The Ras mutant D119N is both dominant negative and activated.

The introduction of mutation D119N (or its homolog) in the NKxD nucleotide binding motif of various Ras-like proteins produces constitutively activated or dominant-negative effects, depending on the system and assay. Here we show that Ras(D119N) has an inhibitory effect at a cell-specific concentration in PC12 and NIH 3T3 cells. Biochemical data strongly suggest that the predominant effect of mutation D119N in Ras-a strong decrease in nucleotide affinity-enables this mutant (i) to sequester its guanine nucleotide exchange factor, as well as (ii) to rapidly bind GTP, independent of the regulatory action of the exchange factor. Since mutation D119N does not affect the interaction between Ras and effector molecules, the latter effect causes Ras(D119N) to act as an activated Ras protein at concentrations higher than that of the exchange factor. In comparison, Ras(S17N), which also shows a strongly decreased nucleotide affinity, does not bind to effector molecules. These results point to two important prerequisites of dominant-negative Ras mutants: an increased relative affinity of the mutated Ras for the exchange factor over that for the nucleotide and an inability to interact with the effector or effectors. Remarkably, the introduction of a second, partial-loss-of-function, mutation turns Ras(D119N) into a strong dominant-negative mutant even at high concentrations, as demonstrated by the inhibitory effects of Ras(E37G/D119N) on nerve growth factor-mediated neurite outgrowth in PC12 cells and Ras(T35S/D119N) on fetal calf serum-mediated DNA synthesis in NIH 3T3 cells. Interpretations of these results are discussed.

Spermine stabilizes the conformation of tRNAPhe in crystals.

Crystals from yeast tRNAPhe were dissolved and compared with tRNAPhe that had not been srystallized. A number of differences were found regarding the interaction with ethidium bromide, the melting point and the circular dichroic signal. These differences were assigned to the presence of spermine in the dissolved crystals indicating a transient stabilization of the conformation of tRNAPhe, probably as a tRNAPhe-spermine complex, after dissolving.

Drosophila ACT88F indirect flight muscle-specific actin is not N-terminally acetylated: a mutation in N-terminal processing affects actin function.

Many eukaryotic proteins are co and post-translationally modified at their N termini by removal of one or two amino acid residues and N(alpha)-acetylation. Actins show two different forms of N-terminal processing dependent on their N-terminal sequence. In class II actins, which include muscle actins, the common primary sequence of Met-Cys-Asp-actin is processed to acetyl-Asp-actin. The functional significance of this in vivo is unknown. We have studied the indirect flight muscle-specific actin, ACT88F, of Drosophila melanogaster. Our results show that ACT88F is N-terminally processed in vivo as a class II actin by removal of the first two amino acid residues (Met and Cys), but that uniquely the N terminus is not acetylated. In addition we show that ACT88F is methylated, probably at His73. Flies carrying the mod(-) mutation fail to complete post-translational processing of ACT88F. We propose that the mod gene product is normally responsible for removing N-acetyl-cysteine from actin. The biological significance of this process is demonstrated by observations that retention of the N-acetyl-cysteine in ACT88F affects the flight muscle function of mod(-) flies. This suggests that the extreme N terminus affects actomyosin interactions in vivo, a proposal we have examined by in vitro motility assays of ACT88F F-actin from mod(-) flies. The mod(-) actin only moves in the presence of methylcellulose, a viscosity-enhancing agent, where it moves at velocities slightly, but significantly, reduced compared to wild-type. These data confirm that N-acetyl-cysteine at the N terminus affects actomyosin interactions, probably by reducing formation of the initial actomyosin collision complex, a process known to involve the actin N terminus.

Ligand-specific state transitions of the membrane-bound acetylcholine receptor.

We have developed a simple, direct and time-resolved method to monitor ligand-induced changes in agonist affinity of the membrane-bound acetylcholine receptor. The assay is based on the quenching of fluorescence of NBD-5-acylcholine observed upon binding of this cholinergic agonist to the receptor. Under conditions of partial saturation with the fluorescent agonist, agonists and local anesthetics but not antagonists can induce an increase in affinity of the receptor for NBD-5-acylcholine. The effect is not observed with receptor fully saturated with the fluorescent agonist. The half-life of the observed change in affinity is independent of the nature of the agonist or local anesthetic applied (t1/2 approximately 60 s at 22 degrees C). We conclude that the same state transition of the receptor can be induced by two groups of cholinergic ligands that are assumed to be non-competitive with each other and to have distinctly different modes of action. The time course of the transition is reminiscent of the slow process of desensitization observed in vivo.

cAPK-phosphorylation controls the interaction of the regulatory domain of cardiac myosin binding protein C with myosin-S2 in an on-off fashion.

Myosin binding protein C is a protein of the myosin filaments of striated muscle which is expressed in isoforms specific for cardiac and skeletal muscle. The cardiac isoform is phosphorylated rapidly upon adrenergic stimulation of myocardium by cAMP-dependent protein kinase, and together with the phosphorylation of troponin-I and phospholamban contributes to the positive inotropy that results from adrenergic stimulation of the heart. Cardiac myosin binding protein C is phosphorylated by cAMP-dependent protein kinase on three sites in a myosin binding protein C specific N-terminal domain which binds to myosin-S2. This interaction with myosin close to the motor domain is likely to mediate the regulatory function of the protein. Cardiac myosin binding protein C is a common target gene of familial hypertrophic cardiomyopathy and most mutations encode N-terminal subfragments of myosin binding protein C. The understanding of the signalling interactions of the N-terminal region is therefore important for understanding the pathophysiology of myosin binding protein C associated cardiomyopathy. We demonstrate here by cosedimentation assays and isothermal titration calorimetry that the myosin-S2 binding properties of the myosin binding protein C motif are abolished by cAMP-dependent protein kinase-mediated tris-phosphorylation, decreasing the S2 affinity from a Kd of approximately 5 microM to undetectable levels. We show that the slow and fast skeletal muscle isoforms are no cAMP-dependent protein kinase substrates and that the S2 interaction of these myosin binding protein C isoforms is therefore constitutively on. The regulation of cardiac contractility by myosin binding protein C therefore appears to be a 'brake-off' mechanism that will free a specific subset of myosin heads from sterical constraints imposed by the binding to the myosin binding protein C motif.

Antipsoriatic anthrones with modulated redox properties. 4. Synthesis and biological activity of novel 9,10-dihydro-1,8-dihydroxy-9-oxo-2-anthracenecarboxylic and -hydroxamic acids.

A novel series of carboxylic and hydroxamic acids based on 1,8-dihydroxy-9(10H)-anthracenone were synthesized from 8-hydroxy-1-methoxy-9,10-anthracenedione as the key intermediate and evaluated both in the bovine polymorphonuclear leukocyte 5-lipoxygenase (5-LO) assay and in the HaCaT keratinocyte proliferation assay for their enzyme inhibitory and antiproliferative activity, respectively. The most potent inhibitors in both assays were the N-methylated hydroxamic acids 5d-8d with straight chain alkyl spacers. Incorporation of these structural features on the anthracenone pharmacophore resulted in increased inhibitory activity against 5-LO while the antiproliferative activity was retained. In addition, prooxidant properties as measured by deoxyribose degradation and cytotoxicity as assessed by LDH release were largely reduced as compared with the antipsoriatic anthralin. Contrary to anthralin, antioxidant properties were observed as documented by the reactivity of the novel compounds against free radicals and inhibition of lipid peroxidation in model membranes.

Simple analogues of anthralin: unusual specificity of structure and antiproliferative activity.

Fifty-nine simple analogues of the antipsoriatic agent, anthralin, have been prepared by modifying the positions of the 1,8-hydroxyl groups, replacement of the hydroxyl groups, substitution at the oxygen functions, introduction of additional functional groups into various positions of the anthracenone nucleus, or removal of particular structural elements. The compounds were evaluated for their antiproliferative action against human keratinocytes and inhibition of the generation of leukotriene B4 in polymorphonuclear leukocytes, which may be useful to resolve the proliferative and inflammatory aspects of psoriasis, respectively. Even though many anthracenones were more potent inhibitors of leukotriene biosynthesis than anthralin, none of the compounds was substantially more effective as this drug in suppressing keratinocyte cell growth. There is an absolute requirement for two hydroxyl groups peri to a hydrogen bond acceptor such as a keto or an imino group for high potency. In addition to further delineating the nature of the pharmacophore for this class of compounds, also naphthalenedione with a peri hydroxyl group was identified as a pharmacophore with antiproliferative activity against keratinocyte growth.

Agonist binding to the nicotinic acetylcholine receptor and probability of channel opening.

Binding of agonists to the nicotinic acetylcholine receptor induces a conformational change by which the integral cation channel is opened. The analysis of this mechanism is commonly based on models which may be classified as either occupational or conformational. Here I summarize results showing that none of these concepts alone is capable of accommodating all experimental observations. A new integrated model based on earlier concepts and the molecular structure of this macromolecule can explain the experiments.

On the kinetics of cholinergic excitation.

We have studied the interaction of NBD-n-acylcholines, fluorescent analogs of acetylcholine with agonist action, with acetylcholine receptor and acetylcholine esterase (EC 3.1.1.7) from Electrophorus electricus. The rates of association of the fluorescent ligands with the two proteins are closely similar during the initial phase of binding, the second order rate constants being of the order of 2 x 10(8) M(?1) s(?1) at physiological salt conditions.

CA 15.3 as a tumour marker in breast cancer.

CA 15.3 is an antigenic determinant associated with human mammary carcinomas. Two murine monoclonal antibodies have been raised against the determinants, and an immunoradiometric assay (IRMA-Kit, Centocor, USA) has been developed to determine the antigen levels in plasma of cancer patients. Based on the 99% confidence limit of healthy women, plasma values above 30 U/ml are considered abnormal. Plasma samples from 357 women were examined in the present study. Healthy females (n = 84) ranged below the cut-off level between less than 10 and 29 U/ml. Higher values were found in 12.5% of benign breast diseases and in 23.6% of breast cancer patients, including all stages. Depending on the stage of the disease, there were elevated levels in 11% of operable breast cancer patients preoperatively, in 7% of the cases with no evidence of disease after primary treatment and in 63.5% of patients with disseminated mammary carcinoma. In metastasized breast cancer the frequency and the degree of abnormal titers were closely related to the extent of the metastatic disease. Follow-up examinations of 63 patients under cytotoxic therapy showed CA 15.3 changes correlating well with the clinical course in up to 90% of the antigen positive cases. The present data indicate that CA 15.3 may be useful in the surveillance of breast cancer patients. However in our study one third of the patients with metastatic breast cancer did not show any increase in CA 15.3 and must be regarded as antigen negative.

Squamous cell carcinoma antigen for monitoring cervical cancer.

The tumour-associated antigen was determined in the plasma of patients with squamous cell carcinoma (SCC) of the uterine cervix by radioimmunoassay. Setting a limit of 2 ng/ml, levels were abnormal in 13.4% of healthy controls, in 14% of patients with carcinoma in situ and in 62% of patients with invasive cervical SCC. The incidence of elevated SCC antigen levels and the absolute antigen plasma concentration were dependent upon the tumour load, increasing significantly with advanced stage disease. Abnormal SCC antigen values in operable cervical cancer declined to normal within one week after radical hysterectomy with pelvic lymphadenectomy. In cases of radiotherapy antigen values took 4-6 weeks after the start of treatment to return to normal. The success of both treatment modalities was announced by an early rise in the SCC antigen in the initial phase of therapy, followed by normalisation. After successful primary treatment and a complete remission during further follow-up SCC antigen in plasma was only increased in 3.8% of the cases. Retrospective evaluations in ten patients with progressive disease showed the reappearance of abnormal SCC titers and further increase preceeding the clinically detectable relapse or progression, with a median interval of 8 weeks. The present study indicates that SCC antigen determination is not useful for the early diagnosis of cervical cancer, but it is a potential means for monitoring the efficacy of individual anticancer therapy of SCC of the uterine cervix and for detecting recurrent disease.

One hundred percent online identity check of pharmaceutical products by near-infrared spectroscopy on the packaging line.

To increase product safety of pharmaceuticals, a new near-infrared (NIR) method for the online identity check of pharmaceutical finished products was validated. The method comprises a new near-infrared device VisioNIR (Uhlmann VisioTec GmbH, Laupheim, Germany) and the appropriate evaluation statistics. The VisioNIR is applied to the packaging line and provides the possibility to perform a 100% product identity check at full line speed. The products were analyzed applying near-infrared spectroscopy (900-1700 nm) in reflectance mode. The scanned products were two widely used pharmaceuticals named Capsule A (containing 300 mg of paracetamol and 250 mg of chlorzoxazone) and Capsule B (containing 500 mg of paracetamol and 30 mg of codeine phosphate). In order to demonstrate the fitness of the VisioNIR the obtained data were compared with the data acquired by Foss NIRSystems 6500 spectrometer (NIRSystems, Silver Springs, MD). The results obtained by the VisioNIR evaluation statistics were compared with the results obtained by the commonly used principal component analysis. The advantages and the suitability of the method are discussed. In this new configuration NIR spectroscopy offers an excellent possibility for non-destructive 100% online quality control of pharmaceutical products.

2-arylalkyl-substituted anthracenones as inhibitors of 12-lipoxygenase enzymes. 1. Structure-activity relationships of the terminal aryl ring.

A series of 2-arylmethyl-substituted anthracenones were synthesized and tested as inhibitors of three types of 12-lipoxygenase isoforms in epidermal homogenate of mice, bovine platelets and porcine leucocytes. Their inhibitory activities were compared with those to inhibit the 5-lipoxygenase enzyme in bovine leucocytes. Structure-activity relationships are described with particular emphasis on modifications of the terminal aryl nucleus. The ability of the compounds to selectively inhibit the 12-lipoxygenase enzymes was dependent on a high overall lipophilicity of the inhibitor, whereas compounds with decreased lipophilicity were also inhibitors of the 5-LO enzyme. Among the more lipophilic inhibitors, the unsubstituted 2-phenylmethyl analogue 6a as well as the carboxylic acid ester 6q appeared to be selective inhibitors of platelet-type 12-LO isoform.

Spontaneous rupture of the spleen: ultrasound patterns, diagnosis and follow-up.

Spontaneous rupture of the spleen is an extremely rare complication usually of infectious diseases or disorders of the haematopoietic system and has been described mostly in case reports. The incidence, symptoms, causes, therapy, and prognosis are poorly defined. From July 1985 to January 2000 41 patients with spontaneous splenic rupture were diagnosed by abdominal ultrasound and confirmed by splenectomy (n=12), CT (n=15), and ultrasound follow up (n=26). An ultrasound grading system was retrospectively established based on the degree of splenic injury (grade 0-2=low grade injury, grade 3=high grade injury) and correlated with surgical procedures. 30 day mortality rate was studied in relation to underlying disorders, ultrasound grades and treatment decisions. 21 patients had underlying malignant disorders (group I) and 20 patients had benign diseases (group II). Between group I and II we observed a highly significant difference in 30 day mortality rates (n=7; 38.1% vs n=1; 5%, p<0.01), but no significant difference in high grade injury rate (n=3; 14.3% vs n=2; 10.0%; p=ns) and surgical treatment rate (n=5; 23.8% vs n=7; 35.0%; p=ns). Depending on ultrasound grades the surgical procedures were 0% for grade 0, 16.7% for grade 1, 30.4% for grade 2, and 60% for grade 3. There were no significant differences between patients, who died within the first 30 days (n=9) and those who survived more than 30 days (n=32) regarding high grade splenic injury rate (n=0; 0% vs n=5; 15.6%; p=ns), and surgical treatment rate (n=2; 22.2% vs n=10; 31.2%; p=ns). Spontaneous rupture of the spleen is an extremely rare event. It is associated with a high mortality rate within 30 days in patients with malignant disease. Sonomorphologic grading is helpful for treatment decisions. 30 day mortality rate is correlated with neither ultrasound grades, nor surgical treatment rates.

Effect of a preventive program based on professional toothcleaning and fluoride application on caries and gingivitis.

The effect of a preventive program was studied in 12-14-yr-old children. 104 children (test group) participated in this program. After four visits in the first 6 wk, the children visited the oral hygienist five times a year to receive professional oral prophylactic treatment and instructions. Additionally a fluoride application (Duraphat) was given two times a year. Children of a control group (n = 117) received no prophylactic treatment. Diet was not controlled in either test or control group. After 2 yr mean caries increment amounted to 2.7 +/- 2.8 new DFS in the test group and 5.0 +/- 4.2 new DFS in the control group, respectively. The most pronounced differences between the two groups could be found on the proximal surfaces. In all, caries was reduced by 46% during the 2-yr period. Plaque accumulation and frequency of gingivitis were significantly reduced only in the test group (67% and 55%, resp.). The data revealed that the program reduced both caries and gingivitis to a remarkable extent.

C-reactive protein is a marker for the diagnosis of adnexitis.

The study reports the C-reactive protein (CRP) plasma concentrations in 115 women with a presumed diagnosis of acute adnexitis. In addition to CRP, blood sedimentation rate, white blood cell count and the body temperature were evaluated and compared with the clinical findings. Diagnosis was confirmed or excluded by laparoscopy (n = 69) or laparotomy (n = 9). Clinical examinations and conventional laboratory examinations were of limited value in the diagnosis of acute adnexitis. In contrast, CRP was a highly sensitive indicator of inflammatory pelvic disease. Furthermore, the CRP determination was superior in assessing the efficacy of an antibiotic treatment.