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R Jordan

Publications and source records attributed to R Jordan.

At least 73 records · Page 4Linked to original sources
PubMed1995

Modulation of the ATPase activity of the molecular chaperone DnaK by peptides and the DnaJ and GrpE heat shock proteins.

Previous studies have demonstrated that the Escherichia coli DnaK, DnaJ, and GrpE heat shock proteins participate in the initiation of bacteriophage lambda DNA replication by mediating the required disassembly of a preinitiation nucleoprotein structure that is formed at the phage replication origin. To gain some understanding in a simpler system of how the DnaJ and GrpE cochaperonins influence the activity of DnaK, we have examined the effect of the cochaperonins on the weak intrinsic ATPase activity of the molecular chaperone DnaK in the presence and absence of peptide effectors. We have found that random sequence peptide chains of 8 or 9 amino acid residues in length yield optimal (10-fold) activation of the DnaK ATPase, whereas peptides with 5 or fewer residues fail to stimulate the ATPase of this bacterial hsp70 homologue. Furthermore, we have discovered that those peptides that interact best with DnaK, as judged by their KA as activators of ATP hydrolysis by DnaK, also act as strong inhibitors of lambda DNA replication in vitro. The inhibitory effect of peptides on lambda DNA replication was overcome by increasing the concentration of DnaK in the replication system. Diminished inhibition was also found when the replication system was supplemented with GrpE cochaperonin, a protein known to increase the effectiveness of DnaK action in lambda DNA replication. These and other results suggest that the peptide-binding site of DnaK is required for its function in lambda DNA replication. Apparently, peptides sequester free DnaK protein and block lambda DNA replication by reducing the amount of DnaK that is free to mediate disassembly of nucleoprotein preinitiation structures. In related studies, we have found that DnaJ, like short peptides, activates the intrinsic ATPase activity of DnaK. DnaJ, however, is substantially more potent in this regard, since it activates DnaK at concentrations 1000-fold below those required for a peptide of random sequence. By itself, the GrpE cochaperonin has no effect on the peptide-independent ATPase activity of DnaK, but GrpE does vigorously stimulate the peptide-dependent ATPase of the DnaK chaperone. Under steady-state conditions, the Vmax of ATP hydrolysis by DnaK was elevated approximately 40-fold by the presence of GrpE and saturating levels of peptides.

Adenosine Triphosphatases↗
PubMed1995

Comparative real-time effects on platelet adhesion and aggregation under flowing conditions of in vivo aspirin, heparin, and monoclonal antibody fragment against glycoprotein IIb-IIIa.

BACKGROUND: A real-time in vitro system of human platelet thrombosis under arterylike flowing conditions similar to those produced in vivo by angioplasty would be useful for the evaluation of potential antiarterial thrombotic agents in association with in vivo trials. Aspirin, heparin, and the chimeric monoclonal antibody antigen-binding fragment 7E3 (c7E3 Fab) directed against platelet glycoprotein (GP) IIb-IIIa have been used in attempts to delay or prevent thrombotic reocclusion of coronary arteries after angioplasty. We compared the effects of these agents administered in vivo on GPIb-mediated platelet adhesion to von Willebrand factor (vWF)/collagen type I (as in atherosclerotic subendothelium) and on subsequent GPIIb-IIIa-fibrinogen/vWF-mediated platelet aggregation under flowing conditions analogous to those in constricted coronary arteries. METHODS AND RESULTS: Citrated whole blood containing mepacrine-labeled platelets from patients and healthy donors was perfused for 1 minute at an abnormally elevated shear rate of 1500 seconds-1 (arterial wall shear stress of 50 to 60 dynes/cm2) at 37 degrees C over collagen I/vWF. The number of adherent fluorescent platelets was quantified every 15 seconds with a low-light-level video camera and epifluorescent microscopy. After 5 healthy donors had ingested 975 mg aspirin, platelet adhesion was unaffected in the aspirin-treated blood compared with the control blood in all experiments (10 of 10), and subsequent aggregation was unchanged in most runs (8 of 10). The blood of 3 aspirin-treated patients undergoing angioplasty was analyzed before and after a 12,000-U heparin injection and 2 minutes, 2 hours, and 24 hours after infusion of 0.25 mg/kg of c7E3 Fab. In these patients, the bolus of heparin did not inhibit either platelet adhesion to collagen I/vWF or subsequent aggregation. In contrast, there was > 50% inhibition of platelet aggregation 2 minutes after the infusion of c7E3 Fab in all 3 patients, and inhibition persisted in 2 of the 3 patients at 2 hours and 24 hours after c7E3 Fab. CONCLUSIONS: In contrast to aspirin or heparin, the in vivo injection of c7E3 Fab considerably reduces platelet aggregate formation mediated by the binding of fibrinogen, vWF, or some other ligand to platelet GPIIb-IIIa under conditions of abnormally increased shear stress analogous to those in narrowed coronary arteries. Platelet adherence to collagen I/vWF is not affected. This study describes an in vitro model of arterial injury (similar to angioplasty) that uses human blood to compare directly, in real time, the precise relative effects of aspirin, heparin, and c7E3 Fab on platelet adhesion and subsequent aggregation.

Abciximab↗
PubMed1995

Shear-induced platelet aggregation is inhibited by in vivo infusion of an anti-glycoprotein IIb/IIIa antibody fragment, c7E3 Fab, in patients undergoing coronary angioplasty.

BACKGROUND: Elevated levels of shear stress such as those that occur in stenotic arterial vessels can directly activate and aggregate platelets and thus contribute to the pathogenesis of acute arterial thrombosis. This shear-induced platelet aggregation (SIPA) is mediated by von Willebrand factor binding to platelet membrane glycoprotein (GP) Ib and GPIIb/IIIa. The chimeric Fab fragment of the monoclonal antibody 7E3 (c7E3 Fab) that binds selectively to GPIIb/IIIa is under clinical evaluation in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). This study was undertaken to investigate the effects on ex vivo SIPA of c7E3 Fab administered to patients undergoing PTCA. METHODS AND RESULTS: Six patients received aspirin (325 mg) and boluses of heparin (12,00o U) followed by c7E3 Fab 0.25 mg/kg. Blood collected from each patient before and after heparin treatment and at various time points after c7E3 Fab administration was subjected to laminar shear stress in a cone-and-plate viscometer. Flow cytometry was used to quantify the extents of platelet aggregation and of antibody binding to GPIIb/IIIa. Results indicate that c7E3 Fab injection resulted in a rapid, extensive blockade of GPIIb/IIIa receptors (98.6 +/- 0.2%) and a 50% inhibition of ex vivo platelet aggregation induced by shear stress. c7E3 Fab also completely abolished the formation of large platelet aggregates ("large" refers to particles > 10 microns in equivalent sphere diameter), which are presumably the aggregates of greatest clinical significance. Partial reversibility of the inhibition was noted within 2 days after drug administration, but even after 1 week, platelet function had not been fully restored. CONCLUSIONS: This study demonstrates that c7E3 Fab is a potent inhibitor of SIPA, which may be an important mechanism of its beneficial effect in the treatment of arterial occlusive diseases and in the prevention of thrombotic complications of coronary artery disease after angioplasty.

Abciximab↗
PubMed1995

Blockade of the human platelet GPIIb/IIIa receptor by a murine monoclonal antibody Fab fragment (7E3): potent dose-dependent inhibition of platelet function.

The platelet glycoprotein (GP) IIb/IIIa receptor can bind fibrinogen, von Willebrand factor, and other adhesive ligands; this binding is the final common pathway mediating platelet aggregation. The purpose of this study was to evaluate the safety and platelet inhibitory characteristics of the Fab fragment of the murine monoclonal anti-GPII/IIIa 7E3 antibody (m7E3 Fab) when administered intravenously as a single bolus dose, as a single and repeat bolus dose, and as a single bolus dose followed by continuous infusions of varying duration. Various dosage regimens of m7E3 Fab were studied in 74 patients with stable angina. Dosage regimens included single doses of m7E3 Fab from 0.1 to 0.3 mg/kg, a single dose of 0.20-0.30 mg/kg, and a repeat dose of 0.05 mg/kg, or a loading dose followed by a continuous infusion of m7E3 Fab for up to 36 hours. To assess the effect of m7E3 Fab on platelet function, quantitative blockade of GPIIb/IIIa receptors, inhibition of ex vivo platelet aggregation, and template bleeding time were measured in all patients. Dose-dependent inhibition of platelet function was evident in response to escalating bolus doses of m7E3 Fab, with maximum inhibition observed at 0.25-0.30 mg/kg body weight; at the 0.30 mg/kg dose, mean (+/- SE) GPIIb/IIIa receptor blockade was 81 +/- 3%, ex vivo platelet aggregation in response to 20 microM ADP was 14 +/- 6% of baseline, and the median bleeding time was > 20 minutes. Although platelet function gradually recovered following a single bolus injection, platelet inhibition could be sustained by continuous, low-dose infusion of the antibody. Platelet inhibition occurred within minutes, but m7E3 Fab that did not bind to platelets cleared rapidly from circulation. Sixteen percent of the m7E3 Fab-injected subjects exhibited low titer, human anti-murine antibody responses. No significant bleeding or allergic reactions were observed in any patients. One of the 74 patients developed transient thrombocytopenia soon after receiving m7E3 Fab. These studies establish that m7E3 Fab can be administered safely at doses that cause profound inhibition of platelet function.

Adult↗
PubMed1995

The immunogenicity of the 7E3 murine monoclonal Fab antibody fragment variable region is dramatically reduced in humans by substitution of human for murine constant regions.

A murine monoclonal antibody (7E3) directed against the platelet glycoprotein IIb/IIIa was engineered to reduce immunogenicity by substituting human for murine constant regions. The chimeric antibody is functionally identical to the murine antibody in vitro. Results from clinical trials with 7E3 Fab antibody fragments, however, show that the 7E3 variable region, which elicits the vast majority of the immune response to murine 7E3 Fab, is rendered dramatically less immunogenic (incidence reduced from 17% to 1%) when the identical variable region is linked to human rather than murine constant regions. Neither murine nor human constant regions were highly immunogenic themselves. We conclude that the constant regions of the Fab fragments are critical in modulating the immune response elicited by the linked 7E3 variable region. Because naturally occurring anti-human Fab fragment antibodies are prevalent both in the normal human population and in the patient population studied here, murine 7E3 Fab and chimeric 7E3 Fab may be fundamentally different in their interactions with the human immune system. This difference may be related to the dramatic difference in immunogenicity observed between murine 7E3 Fab and chimeric 7E3 Fab.

Animals↗
PubMed1995

Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: implications for inhibition of the internal pool of GPIIb/IIIa receptors.

OBJECTIVES: This study sought to describe in detail the pharmacokinetics and pharmacodynamics of chimeric monoclonal 7E3 Fab (c7E3 Fab) and to compare platelet responses to adenosine diphosphate (ADP) and the 11-amino acid thrombin receptor-activating peptide (TRAP [SFLLRNPNDKY-NH2]) in patients undergoing elective coronary angioplasty. BACKGROUND: Inhibition of platelet aggregation with monoclonal antibody c7E3 Fab directed against glycoprotein (GP) IIb/IIIa has been shown to reduce ischemic complications after angioplasty and is being considered for treatment of other acute ischemic syndromes. METHODS: Patients undergoing elective coronary angioplasty received aspirin (325 mg orally), heparin (12,000 U intravenously) and a bolus of c7E3 Fab (0.25 mg/kg body weight). Surface GPIIb/IIIa receptor blockade and aggregation in response to 20 mumol/liter ADP, 5 micrograms/ml collagen and 7.5 and 15 mumol/liter TRAP were assessed. RESULTS: Surface GPIIb/IIIa receptor blockade by c7E3 Fab was 80% 2 h after injection and decreased to 50% at 24 h. Platelet aggregation in response to 20 mumol/liter ADP was inhibited by 73% at 2 h, and this inhibition decreased to 27% at 24 h. Platelet aggregation in response to 7.5 mumol/liter TRAP was inhibited by 53% at 2 h and 30% at 24 h. In contrast, aggregation in response to 15 mumol/liter TRAP was inhibited only 37% at 2 h and 10% at 24 h (p < 0.001 and p = 0.006, respectively vs. 20 mumol/liter ADP). Addition of exogenous c7E3 Fab to platelet-rich plasma led to more complete inhibition of 7.5 mumol/liter TRAP-induced aggregation. CONCLUSIONS: After c7E3 Fab treatment, inhibition of platelet aggregation depends on the agonist and can be overcome by increased thrombin activity but is restored if additional c7E3 Fab is added to block additional GPIIb/IIIa receptors. This phenomenon may be related to an internal pool of GPIIb/IIIa receptors joining the surface membrane and has implications concerning the duration of therapy with c7E3 Fab for patients with unstable angina or acute myocardial infarction.

Abciximab↗
PubMed1995

Immunoglobulin gene rearrangements in lymphoplasmacytic infiltrates of labial salivary glands in Sjögren's syndrome. A possible predictor of lymphoma development.

OBJECTIVES: Sjögren's syndrome is an autoimmune disorder in which patients have a well-recognized risk of developing malignant lymphoma. Although some clinical parameters may herald the onset of lymphoma, few reliable histologic or molecular markers are available that predict progression to a malignant lymphoproliferative disorder. The purpose of this study was to identify the prevalence of immunoglobulin heavy chain monoclonality in labial gland biopsies of patients with Sjögren's syndrome and to compare this to clinical outcome. STUDY DESIGN: The polymerase chain reaction was applied to 76 sequential labial salivary gland biopsies from patients under investigation for Sjögren's syndrome. A seminested polymerase chain reaction technique was used on DNA extracted from formalin-fixed, paraffin-embedded tissue to amplify the V-D-J region of the immunoglobulin heavy chain gene. Thirty-four randomly selected labial salivary glands that showed nonspecific sialadenitis from patients without Sjögren's syndrome were used as controls. RESULTS: Monoclonality, as defined by a single band on polyacrylamide gel electrophoresis was detected in 11 cases (14.5%). Of cases that showed monoclonality, four patients were subsequently diagnosed with extrasalivary lymphoma. In each case the rearranged bands in the lip biopsy and the lymphoma were the same size. In one patient who later developed lymphoma, a monoclonal rearranged immunoglobulin band was not identified. In addition, no cases of the translocation t(14;18) were identified by polymerase chain reaction in any of the lip biopsies showing heavy chain monoclonality or in any of the extrasalivary gland lymphomas. CONCLUSIONS: These results suggest that monoclonal immunoglobulin heavy chain gene rearrangements are a relatively common finding in patients with Sjögren's syndrome and may prove to be a useful marker for predicting the progression to, and early detection of malignant lymphoma.

Adolescent↗
PubMed1995

Different sensitivity to cell killing and chromosome mutation induction by gamma rays in two human lymphoblastoid cell lines derived from a single donor: possible role of apoptosis.

The gamma-ray sensitivity of two human lymphoblastoid cell lines, TK6 and WI-L2-NS, was studied; both cell lines are derived from the same parent, WI-L2. TK6 was more sensitive to the cytotoxic effects of gamma rays, but showed fewer induced chromosome aberrations. There was no difference between the two cell lines in their capacity to rejoin DNA double-strand breaks or in the kinetics of the rejoining process. The kinetics of cell cycle progression following radiation exposure was also similar in TK6 and WI-L2-NS. However, while TK6 cells were very sensitive to apoptosis induction, showing high levels of apoptotic cells within 24 h of exposure, no evidence for any significant levels of apoptosis was found for WI-L2-NS within 30 h of irradiation, when chromosome aberration frequency was determined. Evidence of apotosis were seen at later times in WI-L2-NS cells, but the levels were significantly lower than comparably treated TK6 cells. The results support a hypothesis that the lower survival and induced aberration frequencies in TK6 cells are due to selective removal of damaged cells by apoptotic processes prior to analysis of chromosome aberration frequencies in mitosis.

Apoptosis↗
PubMed1994

Early detection of lymphomas in Sjögren's syndrome by in situ hybridisation for kappa and lambda light chain mRNA in labial salivary glands.

Sjögren's syndrome (SS) is an autoimmune disease characterised by a generalised lymphoproliferation. Patients have an increased risk of developing lymphomas which are usually of the type associated with mucosa-associated lymphoid tissue (MALT). Histological examination of the minor salivary glands of the lower lip is a common and useful diagnostic test for SS but has not been able to provide information with regard to potential malignant change. In this study, a sensitive in situ hybridisation technique for the detection of kappa and lambda immunoglobulin light-chain mRNA was applied to labial salivary glands of 14 patients with SS. 7 cases showed light chain restriction, in 5 cases this was kappa(kappa:lambda ratio > 8.0) and in 2 it was lambda(kappa:lambda ratio < 0.6). Of these 7, 5 developed lymphomas--4 were low grade lymphomas of MALT type and the fifth patient died of disseminated lymphoma. The finding of light chain restriction in lip minor salivary glands is strong evidence of a monoclonal population of B-cells at this site. It is concluded that in patients with SS who develop lymphomas, dissemination of malignant cells may result in detectable disease in the minor salivary glands. Determination of kappa:lambda ratios in labial minor salivary glands may thus provide important prognostic information.

Autoimmune Diseases↗
PubMed1994

Anti-inflammatory and bronchodilator properties of RP 73401, a novel and selective phosphodiesterase type IV inhibitor.

1. We have investigated the effects of RP 73401, a novel, potent and highly selective cyclic nucleotide phosphodiesterase (PDE) type IV inhibitor, in guinea-pig and rat models of bronchoconstriction and allergic inflammation. In some models, the effects of RP 73401 have been compared with those of the standard PDE type IV inhibitor, rolipram. 2. RP 73401 (0.4-400 micrograms kg-1, intratracheally (i.t.) on lactose) inhibited antigen-induced bronchospasm in previously sensitized conscious guinea-pigs (ID50: 7 +/- 1 micrograms kg-1) and in anaesthetized rats (ID50: 100 +/- 25 micrograms kg-1). Rolipram inhibited the antigen-induced bronchospasm in guinea-pigs with an ID50 of 5 +/- 1 micrograms kg-1. In guinea-pig bronchoalveolar lavage (BAL) fluid, total inflammatory cell and eosinophil numbers were reduced by RP 73401 (ID50s: 3.9 +/- 0.8 micrograms kg-1 and 3.2 +/- 0.7 micrograms kg-1, respectively). In the rat, inflammatory cell numbers are less affected. Only the highest dose of RP 73401 (400 micrograms kg-1) significantly inhibited eosinophil influx (41 +/- 16% inhibition). 3. RP 73401 (0.02-100 micrograms kg-1, i.v.) inhibited PAF-induced bronchial hyperreactivity to bombesin in the anaesthetized guinea-pig (ID50: 0.09 +/- 0.03 micrograms kg-1) and inhibited (0.4-40 micrograms kg-1, i.t.) histamine-induced airway microvascular leakage in the anaesthetized guinea-pig by approximately 60% at all doses. 4. RP 73401 relaxed guinea-pig isolated trachea under basal tone (EC50: 9 nM) and when precontracted with histamine (IC50: 2 nM), methacholine (IC50: 29 nM) or leukotriene D4 (LTD4, IC50: 4 nM). 5. RP 73401 (0.4-100 microg kg-1, i.t.) inhibited bronchospasm induced by histamine (ID.%: 34 +/- 6 microg kg-1), methacholine (ID50: 66 +/- 12 pg kg-1) and LTD4 (ID50: <4 microg kg-1) in the anaesthetized guinea pig.Against these same bronchoconstrictors, rolipram (i.t.) had ID5o values of 44 +/- 4, 72 +/- 18 and<4 pg kg- respectively. RP 73401 (4 and 40 pg kg-, i.t.) increased the magnitude and duration of bronchodilatation produced by salbutamol in the anaesthetized guinea-pig. At doses producing significant bronchodilatation, RP 73401 was without effect on heart rate or blood pressure in the anaesthetized guinea-pig. RP 73401 (0.01 -0.25 mg kg-1, i.v.) did not affect heart rate and produced only a small fall in blood pressure in the anaesthetized rat.6. These data demonstrate that RP 73401 and rolipram inhibit antigen- and mediator-induced bronchospasmin guinea-pigs with the same potency. Furthermore, RP 73401 administered directly into the airways, protects against allergic airway inflammation. These results indicate the importance of PDE IV in regulating smooth muscle and inflammatory cell activity. At doses suppressing the inflammatory response in the lung, RP 73401 had little effect in the cardiovascular system. RP 73401 may have a role as a bronchodilator and, more importantly, as a prophylactic anti-inflammatory agent in the treatment of asthma.

Albuterol↗
PubMed1994

Demonstration of c-erbB-2 oncogene overexpression in salivary gland neoplasms by in situ hybridization.

The level of c-erbB-2 cellular mRNA in 18 salivary gland tumours and in 7 normal salivary glands was determined by in situ hybridization using [35S] labelled RNA probes. Computer assisted quantitation of the autoradiographic signal indicated a significantly higher c-erbB-2 expression in the tumour group (22.64 grains per cell +/- 3.79; 95% CI) as compared to the non-neoplastic salivary gland tissue (4.11 +/- 0.90; 95% CI). The c-erbB-2 expression as measured by grain counts per cell for the pleomorphic adenomas (16.29 +/- 1.87; 95% CI), mucoepidermoid carcinomas (31.52 +/- 0.08; 95% CI) and the acinic cell carcinomas (44.24 +/- 17.11; 95% CI) were significantly greater than the expression for the normal group. The acinic cell carcinomas exhibited the greatest level of expression. As observed at the individual cell level, the autoradiographic signal was distributed uniformly in the neoplastic tissues, regardless of the cell type. This study confirms the hypothesis that the c-erbB-2 oncogene is overexpressed at the mRNA level in salivary gland tumours.

Adenoma, Pleomorphic↗
PubMed1994

Propofol, seizures and generalized paroxysmal fast activity in the EEG.

Propofol is a relatively new anesthetic agent used in outpatient surgery. Some investigators use it in the treatment of status epilepticus and in epilepsy surgery and have concluded that propofol has an anticonvulsant effect. Cases of seizure-like behaviors, myoclonus and opisthotonus following propofol anesthesia have been reported. Although rare, official warnings about this association have been issued. Different EEG abnormalities, and no abnormality, have been associated with propofol. We report a case of a healthy man who developed nonconvulsive seizures and generalized paroxysmal fast activity in his EEG following use of propofol for anesthesia.

Adult↗
PubMed1994

Noninvolvement of the X chromosome in radiation-induced chromosome translocations in the human lymphoblastoid cell line TK6.

Fluorescence in situ hybridization procedures were used to examine the influence of chromosome locus on the frequency and type of chromosome aberrations induced by 60Co gamma rays in the human lymphoblastoid cell line TK6. Aberrations involving the X chromosome were compared to those involving the similarly sized autosome chromosome 7. When corrected for DNA content, acentric fragments were induced with equal frequency in the X and 7 chromosomes. Dose-dependent increases in chromosomal interchanges involving chromosome 7 were noted and the frequencies of balanced translocations and dicentrics produced were approximately equal. Chromosome interchanges involving the X chromosome were rare and showed no apparent dose dependence. Thus, while chromosomes 7 and X are equally sensitive to the induction of chromosome breaks, the X chromosome is much less likely to interact with autosomes than chromosome 7. The noninvolvement of the X chromosome in translocations with autosomes may reflect a more peripheral and separate location for the X chromosome in the mammalian nucleus.

Cell Line↗
PubMed1993

Thermodynamic and structural analysis of the folding/unfolding transitions of the Escherichia coli molecular chaperone DnaK.

The thermal unfolding of the Escherichia coli 70 kDa heat shock protein, DnaK, exhibits three well defined transitions. At pH 7.6, these transitions are centered at 45.2, 58.0 and 73.3 degrees C. High sensitivity calorimetric scans as a function of pH indicate that the folding/unfolding behavior is well described by a four-state model which includes a delta H, tm and delta Cp for each state. Calorimetric scans of a 44 kDa N-terminal proteolytic fragment show a major transition centered at 47.5 degrees C (N1) and a minor transition at 79.4 degrees C (N2). A calorimetric scan of a 23 kDa C-terminal proteolytic fragment exhibits a low temperature peak at 58.5 degrees C (C1) and a high temperature peak at 70.6 degrees C (C2). Deconvolution analysis of the low temperature peak reveals that it is actually composed of two transitions of roughly equal delta H centered at 50.4 degrees C (C1a) and 58.2 degrees C(C1b). These experiments have allowed us to assign the transitions of the intact protein as follows. The low temperature transition of DnaK can be assigned to the N-terminal region on the basis of the similarity between the delta H and tm values for the low temperature transition and those obtained for the N1 transition of the isolated N-terminal fragment. This assignment is also supported by measurements of the intrinsic fluorescence emission as a function of temperature. DnaK contains a single tryptophan localized at residue 102 in the N-terminal domain of the protein. Additionally, calorimetric scans show that the tm of the low temperature transition increases by 9.2 degrees C in the presence of excess ADP, which is known to bind to the N-terminal domain. The middle transition can be assigned to the C1a and C1b transitions of the C-terminal fragment on the basis of the similarity of delta H and tm. In the intact protein C1a and C1b form a single cooperative unit; however, the cooperative interactions between these folding/unfolding domains are disrupted in the isolated fragment. The high temperature transition of the intact protein is composed of contributions from both the N-terminal and C-terminal regions of the protein. These studies have allowed us to develop a quantitative model of the folding/unfolding behavior of DnaK.

Bacterial Proteins↗
PubMed1993

Control of intrinsic buccal muscles by motoneurons B11, B15, and B16 in Aplysia californica.

To further define the control of intrinsic buccal muscles by identified motoneurons B11, B15, and B16 in Aplysia californica, we made physiological recordings from semi-intact and isolated preparations of the buccal ganglia and buccal mass. B15 and B16 were previously known to innervate muscle 15 (also called the accessory radula closer muscle). We show here that these motoneurons also innervate other buccal muscles ipsilateral to their cell bodies; for example, B15 reliably innervates and can cause contraction in intrinsic muscle I4. Likewise, B11 appears to innervate all of the intrinsic buccal muscles, but elicits contractions only in I4. The form and time course of synaptic plasticity displayed by these motoneurons is common to all the muscles they innervate. No evidence for electrical coupling between fibers of adjacent muscles could be obtained, suggesting that all recorded innervation is direct. Like B15 and B16 (Cohen et al., '78), B11 neuromuscular synapses appear to be cholinergic, since they are reversibly antagonized by hexamethonium and d-tubocurarine (acetylcholine [ACh] receptor antagonists). In addition, direct application of ACh elicits depolarizing responses and contractions in I4 which are also blocked by hexamethonium. Application of glutamate and GABA do not elicit depolarizations or contractions in I4 or I5. We conclude that these motoneurons control radula position through complex synaptic actions on multiple intrinsic buccal muscles, using ACh as their conventional transmitter.

Acetylcholine↗