question of the possible identity of catalytic and regulatory proton pathways in the chloroplast FoF1 ATPase has been studied using different energy-transfer inhibitors. Venturicidin, a reversible inhibitor of Fo, affects neither the delta mu H(+)-dependent thiol reduction of the membrane-bound chloroplast ATPase nor its ability to be activated by the proton gradient. It seems therefore to block only the proton flow required by the catalytic function of the enzymes. Venturicidin, however, also slows down the deactivation of the thiol-reduced ATPases during uncoupled ATP hydrolysis, following a delta mu H+ activation, but phloridzin, a reversible F1 inhibitor, has the same effect. Tentoxin, an irreversible F1 inhibitor, decreases the rate of ATP hydrolysis but does not affect the rate of deactivation. These findings suggest that catalytic and regulatory H(+)-binding sites are different. No distinction can be made, if any, between protons involved in unmasking the thiol-sensitive groups of F1 and in activating the enzyme. The effect of venturicidin and phloridzin on the deactivation is consistent with an inhibitory effect of newly formed--by ATP hydrolysis--ADP molecules, which might affect the enzyme without passing through the medium. Phosphate at millimolar concentration has an effect similar to low concentrations of phloridzin and venturicidin, probably by a simple back-reaction effect.


Photosynthetic electron-transfer reactions in the green sulfur bacterium Chlorobium vibrioforme: evidence for the functional involvement of iron-sulfur redox centers on the acceptor side of the reaction center. The green sulfur bacterium Chlorobium vibrioforme was cultured in the presence of ethylene to selectively inhibit the synthesis of the chlorosome antenna BChl d. Use of these cells as starting material simplified the isolation of a photoactive antenna-depleted membrane fraction without the use of high concentrations of detergents. The preparation had a BChl alpha/P840 of 50, and the spectral properties were similar to those of preparations isolated from cells grown with a normal complement of chlorosomes. The membrane preparation was active in NADP+ photoreduction. This indicated that the fraction contained reaction centers with complete electron-transfer sequences which were then characterized further by flash kinetic spectrophotometry and EPR. We confirmed that cytochrome c553 is the endogenous donor to P840+, and at room temperature we observed a recombination reaction between the reduced terminal acceptor and P840+ with a t1/2 = 7 ms. Oxidative degradation of iron-sulfur centers using low concentrations of chaotropic salts introduced a faster recombination reaction of t1/2 = 50 microseconds which was lost at higher concentrations of chaotrope, indicating the participation of another iron-sulfur redox center earlier than the terminal acceptor. Cluster insertion using ferric chloride and sodium sulfide in the presence of 2-mercaptoethanol restored both the 50-microseconds and 7-ms recombination reactions, allowing definitive assignments of these centers as iron-sulfur centers. Following the suggestion of Nitschke et al. [(1990) Biochemistry 29, 3834-3842], we associate these two kinetic phases to back-reactions between P840+ and iron-sulfur centers FX and FAFB, respectively. The iron-sulfur cluster degradation and reconstitution protocols also led to inhibition and restoration of NADP+ photoreduction by the membrane preparation, providing unequivocal evidence for the function of the centers FX and FAFB in the physiological electron-transfer sequence on the acceptor side of the Chlorobium reaction center. At 77 K we observed a recombination reaction of t1/2 = 20 ms that we suggest occurs between Fx- and P840+. Degradation of the iron-sulfur clusters resulted in replacement of the 20-ms phase with a faster reaction of t1/2 = 80 microseconds that was most likely a recombination between the early acceptor A1- and P840+ or decay of 3P840. Analysis of the iron-sulfur centers in the preparation by EPR at cryogenic temperature supports the optical measurements. EPR signals originating from the terminal acceptor(s) were not observed following treatment of the membrane preparation by chaotropes, and a modified signal was restored following cluster reinsertion.(ABSTRACT TRUNCATED AT 400 WORDS)


Is bicarbonate in Photosystem II the equivalent of the glutamate ligand to the iron atom in bacterial reaction centers? Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, QA and QB. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g., Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered GluM234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P+) in the absence of exogenous donor, i.e., the recombination of P+Q-A or P+Q-B, respectively; (b) the kinetics of electron transfer from Q-A to QB; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P+ (QB turnover). The absence of a formate-bicarbonate effect in these mutants suggests that GluM234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of QA. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (tau = 3.5 ps), followed by reduction of the bacteriopheophytin (tau = 0.9 ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of QA was slowed by a factor of 2 (tau = 410 +/- 30 and 47 +/- 30 ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type).(ABSTRACT TRUNCATED AT 400 WORDS)


Oxidation-reduction properties of the ferredoxin-linked glutamate synthase from spinach leaf. Oxidation-reduction titrations have been conducted to determine the midpoint potential (Em) values of the three electron-carrying prosthetic groups of the ferredoxin-linked glutamate synthase isolated from spinach leaves. Titrations using electron paramagnetic resonance (EPR) signals to monitor the oxidation state of the [3Fe-4S]+,0 cluster found in the enzyme, indicated the presence of a single n = 1 component with Em = -170 mV at pH 7.7. Titrations using absorbance changes in the visible region to monitor the oxidation states of the FAD and FMN groups present in the enzyme could be fit to a single n = 2 Nernst curve with Em = -180 mV at pH 7.7. The magnitude of the absorbance change observed during this titration accounts for all of the FMN and FAD found in the enzyme, indicating that the two flavins are either isopotential or differ in Em by less than about 30 mV. Neither optical nor EPR titrations gave any evidence for the presence of stable flavin free radicals. These results represent the first characterization of the redox properties of the prosthetic groups of a ferredoxin-dependent glutamate synthase.


Electron transfer from excited tryptophan to cytochrome c: mechanism of phosphorescence quenching? Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm.


Rates of cyanide binding to the catalytic intermediates of mammalian cytochrome c oxidase, and the effects of cytochrome c and poly(L-lysine). Rate constants of cyanide binding to 'fast' oxidase have been measured in the fully-oxidised (O), peroxy (P) and ferryl (F) states at pH 8.0. Values of 2.2, 8 and 10 M-1 s-1, respectively, were obtained. Thus, none of these states appears to exhibit a rate that would identify it as the species responsible for the extremely rapid cyanide binding observed during turnover. On the other hand, with 'oxidised' enzyme as prepared, containing a very small fraction of one-electron-reduced (E state) oxidase, a corresponding fraction of enzyme exhibited spectral changes consistent with cyanide binding with a rate constant in excess of 10(4) M-1 s-1. Evidence is presented suggesting that mediation of electron transfer from one-electron-reduced, cyanide-liganded enzyme to free, ferric oxidase, rather than a global protein conformational change of the enzyme, is responsible for the greatly enhanced cyanide binding rates seen in the presence of cytochrome c or poly(L-lysine). Inter-oxidase electron exchange in 'oxidised' enzyme can result in a complicated dependence of the binding rate on cyanide concentration. We have demonstrated that this may give rise to a saturation of the rate of cyanide binding.


Determination of the position of the Qi.- quinone binding site from the protein surface of the cytochrome bc1 complex in Rhodobacter capsulates chromatophores. The technique of distance measurement, utilizing spin relaxation enhancement by an external probe, has been extended to the study of intrinsic semiquinone radicals through the use of holmium-EDTA complexes and continuous wave electron paramagnetic resonance spectroscopy. This technique has been used to determine the distance of the semiquinone anion, Qi (also designated as Qn.- or Qc.-), from the surface of the ubiquinone cytochrome c oxidoreductase, consisting of only three subunits, in membrane particles from Rhodobacter capsulates. The location of the semiquinone anion is 6-10 A from the N side protein, establishing that there are two separate quinone reaction sites, i.e., 'Qi' and 'Qo', within this complex on opposite sides of the membrane. The results are discussed in relation to reported ENDOR, EPR, and optical studies of the mitochondrial counterpart.


Stimulation of the catalytic cycle of the Ca2+ pump of porcine plasma-membranes by negatively charged phospholipids. The (Ca(2+)+Mg2+)-ATPase of the plasma membrane is activated by negatively charged phospholipids. The mechanism of this activation was investigated by studying the effect of negatively charged phospholipids on the steady-state phosphointermediate level and on the p-nitrophenylphosphatase activity. Both parameters were differentially affected by different acidic phospholipids. The level of phosphoprotein intermediate was not affected by phosphatidylserine (20% of total phospholipid), but it was increased by 60% by phosphatidylinositol 4-phosphate. Phosphatidylserine increased the p-nitrophenylphosphatase activity, whereas phosphatidylinositol 4-phosphate had no significant effect. It is suggested that phosphatidylinositol 4-phosphate mainly affects a reaction step which leads to accelerated formation of the phosphointermediate, whereas the action of phosphatidylserine would affect two reaction steps, one upstream and one downstream of the phosphointermediate.


Binding of scatter factor to epithelial cell membrane protein: identification of its receptor. Binding of scatter factor (SF) to the surface protein of Madin-Darby canine kidney (MDCK) cells was investigated. The factor has a specific affinity for membrane proteins of MDCK cells and could be purified 10-20-fold using a membrane protein-affinity chromatographic procedure. The binding was pH- and salt-dependent. The factor did not bind to columns prepared with membrane proteins from non responder cells or with bovine serum albumin. Further purification to homogeneity was achieved using reverse phase and immunoaffinity chromatography. The factor dissociated into 92, 62 and 34/32 kDa bands on SDS-PAGE under reducing conditions. A 230 kDa protein band, the receptor-SF complex, was observed when radiolabeled SF was crosslinked to surface proteins of MDCK cells and the complexes were subjected to electrophoresis. The binding of radiolabeled SF to the MDCK cells was decreased in presence of excess unlabeled SF. These observations suggest that the binding of SF to surface proteins of MDCK cells is specific and occurs predominantly to a 150 kDa protein.


Transmembrane redistribution of phospholipids of the human red cell membrane during hypotonic hemolysis. The transmembrane distribution of spin-labeled phospholipids was measured in human erythrocytes before and after hypotonic hemolysis by electron paramagnetic resonance. With a first series of partially water soluble probes a complete randomization of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin analogues was achieved when cells were resealed in the absence of Mg-ATP or when the aminophospholipid translocase was inhibited by vanadate or calcium. If the ghosts were resealed with Mg-ATP inside, the transmembrane asymmetry of the aminophospholipids was reestablished. With long chain insoluble spin-labeled lipids complete randomization was obtained with the phosphatidylcholine analogue but even in the presence of vanadate only a small percentage (approx. 15%) of the spin-labeled phosphatidylserine flopped to the outer monolayer and comparable percentage of the spin-labeled sphingomyelin flipped to the inner monolayer, indicating a hierarchy in the phospholipid redistribution for these water insoluble lipids during hemolysis. The mechanism by which a selective randomization takes place is not known. It may involve phosphatidylserine-protein interactions in the inner leaflet and sphingomyelin-cholesterol or sphingomyelin-sphingomyelin interaction in the outer leaflet.


Cyclic AMP stimulates Ca(2+)-ATPase-mediated Ca2+ extrusion from human platelets. The effect of cAMP on active Ca2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca2+ in the cytoplasmic compartment ([Ca2+]cyt). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca2+]cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147-163). Forskolin, at a maximally effective concentration of 10 microM, was shown to stimulate Ca2+ extrusion by increasing by a factor of 1.6 +/- 0.5 the Vm of a saturable component, previously identified with a Ca(2+)-Mg(2+)-ATPase located in the plasma membrane. Neither the Km (80 nM) or Hill coefficient (1.7 +/- 0.3) of the Ca(2+)-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na+/Ca2+ exchanger) over the [Ca2+]cyt range examined (50-1500 nM). Dibutyryl-cAMP (Bt2-cAMP, 1 mM) stimulated the Ca(2+)-Mg(2+)-ATPase component of Ca2+ extrusion by a factor of 2.0 +/- 0.6. Separate experiments showed that 10 microM forskolin reduces the resting [Ca2+]cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in Vm of the pump, countered by a 37-74% increase in the rate constant for passive Ca2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca2+]cyt and (b) increasing the rate of Ca2+ extrusion after the initial influx or triggered release event.


Stimulation of dense tubular Ca2+ uptake in human platelets by cAMP. Elevation of intracellular cAMP is shown to increase the rate (V) and maximal extent of Ca2+ uptake by the dense tubules in intact human platelets. Elevation of [cAMP] was accomplished by preincubation with the adenylate cyclase activator forskolin or with dibutyryl-cAMP (Bt2-cAMP). The free concentration of Ca2+ in the dense tubular lumen ([Ca2+]dt) was monitored using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. The free cytoplasmic Ca2+ concentration ([Ca2+]cyt) was monitored in parallel experiments with quin2. Both [Ca2+]cyt and [Ca2+]dt were analyzed in terms of competition between pump and leak mechanisms in the plasma membrane (PM) and dense tubular membrane (DT). When platelets are incubated in media with approx. 1 microM external Ca2+, [Ca2+]cyt is approx. 50 nM and [Ca2+]dt is very low. When 2 mM external Ca2+ is added, [Ca2+]cyt rises to approx. 100 nM and the process of dense tubular Ca2+ uptake can be resolved. Forskolin (10 microM) and Bt2-cAMP increase the rate of dense tubular Ca2+ uptake (V) to 2.1 +/- 0.60 and 1.70 +/- 40 times control values (respectively). The agents also increase the final [Ca2+]dt to 1.70 +/- 0.21 and 1.72 +/- 0.60 times control values (respectively). Titrations with ionomycin (Iono) showed that the increase was due to an increase in the Vm of the dense tubular Ca2+ pump. With [Iono] = 500 nM, [Ca2+]cyt was raised to greater than or equal to 1.0 microM and Vm of the dense tubular pump was elicited. (At [Iono] = 1.0 microM, the final [Ca2+]dt values were degraded 15% due to shunting of Ca2+ uptake.) Analysis showed that forskolin (10 microM) and Bt2-cAMP (1 mM) increase the Vm by a factors of 1.56 +/- 40 and 1.56 +/- 40, respectively. Analysis showed that neither agent changed the Km of the pump significantly from its control value of 180 nM. Neither agent changed the rate constant for passive leakage of Ca2+ across the DT membrane (1.7 min-1).


Cyclic GMP increases the rate of the calcium extrusion pump in intact human platelets but has no direct effect on the dense tubular calcium accumulation system. Sodium nitroprusside (SNP) and other agents that elevate cGMP levels are known to inhibit the aggregation of human platelets. Published data suggest that cGMP attenuation of agonist-induced Ca2+ transients is involved in this effect. The present study shows that elevation of cGMP increases the rate of the Ca2+ extrusion pump located in the plasma membrane (PM) but does not have a direct effect on the Ca2+ accumulating pump of the dense tubules (DT). The study verifies that SNP can specifically elevate the cGMP level in the platelet. The kinetics of the Ca2+ extrusion system were studied in situ in platelets overloaded with the cytoplasmic Ca2+ indicator quin2 according to a published protocol developed in this laboratory. Elevation of cGMP by means of (10 microM) SNP increased the Vm of the Ca(2+)-ATPase pump by 63%, without affecting its Km (66-80 nM) or Hill coefficient (1.6-1.8). Dibutyryl-cGMP (Bt2-cGMP), preincubated for 45 min at 1 mM, increased the Vm by a factor of 2.2 +/- 0.4. The experiments did not give any indication that SNP or Bt2-cGMP change the rate of the Na+/Ca2+ exchanger which makes a minor contribution to Ca2+ extrusion in the studied [Ca2+]cyt range. The rate constant for passive leakage of Ca2+ across the PM was increased by 32 +/- 4% by SNP and 90 +/- 34% by Bt2-cGMP. The net result is that the free Ca2+ in the cytoplasm ([Ca2+]cyt) at 'rest' is lowered from control values of 112 nM to 89 nM or 80 nM, respectively. The kinetics of Ca2+ uptake by the dense tubules were determined in situ using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. Analysis showed that SNP and Bt2-cGMP had no effect on the Vm or Km of the dense tubular pump, and did not affect the rate constant for passive leakage. The agents did decrease resting [Ca2+]dt by 25% or 30%, respectively, but this result can be explained purely in terms of the reduced [Ca2+]cyt. The effects of cGMP (vs. cAMP) on the PM and DT pumps are closely correlated with reported effects of cGMP/cAMP induced phosphorylation of a protein of the molecular weight of the PM pump and a 22 kDa activator of the DT pump. Cyclic AMP increases the rate of both the PM and the DT pumps, whereas cGMP increases the rate of the PM pump only.(ABSTRACT TRUNCATED AT 400 WORDS)


Effects of magnesium and ATP on pre-steady-state phosphorylation kinetics of the Na+,K(+)-ATPase. The aim of the present work was to elucidate the role played by ATP and Mg2+ ions in the early steps of the Na+,K(+)-ATPase cycle. The approach was to follow pre-steady-state phosphorylation kinetics in Na(+)-containing K(+)-free solutions under variable ATP and MgCl2 concentrations. The experiments were performed with a rapid mixing apparatus at 20 +/- 2 degrees C. The concentrations of free and complexes species of Mg2+ and ATP were calculated on the basis of a dissociation constant of 0.091 +/- 0.004 mM, estimated with Arsenazo III under identical conditions. A simplified scheme were ATP binds to the ENa enzyme, which is phosphorylated to MgEPNa and consequently dephosphorylated returning to the ENa form, was used. In the absence of ADP and phosphate four rate constants are relevant: k1 and k-1, the on and off rate constants for ATP binding; k2, the transphosphorylation rate constant and k3, the constant that governs the dephosphorylation rate. The values obtained were: k1 = 0.025 +/- 0.003 microM-1 ms-1 for both free ATP and ATPMg; k-1 = 0.038 +/- 0.004 ms-1 for free ATP and 0.009 +/- 0.002 ms-1 for ATPMg; k2 = 0.199 +/- 0.005 ms-1; k3 = 0.0019 +/- 0.0002 ms-1. The model that seems best to explain the data is one where (i) the role of true substrate can be played equally well by free ATP or ATPMg, and (ii) free Mg2+, an essential activator, acts by binding to a specific Mg2+ site on the enzyme molecule.


Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study. Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847-851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.


Effect of graded hypoxia on brain cell membrane injury in newborn piglets. Alterations in brain cell membrane structure and function during cerebral hypoxia were investigated by measuring Na+,K(+)-ATPase activity and levels of lipid peroxidation products in brain cell membranes obtained from newborn piglets following exposure to 60 min of hypoxic hypoxia in vivo. Cerebral hypoxia was documented as a decrease in the ratio of phosphocreatine to inorganic phosphate (PCr/Pi) using 31P-NMR spectroscopy. During hypoxia (FiO2 0.07-0.11), PCr/Pi decreased 28-47% compared to the corresponding baseline value without a decrease in cerebral ATP levels. No change in brain cell membrane Na+,K(+)-ATPase activity was observed for changes in PCr/Pi of less than 30%. When PCr/Pi was at least 30% lower than baseline, Na+,K(+)-ATPase activity decreased linearly as a function of the decrease in PCr/Pi (r = 0.95). Levels of lipid peroxidation products (conjugated dienes and fluorescent compounds) increased significantly as PCr/Pi decreased. These data suggest that below a critical threshold value of oxidative metabolism there are progressive changes in brain cell membrane structure and function during cerebral hypoxia, and demonstrate that membrane alterations occur prior to changes in cellular ATP levels.


Brain cell membrane dysfunction following acute asphyxia in newborn piglets. Brain cell membrane function during and following single and repeated episodes of asphyxia was investigated. Asphyxia in 24 anesthetized, paralyzed, mechanically-ventilated newborn piglets was produced by stopping ventilation for 2-3 min followed by recovery with reventilation. Measurements of cerebral Na+,K(+)-ATPase activity and of lipid peroxidation products, conjugated dienes and fluorescent compounds, were made during control (n = 12), asphyxia (n = 5), recovery after a single asphyxia event (n = 4), and recovery following 7 repeated asphyxia episodes (n = 3). Cerebral Na+,K(+)-ATPase activity remained unchanged from control during asphyxia (14.57 +/- 2.43 compared to 15.33 +/- 4.27 mumol Pi/mg protein/h, mean +/- SD), but was significantly reduced both during recovery after single (3.87 +/- 1.66) and after repeated (2.59 +/- 1.58) asphyxias, representing a 73 and 82% reduction in enzyme activity, respectively. Conjugated dienes and fluorescent compounds were similarly unchanged during asphyxia compared to control, but increased during recovery from single and from repeated episodes. Decreased cerebral Na+,K(+)-ATPase activity, simultaneous with an increase in lipid peroxidation products, reflects significant cellular membrane damage consistent with oxygen free radical formation during the recovery from acute asphyxia in the newborn piglet.


Interface between hydroxyapatite and mandibular human bone tissue. Samples from intraosseous dental implants, removed from patients for mechanical failures, were examined to analyse the interaction between hydroxyapatite as plasma sprayed coating on titanium supports and human bone. The implantation time varied up to 8 years. No failures had arisen from problems at the interface between the hydroxyapatite coating and bone. The number of samples examined and the implantation times give good statistical conclusions. Histological and microchemical studies showed the good performance and compatibility of this sprayed hydroxyapatite. We present evidence from the best samples which show close bonding with the surrounding bone tissue. New bone is seen all around the coated implant. The composition of the calcium phosphate deposited on the hydroxyapatite and cellular approach were determined, and demonstrate the efficiency of the interaction between this plasma sprayed hydroxyapatite and the bone.


Toxicity of silica-containing calcium phosphate glasses demonstrated in mice. Suspensions of calcium phosphate glass containing various concentrations of silica (glass composition (moles): 100 Ca(PO3)2 to x SiO2,x = 0, 5, 10, 15 or 40) dispersed in normal saline were injected intraperitoneally into C57BL/6 mice to determine the mortality within 30 days. The mortality was 0/10, 3/10, 9/10, 10/10 and 10/10 at x = 0, 5, 10, 15 and 40 mol of silica, respectively. By means of inductively coupled plasma analysis, the amount of dissolved silica (Si4+) in water at 37 degrees C from the calcium phosphate glass depended on the amount of silica in the glasses. The mortality of mice was directly proportional to the silica content of the glass injected intraperitoneally. These results clearly show that the dissolved silica (Si4+) from the glass, monomeric or low molecular silicic anion, is highly toxic. The SiO2 component in biomaterials has toxic potential when dissolved in the body.


Theoretical prediction of the gel electrophoretic retardation changes due to point mutations in a tract of SV40 DNA. The changes of gel electrophoretic retardation due to single base substitutions in a 173 bp fragment of Sv40 DNA were predicted by using a theoretical model based on conformational energy calculations. As described in previous papers, this model allows successful prediction of the gel electrophoretic retardation of synthetic as well as natural DNAs reported in literature. The experimental retardations related to 195 point-mutated DNAs were reproduced with a standard deviation of 0.05 comparable with the experimental one of 0.04. This result, which represents a very critical test for the proposed model, indicates that DNA superstructures can be satisfactorily predicted on the simple physical basis of the integration of the nearest-neighbour perturbations in the dinucleotide steps. Thus, cooperative effects appear, in the majority of cases investigated, to play a second order role.


Cystectomy and substitution enterocystoplasty: alternative primary treatment for T2/3 bladder cancer. The optimal treatment for invasive bladder cancer remains controversial. Although external beam radiotherapy is able to eradicate the disease in a number of patients, the difficulty is selecting those who will respond. Those who do develop a local recurrence will require a salvage cystectomy combined with urinary diversion. The results of performing cystectomy and bladder reconstruction as a primary procedure are presented and the concept of combining this with chemotherapy as an alternative strategy for the management of bladder cancer is discussed.


Comparison of DMSA scintigraphy with intravenous urography for the detection of renal scarring and its correlation with vesicoureteric reflux. A series of 208 patients was prospectively assessed for reflux nephropathy by intravenous urography (IVU) and 99mTc-dimercaptosuccinate (DMSA) scintigraphy. All patients were studied at least 3 months after their most recent urinary tract infection and micturating cystourethrography (MCU) was performed prior to the scintigraphic studies. DMSA scintigraphy detected significantly more cortical abnormalities than did IVU. There was also a correlation between cortical abnormalities in the DMSA studies and the degree of reflux on MCU. The validity of DMSA as a cortical imaging agent is evaluated and the histological evidence for its efficacy derived from the animal model is reviewed, lending weight to its establishment as the "gold standard" for renal cortical scarring.


GLOBIC: a very fast microcomputer program for fingerprinting, characterization and comparison of long nucleotide sequences. This paper describes the program GLOBIC, which compares, characterizes and fingerprints even 0.1 Mbase sequences in a few minutes with the aid of an IBM-AT microcomputer. Instead of the nucleotide sequences themselves, GLOBIC compares the local nucleotide or short oligonucleotide compositions. GLOBIC presents two-dimensional maps of contour lines depicting the similarity of two different sequences, a sequence compared to itself, to its complementary sequence or to a random sequence. A vocabulary is presented to translate the typical patterns appearing in the two-dimensional maps into their meanings as relationships between the compared sequences. The application of GLOBIC is demonstrated using several examples from the genomic nucleotide sequences of bacteriophage T7, adenovirus type-2 and Epstein-Barr virus.