In an attempt to determine which conformational parameters are important for the biological activity of ovine corticotropin-releasing factor (oCRF), we have synthesized in significant amounts (50-200 mg) and characterized chemically, structurally (CD), and biologically, oCRF analogues with substitution of each amino acid by its corresponding D-isomer. Out of 37 of these analogues, three were found to be equipotent to, or twice as potent as, oCRF, 13 had potencies in the range from 10 to 60%, 17 had potencies ranging from 1 to 10%, and the four others had potencies less than 0.5%. None of the analogues antagonized oCRF-induced release of ACTH in vitro at concentrations > or = 1000 oCRF. Since antagonists to CRF action can be generated by deletion of the first 8-14 residues, a series of CRF antagonists which exhibit significantly higher in vitro and in vivo biological potency than [Met18,Lys23,Glu27,29,40,Ala32,41,-Leu33,3 6,38] h/rCRF, [alpha-helical-CRF9-41], is also described. [D-Phe12,Nle21,38,Arg36]h/rCRF, in particular, was found to be ca. 15 times more potent than alpha-helical-CRF9-41 in vitro. In the rat, however, this analogue was about as effective as alpha-helical-CRF9-41 in blocking CRF-induced decrease in mean arterial blood pressure and increase in heart rate. Its potency in blocking epinephrine release by CRF was not significantly different from that of alpha-helical-CRF9-41. In the adrenalectomized rat, [Lys36]alpha-helical-CRF(9-41) (1.7 mg/kg) blunted the effect of endogenous CRF over a 90-min period; by comparison, a similar dose of alpha-helical-CRF9-41 was effective for less than 1 h.

Adrenocorticotropic Hormone↗

PubMed1993

In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells.

In the murine spleen, germinal centers are the anatomic sites for antigen-driven hypermutation and selection of immunoglobulin (Ig) genes. To detail the kinetics of Ig mutation and selection, 178 VDJ sequences from 16 antigen-induced germinal centers were analyzed. Although germinal centers appeared by day 4, mutation was not observed in germinal center B cells until day 8 postimmunization; thereafter, point mutations favoring asymmetrical transversions accumulated until day 14. During this period, strong phenotypic selection on the mutant B lymphocytes was inferred from progressively biased distributions of mutations within the Ig variable region, the loss of crippling mutations, decreased relative clonal diversity, and increasingly restricted use of canonical gene segments. The period of most intense selection on germinal center B cell populations preceded significant levels of mutation and may represent a physiologically determined restriction on B cells permitted to enter the memory pathway. Noncanonical Ig genes recovered from germinal centers were mostly unmutated although they probably came from antigen-reactive cells. Together, these observations demonstrate that the germinal center microenvironment is rich and temporally complex but may not be constitutive for somatic hypermutation.

Animals↗

PubMed1993

Potassium selectivity in proteins: oxygen cage or pi in the face?

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Binding Sites↗

PubMed1993

Cystic fibrosis. Sickly channels in mild disease.

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Animals↗

PubMed1993

Enhanced stimulation of myosin subfragment 1 ATPase activity by addition of negatively charged residues to the yeast actin NH2 terminus.

We examined the effects of yeast actin NH2-terminal mutations on actomyosin interactions and the function of actin in vivo through measurements of actin-activated ATPase activity, cosedimentation with rabbit muscle myosin subfragment 1 (S-1), in vitro motility, and invertase secretion assays. As reported earlier (Cook, R. K., Blake, W., and Rubenstein, P. A. (1992) J. Biol. Chem. 267, 9430-9436), elimination of NH2-terminal acidic residues from yeast actin results in an increased actin bundling, decreased actin-activated S-1 ATPase, and complete inhibition of actin filament sliding over myosin. Here we show that the addition of 2 new acidic residues to the NH2 terminus of yeast actin increased the Vmax value and the catalytic efficiency of the actin-activated ATPase activity of S-1. However, the binding of actin to S-1 in the presence of ATP and the velocities of actin sliding over myosin in the in vitro motility assays were not affected by this mutation. Thus, the number of actin NH2-terminal negative charges is important for actin activation of myosin S-1 ATPase activity, while only a minimum number of acidic residues is required for actin sliding over myosin in vitro. The number of actin NH2-terminal negative charges therefore appears to determine the efficiency with which the energy from ATP hydrolysis is converted to filament sliding.

Actins↗

PubMed1993

Total synthesis, purification, and characterization of human [Phe(p-CH2SO 3Na)52, Nle32,53,56, Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58, and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58.

The synthesis of [Phe(p-CH2SO3Na)52, Nle32,53,56 Nal55]-CCK20-58, [Tyr52, Nle32,53,56, Nal55]-CCK-58 and of [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 using the (9-fluorenylmethyloxy)-carbonyl (Fmoc) strategy on a 2,4-DMBHA resin is described. The crude peptide preparations were extremely complex when analyzed by RP-HPLC, capillary zone electrophoresis (CZE), and ion-exchange chromatography (IE-FPLC). We found that the most effective strategy for purification included cation-exchange chromatography followed by a RP-HPLC desalting step. The highly purified peptides (purity greater than 90%) were characterized by RP-HPLC, size exclusion HPLC (SEC), IE-FPLC, CZE, mass spectrometry, amino acid analysis, and Edman sequence analysis (for [Tyr52, Nle32,53,56, Nal55]-CCK-58). The results demonstrate the applicability of the 2,4-DMBHA resin for Fmoc solid-phase synthesis of long peptides amides (58 residues in length in this case) as well as the efficacy of an FPLC/RP-HPLC approach for the purification of very long, heterogeneous crude peptides, allowing a true assessment of the biological properties of these analogs to be carried out. [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK20-58 was less than 1% as potent as CCK-8 while [Tyr52, Nle32,53,56, Nal55]-CCK-58 and [Phe(p-CH2SO3Na)52, Nle32,53,56, Nal55]-CCK-58 were inactive at the doses tested (< 0.01%).

Amino Acid Sequence↗

PubMed1993

Effects of PEG-electrolyte (Colyte) lavage on serum acetaminophen concentrations. A model for treatment of acetaminophen overdose.

The purpose of this study was to evaluate whole gut lavage with polyethylene glycol electrolyte solution (Colyte), as a potentially adjunctive measure in lowering serum acetaminophen levels. The effect of bowel lavage was evaluated on serial serum acetaminophen concentrations after 2-g and 4-g doses in 7 and 12 male patients, respectively. Mean peak level of serum acetaminophen after 2 g (60 min after intake) was not significantly lowered by bowel lavage. After 4 g, peak acetaminophen serum levels were significantly lower after bowel lavage (65.4% of controls, P < 0.001). Urinary concentrations of the mercapturic acid conjugate of the toxic metabolite were also significantly reduced by lavage (55% after 2 g and 45% after 4 g, P < 0.01). Activated charcoal given orally after administration of 4 g of acetaminophen had no significant effect on peak serum levels and had no additive effect on lavage. These studies suggest that rapid, complete bowel lavage with a polyethylene glycol electrolyte solution may be beneficial as an adjunct to the treatment of the acetaminophen intoxication.

Acetaminophen↗

PubMed1993

Simultaneous recording of brainstem and cortical acoustic evoked potentials in children: methodical aspects and normative data.

Early and late acoustic evoked responses were simultaneously recorded from 42 children aged 3-17 years by means of a computer-based device using self-developed software. Filling the interstimulus intervals of late acoustic evoked potentials with clicks leads to an increase of latencies and a decrease of amplitudes of N1 and P2 components. Age-dependent normal values for various recording conditions are given.

Adolescent↗

PubMed1993

Pancreatic xanthomatous neuropathy associated with hyperlipidemia: a cause of abdominal pain mimicking chronic pancreatitis.

"Hyperlipidemic crisis" is a term used to describe episodic abdominal pain in patients with hyperlipidemia. The morphologic correlates of this phenomenon have not been investigated and the etiology of the disorder is uncertain. We report a unique histologic finding in the pancreas of a 34-year-old woman with a 17-year history of episodic abdominal pain, sometimes accompanied by hyperamylasemia. At the age of 18 years, grossly elevated cholesterol and triglyceride levels were documented and type V hyperlipidemia was diagnosed. At the age of 34 years, subtotal pancreatectomy was performed for intractable abdominal pain. Histologic examination identified an increased number of enlarged pancreatic nerves that were infiltrated by foamy macrophages and encircled by fibrous tissue; endoneurial infiltration with macrophages occasionally split nerves into individual fascicles. Otherwise, the pancreas had only minimal fibrosis, nesidioblastosis, mucinous metaplasia of some pancreatic ducts, and scattered small collections of chronic inflammatory cells, subtle features suggesting very mild chronic pancreatitis. We propose that this novel xanthomatous neuropathy mimicked pancreatitis and was one of the underlying pathophysiologic mechanisms of abdominal pain in this patient. Further studies are necessary to document the prevalence of this new entity in patients with hyperlipidemia and to correlate its occurrence with "hyperlipidemic crisis" in those individuals.

Abdominal Pain↗

PubMed1993

Mechanism of charybdotoxin block of a voltage-gated K+ channel.

Charybdotoxin block of a Shaker K+ channel was studied in Xenopus oocyte macropatches. Toxin on rate increases linearly with toxin concentration in an ionic strength-dependent fashion and is competitively diminished by tetraethylammonium. On rate is insensitive to transmembrane voltage and to K+ on the opposite side of the membrane. Conversely, toxin off rate is insensitive to toxin concentration, ionic strength, and added tetraethylammonium but is enhanced by membrane depolarization or K+ (or Na+) in the trans solution. Charge neutralization of charybdotoxin Lys27, however, renders off rate voltage insensitive. Our results argue that block of voltage-gated K+ channels results from the binding of one toxin molecule, so that Lys27 enters the pore and interacts with K+ (or Na+) in the ion conduction pathway.

Animals↗

PubMed1993

Strength training by electrostimulation conditions for efficacy.

The overload imposed on the neuromuscular system under Electrostimulation (ES) can be expressed by applied current intensity or by Electrically Evoked Torque (EET). The aim of this study was to discern which of these two parameters is the one which is determinant for the efficacy of training by ES. Test and training involved isometric contraction of the flexion maintained at a joint angle of 25 degrees (0 degree = extension). The 16 trained subjects received 15 sessions of 25 electrically evoked contractions, using a monophasic rectangular waveform current at a frequency of 2500 Hz modulated at 90 Hz. Each stimulation lasted for 5 sec at the maximal tolerable current. Contrary to the control group (n = 16), the trained group significantly increased their MVIC (15.6%). The individual strength gains ranged from -5% to 49%. No correlation was found between current level and strength modifications. A direct relation was established between the EET and the strength gains (a minimum threshold of EET must be reached during at least 8 sessions to induce strength increases). The "overload principle", previously described for voluntary contraction strengthening, seems to be suitable for electrical stimulation and concerns the EET shown on the ergometer as the effect of the contraction of agonist and antagonist muscles.

Adult↗

PubMed1993

FK506 versus cyclosporine as primary immunosuppressive agent for orthotopic liver allograft recipients. Histologic and immunopathologic observations.

We investigated possible explanations for the common occurrence of perivenular lesions in liver allografts of patients on FK506 within a few weeks to several months after OLT. Hematoxylin and eosin-stained sections of pre- and postperfusion biopsy specimens and day 7 post-transplant protocol biopsy specimens from 31 patients, randomly assigned to either FK506 or CsA as primary immunosuppressive agent, were reviewed, and immunohistochemical stains for HLA-DR antigen and S-100 protein were performed by the avidin-biotin peroxidase complex method. The histologic features of cellular rejection in the portal tracts of day 7 posttransplant allograft biopsy specimens from patients on FK506 were milder than those from patients on CsA. Immunohistochemical stains for HLA-DR showed intense positivity in a variety of cell types in day 7 posttransplant specimens from both groups, including sinusoidal-lining cells, bile duct epithelial cells, vascular endothelial cells, inflammatory cells, and occasional injured hepatocytes. Although diffuse lobular staining was seen in the majority of cases in both groups, either with or without rejection, liver biopsy specimens from patients on FK506 showed concentration of positively stained cells in perivenular regions more often, and at a lower overall histologic grade of rejection, than specimens from patients on CsA. There were no differences in the number and distribution of S-100 protein-positive dendritic APC between biopsy specimens from FK506 versus CsA-treated patients, or between specimens with and without cellular rejection in either group. It is suggested that the development of perivenular injury, which is seen frequently in allograft biopsy specimens from patients on FK506 obtained at various intervals after transplantation, may be related to drug toxicity rather than to the process of allograft rejection.

Cyclosporine↗

PubMed1993

Intravenous flunitrazepam in the treatment of alcohol withdrawal delirium.

Alcohol withdrawal delirium (AWD) requires treatment with an adequate sedative, anticonvulsant, and antipsychotic agent next to general intensive care measures. Optimal medication should have a rapid onset of action and the possibility of parenteral application. A specific antagonist should be available. Flunitrazepam is a benzodiazepine that fulfills all these criteria. Twenty five patients suffering from AWD (mean age 45 years) took part in an open trial and underwent treatment with infusions of flunitrazepam (concentration: 8 mg/250 ml NaCl; speed, 250 ml/hr). Psychopathological, vegetative, and vital parameters were assessed every hour. All patients survived. They were treated with a mean total dose (SD) of 83.9 (45.4) mg of flunitrazepam (1.3 mg/kg body weight), which induced sedation 13.2 (5.3) min after the initiation of intravenous treatment. The mean duration of AWD (85.1 +/- 39.4 hr) corresponded to other studies, whereas the frequency of preexisting and concomitant diseases was higher (92%) in our patients. A patient who suffered from bronchitis and had a nasopharyngeal tamponade showed severe respiratory depression after having received 4 mg of flunitrazepam. This complication remitted immediately when 0.5 mg of flumazenil was given intravenously. No epileptic manifestation was observed during the treatment or after discontinuation of flunitrazepam. Vegetative and psychopathological symptoms (tremor, sweating, hallucinations, confusion, and restlessness) remitted rapidly. Our data suggest that intravenous flunitrazepam can be an efficacious and safe alternative to traditional treatment strategies of AWD.

Adult↗

PubMed1993

Frameshifting in the expression of the Escherichia coli trpR gene is modulated by translation initiation.

The Escherichia coli trpR gene encodes the 108-amino-acid-long Trp repressor. We have shown previously that a +1 frameshifting event occurs during the expression of trpR, resulting in the synthesis of an additional (+1 frame) polypeptide. Using trpR-lac'Z fusions, we have recently found that the transition from the 0 to the +1 frame occurs via the bypassing of a 55-nucleotide-long segment of the trpR+1-lac'Z mRNA (I. Benhar, and H. Engelberg-Kulka, Cell 72:121-130, 1993). Here we show that the frequency of trpR frameshifting (or bypassing) can be regulated both in vivo and in vitro. This frequency is inversely proportional to the rate of initiation of translation of the trpR gene. Hence, modulating the level of translation initiation affects the frequency of frameshifting.

Bacterial Proteins↗