Novel mechanisms of Escherichia coli succinyl-coenzyme A synthetase regulation

Low concentrations of ADP are shown to increase the rate of phosphoenzyme formation of E. coli succinyl-coenzyme A (CoA) synthetase (SCS) without altering the fraction of phosphorylated enzyme. This is true when either ATP or succinyl-CoA and Pi are used to phosphorylate the enzyme. The stimulatory effect of ADP is not altered by sample dilution, is retained upon partial purification of the enzyme, and reflects the binding of ADP to a site other than the catalytic site. GDP also alters the phosphorylation of the E. coli SCS but does so primarily by enhancing the level of the phosphoenzyme and only when ATP is used as the phosphate donor. GDP appears to function by neutralizing the action of a specific inhibitory protein. This inhibitor of SCS allows for interconversion of succinate and succinyl-CoA in a manner dissociated from changes in ATP-ADP metabolism. These previously unidentified and varied mechanisms by which SCS is regulated focus attention on this enzyme as an important control point in determining the cell's potential to meet its metabolic demands.     

Leukocytes in the cervix: a quantitative evaluation of cervicitis

OBJECTIVE: To quantify the numbers of leukocytes in the normal cervix and relate these numbers to the diagnosis of cervicitis. METHODS: Isolated cell suspensions were prepared from cervical tissue recovered at hysterectomy from 37 women who had no obvious cervical disease. The percentages of CD45+ cells (leukocytes) in these preparations were determined using immunofluorescence-based flow cytometric analysis. These percentages were compared with the pathologist's assessment of cervicitis. RESULTS: Leukocytes were present in all cervical samples tested. For endocervical samples, the mean (+/- standard error of the mean [SEM]) percentage of CD45+ cells was 12.4 +/- 1.9% of cells in patients with a diagnosis of cervicitis (n = 16) and 9.1 +/- 1.1% in patients without cervicitis (n = 17). For ectocervical samples, the mean (+/- SEM) percentage was 14.8 +/- 3.0% in those with cervicitis (n = 16) and 9.5 +/- 1.6% in those without cervicitis (n = 19). The differences between samples from patients with cervicitis and those without cervicitis were not statistically significant at the .05 level. Intra- and interassay variabilities were 5.7 +/- 1.2% and 7.3 +/- 1.6%, respectively. CONCLUSION: Our study demonstrates there is a resident population of leukocytes in the cervix. Leukocyte number did not relate clearly and consistently to the diagnosis of cervicitis made by the pathologist. We suggest that the resident population of leukocytes, in the absence of other indicators of infection, may confuse determinations of cervicitis.     

The problem of Q fever in zoo ungulates]

Antibodies to Coxiella burnetii were recorded from approximately 13% of 469 ungulates which had been examined by means of micro-CFT (complement fixation test). Particular reference is made to a Q-fever situation in a musk ox herd. The results obtained from these investigations are likely to support the conclusion that latent Q-fever infection may be expected to occur in ungulates and to grow manifest in stress situations.     

Intracerebroventricular growth-hormone-releasing peptide-6 stimulates eating without affecting plasma growth hormone responses in rats

The purpose of this study was to determine the effect of intracerebroventricular (i.c.v.) injections of a synthetic, opioid-related hexapeptide, growth-hormone-releasing peptide-6 (GHRP-6), on stimulation of eating by rats and to correlate this aspect of feeding behavior with the peripheral plasma growth hormone (GH) response to the administered peptide. GHRP-6 dissolved in 5 microL of saline was injected into the lateral ventricles of sated, adult, male, Sprague-Dawley rats in doses from 0 pmol (saline only) to 1000 pmol. For 1 hour after injection, the occurrence of eating was noted, and specimens of arterial blood were collected at 0, 15, 30, and 60 minutes. The plasma was assayed for GH. A nearly linear, statistically significant (p < 0.01) dose-response relationship between the dose of GHRP-6 and the incidence of eating was noted. The mean change from baseline of plasma GH during the 60 minutes after injection was not dose-related (p > 0.2, p > 0.1, and p > 0.1 at 15, 30, and 60 minutes, respectively). We conclude that GHRP-6 given intracerebroventricularly to sated, adult, male, Sprague-Dawley rats stimulates eating and suggest that it does so by some mechanism that is independent of its GH-releasing property.     

Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells

Originally purified as a major lipid component of a strain of the cyanobacterium Lyngbya majuscula isolated in Cura?ao, curacin A is a potent inhibitor of cell growth and mitosis, binding rapidly and tightly at the colchicine site of tubulin. Because its molecular structure differs so greatly from that of colchicine and other colchicine site inhibitors, we prepared a series of curacin A analogs to determine the important structural features of the molecule. These modifications include reduction and E-to-Z transitions of the olefinic bonds in the 14-carbon side chain of the molecule; disruption of and configurational changes in the cyclopropyl moiety; disruption, oxidation, and configurational reversal in the thiazoline moiety; configurational reversal and substituent modifications at C13; and demethylation at C10. Inhibitory effects on tubulin assembly, the binding of colchicine to tubulin, and the growth of MCF-7 human breast carcinoma cells were examined. The most important portions of curacin A required for its interaction with tubulin seem to be the thiazoline ring and the side chain at least through C4, the portion of the side chain including the C9-C10 olefinic bond, and the C10 methyl group. Only two modifications totally eliminated the tubulin-drug interaction. The inactive compounds were a segment containing most of the side chain, including its two substituents, and analogs in which the methyl group at the C13 oxygen atom was replaced by a benzoate residue. Antiproliferative activity comparable with that observed with curacin A was only reproduced in compounds that were potent inhibitors of the binding of colchicine to tubulin. Molecular modeling and quantitative structure-activity relationship studies demonstrated that most active analogs overlapped extensively with curacin A but failed to provide an explanation for the apparent structural analogy between curacin A and colchicine.     

Treatment of humoral rejection after heart transplantation

BACKGROUND: Until a few years ago, the incidence of humoral rejection after heart transplantation was underestimated. These episodes were frequently very aggressive and often fatal, because the maintenance and emergency immunosuppression available at the time only inadequately covered the humoral branch of the immune response. In spite of individual case reports, the effects of blood purification procedures or cyclophosphamide in this situation can only be insufficiently estimated. METHODS: To evaluate this therapy concept, 20 dog-lymphocyte-antigen-matched dogs underwent heterotopic neck-heart transplantation. Fourteen dogs underwent transplantation after having been previously sensitized through multiple skin transplantations, 6 dogs were not sensitized (control). The animals received an induction with 3x 250 mg prednisolone, as well as triple immunosuppression (cyclosporine, azathioprine, and cortisone). Biopsy (light microscopy, immunofluorescence), intramyocardial voltage, electric myocardial impedance (>200 kHz, <10 kHz), and echocardiographic (left ventricular wall thickness, diastolic relaxation velocity) examinations were performed daily to monitor rejection. Rejection therapy was continued for 3 days according to the following regimen: apheresis, cortisone boluses (CB), and cyclophosphamide in group A1 (n = 4), apheresis and CB without cyclophosphamide in group A2 (n = 4), and CB only in group C (n = 6). The subsequent course under triple immunosuppression was then observed. RESULTS: In the sensitized animals the onset of severe humoral rejection on the fifth day deteriorated cardiac function down to 75% (70% to 80%) of the initial values. In groups A1 and A2, apheresis resulted in recovery to near-control values (89% to 94%) within two hours, and indeed to complete recovery (97% to 101%) after the second apheresis, that is, within 1 day. In group C recovery was delayed (2 days) and incomplete (84% to 91 %). After therapy was discontinued, rejection-related functional deterioration recurred immediately in group C, and from 2 to 3 days after apheresis, regardless of whether cyclophosphamide therapy was performed (group A1) or not (group A2). In the control group all animals showed a rejection-free posttransplantation course. CONCLUSIONS: By diluting inflammatory mediators, apheresis leads to a rapid improvement in cardiac function during severe humoral rejection after head transplantation. Neither apheresis nor cyclophosphamide therapy are able to have an immediate positive influence on the activation of the immune cascade and to prevent an ongoing rejection.     

Surgical aspects in the multidisciplinary treatment of soft tissue sarcomas

Sarcomas are rare malignant tumors with a large variety of histologic subtypes. The surgical approach depends more on the histologic grade, the size and the site of the tumor. Radiologic diagnosis relies predominantly on MR-imaging. Discernible improvements have taken place in soft tissue sarcoma patient survivorship and quality of life over the past 20 years, with overall 5-year survival currently at approximately 50-80%. The place of surgery in the treatment of soft-tissue sarcoma is defined in the light of a review of the recent literature. Radical surgical resection is the mainstay of therapy. Local recurrence is the most common type of failure. Local recurrence is resectable and limb preservations possible in the majority of patients. Survival after treatment of local recurrence is determined mainly by the grade and secondarily by the size of the tumor. The essential risk factor for local recurrence is the quality of surgical resection, defined by the definitive resection margins. A lateral safety margin of 5 cm and of 2 cm to the depth should be respected. In sarcoma of the extremity the compartment is defined based on clinical, radiographic, histopathologic and operative findings. The use of muscle flaps to fill the surgical defects can improve the functional result and reduce the complication rate. Only about 5% of the patients need amputation. Evaluation of functional results must be based on objective criteria. In retroperitoneal sarcoma the significant factors for determining prognosis are grade and completeness of exzision. Multidisciplinary treatment according to common protocols is essential. Shifts in treatment have taken place over the past decade, from single-modality treatment involving radical surgery with compartment resection to sophisticated limb-salvage strategies combined with radiation therapy. In case of inadequate surgery e.g. in a large tumor with positive margins in high-grade soft tissue sarcomas the addition of radiotherapy can improve local control, but cannot ensure that obtained by adequate surgery. Patients with large (greater than 5 cm), high grade soft tissue sarcoma are at high risk for distant recurrence and disease-related mortality. Investigations of combined modality therapy with newer chemotherapy agents and dose intensification treatment strategies are warranted.     

Prenatal diagnosis of Apert syndrome

Apert syndrome (AS) is clinically characterized by typical facial features and symmetrical syndactyly of the digits. AS is inherited as an autosomal dominant trait. Recently, a fibroblast growth factor receptors 2 (FGFR2) mutation, either C934G or C937G, was identified in exon IIIa. Our report documents an affected mother and son in whom one of the two mutations in AS had occurred sporadically in the mother. The diagnosed of AS was based on associated abnormal physical features and on molecular genetic analysis. A C-to-G transversion at position 937 of the cDNA resulting in a proline-to-arginine substitution at codon 259 was found in the mother. In her second pregnancy, prenatal diagnosis by both restriction analysis and direct sequencing was undertaken and this showed that the female fetus had not inherited the mutation.