Enamel defects in primary canines related to traditional treatment of teething problems in Sudan.

In parts of Sudan and some other countries, teething is thought to be the cause of severe health problems in infants, and a traditional treatment involves lancing the alveolar process over the unerupted canines with a heated needle, a procedure known as 'haifat'. Three hundred and ninety-eight children aged 4-8 years were examined for the presence of enamel defects on primary canines, and their parents or guardians were questioned regarding past teething problems and their treatment. Two hundred and fifty-eight (65%) of the children had experienced health problems that had been attributed to teething, and 89 (22%) had been subjected to 'haifat'. 'Haifat' had been practised by all socio-economic groups, but was most prevalent in the lower groups. Enamel defects on the buccal surface of the primary canines were found in 25 (28%) of the children in the 'haifat' group and in 25 (8%) of the other children.


The effect of air-polishing occlusal surfaces on the penetration of fissures by a sealant.

An in vitro study was performed to assess the depth of penetration of a fissure sealant after cleaning the occlusal surfaces with an air-polishing unit, and to compare the results with those obtained after cleaning with pumice and water and after no cleaning. Forty-six clinically non-carious premolars and permanent molars were randomly distributed into three groups. Following cleaning, etching and fissure sealing, the teeth were sectioned. Each section was examined under a light reflecting microscope and the depth of resin penetration was determined. Fissure cleaning with an air-polishing unit produced a statistically significant increase in depth of penetration of sealant resin, and its use as a standard cleaning method before fissure sealing is recommended.


A study to compare the effectiveness of temazepam and a chloral hydrate/hydroxyzine combination in sedating paediatric dental patients.

The study compared the effectiveness of temazepam and a mixture of chloral hydrate and hydroxyzine in sedating 20 young children aged 20 to 60 months (mean age 38.7 months). All the children exhibited negative behaviour during a screening visit and required at least two visits for restorative treatment with the use of sedation. The children were assigned randomly to receive either 50 mg/kg of chloral hydrate with 25 mg of hydroxyzine or 0.3 mg/kg of temazepam for the first visit, and the alternate regimen for the second visit, in a double-blind manner. Pulse rate and blood oxygen saturation levels were monitored before, during and after the operative procedures. All the treatment sessions were video-recorded and evaluated independently by three paediatric dentists for the degree of crying, movement, sleep and overall behaviour during specific procedures and at specific time intervals. The results showed no statistically significant differences between the two pharmacologic regimens with regard to crying, movement, sleep and overall behaviour. No significant difference in behaviour was found related to either the order of administration of the drugs or to the sex of the patients. It was concluded that 0.3 mg/kg temazepam and a mixture of 50 mg/kg chloral hydrate with 25 mg hydroxyzine had similar sedative effects on the children receiving dental treatment.


[Adolph Witzel, 100 years of the teaching of dentistry in Jena].

Dr. med. Adolph Witzel has founded 100 years before the dental school of the university Jena. His personality, work and scientific performance are presented.


[Periodontology in Jena--25 years of teaching, research and therapy].

Periodontology is a young department and has developed in Jena in the last 25 years; since 1982 it is a part in state examination. Maintenance of teeth is possible until old age and requires periodontology as a part of therapy. Biochemically, microbiologically and immunologically methods make it possible to differentiate unlike forms of periodontitis and their special therapy.


[Maxillary development after early secondary osteoplasty in complete unilateral cheilognathopalatoschisis].

The paper presents the retrospective investigation of 99 cleft palate cases with unilateral clefts. The effects of two different lip closures procedure on the development of nasal, labial and maxilla region were investigated. In 50 cases the Le Mesurier lip closure procedure was followed, and in 49 cases the Millard procedure. In the Le Mesurier group a lengthening of lip was observed, while in the Millard group lip length was symmetrical. The cast analysis shows an even development of the anterior maxilla width. The cephalometric analysis also shows a favourable development in the Millard group.


[Age- and sex-specific tooth and jaw measurements and their relation in the 1st mixed dentition phase--a longitudinal study on Jena children at the age of 7 1/2 to 8 1/2 years].

In a longitudinal study consisting of three single examinations dental findings of 89 individuals (40 boys and 49 girls) were recorded. The aim of the study was to check up the process of dental growth and development of 7.6- to 8.6-year-old children. Age- and sex-specific differences could be established in the three occlusal dimensions by means of clinical findings and 3-d-analysis of the plaster casts. The rate of DMF teeth was remarkably low in girls and boys; the presence of malocclusions was surprisingly high.


[An examination of the prognostic value of biological parameters exemplified by the crucial prognosis-relevant factors of the TPI in the patient load of the Jena Clinic in 1968 to 1991. Therapy-dependent Prognosis Index].

The data of 240 patients with non-pretreated oral squamous cell carcinoma were analyzed in dependence on the tumor size (T-category), on the histologic checked lymph node status and the histopathologic grade of malignancy. There is a conformity to the results published by the DOSAK regard to the tumor size (T-category). The comparison of the survival of our patients with the estimated survival rate of the TPI shows also conformable results. Differences exist in the prognostic valuation of the lymph node status by the DOSAK. The histologic verificated lymph node status and the malignancy grade will recommend as decisionsive prognostic relevant parameters.


[Experimental studies of electroplating-resin bonding systems].

Optimized conditioning of galvanically produced gold frameworks (Auro-Galva-Crown-System) by means of Silicoater, Silicoater MD and Rocatec techniques gives rise to a reliable bond to veneering resins. After experimental stress (thermocycling) the strength of the compound could be found between 17 and 26 MPa and SEM investigations of the contact zone showed no crevices.


[Inlays made from the glass ceramic Bioverit II. A clinical and scanning electron microscopic study].

Inlays made of glass ceramics Bioverit II were proved just after inserting with a composite, and after 6 month's and one year. No marginal clefts were observed at clinical controls. The scanning study demonstrated excellent compound enamel-composite-glass ceramics just after inserting. A 15 to 20 microns deep furrow within the composite at most of the proved teeth was recognized one year later. The margins of enamel and inlays were yet covered with composite.


[The strategy in decision making for the development of an orthodontic consultation system].

The high increase of information mainly because of new exact methods of examination makes it necessary to search for new possibilities of data storage, data processing and data analyzing. Orthodontic cases retrospectively assessed are taken as a knowledge-based system, and the individual case is classified by objective criteria metrically determinable as well as subjective criteria. After classification a plan of treatment can be elaborated.


[Experimental studies on tissue expansion in reconstructive surgery].

In the present study, a porcine model for controlled tissue expansion was investigated under the conditions or formation of extreme enlarged randomized flaps. The circulatory disturbances and necrotizing processes to be expected were evaluated immediately after flap formation by intravenous injection of fluorescein dye to predict the surviving area. Lectin histochemistry and immunohistology were performed using either the direct and indirect immunofluorescence technique (DIFT, IIFT) or immunoperoxidase techniques (POX). Over all, the expanded randomized flaps healed without complications in all animals. The acute randomized flaps showed clear necroses already after 10 days. In the thoracic area they averaged 60.3% (SD = 8.17%) and in the pelvic area 47.32% (SD = 14.11%) of the flap. The use of the fluorescein dye test in the present experiment demonstrated that the flap surviving area is significantly greater than the flap staining area. The regular expression of suprabasal keratins and epidermal lectin binding sites provides evidence for a normal epidermal cell differentiation.


[The results of caries prevention in kindergartens].

It was proved some years, if the program of the WHO for the oral healthy of preschool children could be realized through a complex caries prevention in 5 kindergartens of the town Jena. Since 1986 we found in 50 per cents of six years old children dentures free of caries after three years of caries prevention.


Pattern of expression of transforming growth factor-beta 4 mRNA and protein in the developing chicken embryo.

Expression of TGF-beta 4 mRNA and protein was studied in the developing chicken embryo using specific cDNA probes and antibodies for chicken TGF-beta 4. Expression of TGF-beta 4 mRNA was detected by day 4 of incubation (Hamburger and Hamilton stage 22, E4) by RNA Northern blot analysis and increased with developmental age until day 12 of incubation (stage 38, E12) where it was detected in every embryonic tissue examined, with expression being highest in smooth muscle and lowest in the kidney. The steady-state level of expression of TGF-beta 4 mRNA remained relatively constant in most embryonic tissues through day 19 (stage 45, E19). In situ hybridization analysis detected TGF-beta 4 mRNA as early as the "definitive primitive streak" stage (stage 4); during neurulation (stage 10), TGF-beta 4 mRNA was detected in all three germ layers, including neuroectoderm. Following neurulation, TGF-beta 4 mRNA was detected in the neural tube, notochord, ectoderm, endoderm, sclerotome, and myotome, but not dermotome at stage 16. By day 6 of incubation (stage 29, E6), TGF-beta 4 mRNA was localized in several tissues including heart, lung, and gizzard. Immunohistochemical staining analysis also showed expression of TGF-beta 4 protein in all three germ layers as early as stage 4 in various cell types in qualitatively similar locations as TGF-beta 4 mRNA. These results suggest that TGF-beta 4 may play an important role in the development of many tissues in the chicken.


Cell interaction and its role in mesoderm cell migration during Xenopus gastrulation.

In the Xenopus gastrula, the mesoderm moves as a coherent cell aggregate across the blastocoel roof toward the animal pole. We show that the cohesion of the mesoderm is not only mechanically necessary, but that aggregate formation has profound effects on the migratory behavior of mesoderm cells. Whereas isolated mesoderm cells are bi- or multipolar, move stepwise and change their direction of movement frequently, aggregated mesoderm cells migrating on their in vivo substrate appear unipolar and move continuously and persistently. Moreover, only mesoderm cell aggregates, but not single cells, can follow guidance cues present in the extracellular matrix of the blastocoel roof substrate. Thus, the cohesion of the mesodermal cell mass is an essential feature of mesoderm migration during Xenopus gastrulation. We show that the Ca(2+)-dependent cell adhesion molecule U-cadherin is involved in mediating this cohesion.


Detection of soluble antigens by the reverse passive haemagglutination assay.

A series of parameters that influence coupling of antibodies to the surface of erythrocytes with Cr3+ as well as the sensitivity and specificity of the reverse passive haemagglutination (RPH) assay in various antigen-antibody systems have been studied. The optimum coating of erythrocytes with antibodies was achieved at a final concentration of 30 micrograms CrCl3/ml with a protein antibody/CrCl3 ratio of 3.66 for most antibody IgG tested (anti-human IgG, anti-human albumin, anti-human hemoglobin and anti-rabbit IgG) and of 3.16 for anti-human myoglobin. The temperature and incubation time were of 30 degrees C and 60 min, respectively. Five soluble antigens were assayed by RPH: myoglobin, hemoglobin, IgG, human albumin and rabbit IgG. The minimum detectable analyte amount ranged between 1.75 ng/ml for human myoglobin and 17.16 ng/ml for human albumin. The reaction specificity was demonstrated by the absence of cross-reactions against heterologous antigens and the RPH inhibition reaction with immune sera. By stabilization with glutaraldehyde of antibody-coated erythrocytes the shelf-life was prolonged from seven days (non-stabilized erythrocytes) up to 300 days. The reverse passive haemagglutination assay is quite simple, rapid, easy in execution and by its high sensitivity is comparable to RIA and ELISA.


[Molecular analysis of breast cancers: recent developments].

The etiology of cancer is a complex interplay of various factors, including genetic alterations. Multiple studies have been carried out to identify and characterize mutations that frequently occur during tumorigenesis. In human breast cancer, amplification of proto-oncogenes (c-myc, c-erbB-2/neu) and chromosome 11q13, mutation of p53 and loss of heterozygosity (chromosomes 1, 3p, 6q, 7q, 11p, 13q, 16q, 17, 18q and 22q) represent the major types of genetic abnormalities that have been frequently observed in tumor DNAs. The genetic deletions and mutations could inactivate tumor-suppressor genes. In some studies, specific alterations have been associated with some clinical parameters. Recently, linkage analyses, on large families with a predisposition to breast cancer, have been performed to map putative breast cancer susceptibility genes. The survey of high risk patients should be organised to make an earlier diagnosis.


[Psychological disorders encountered during the post-transplantation period of bone marrow].

Psychological issues encountered during the procedure of a bone marrow transplantation (BMT) have already been described. Anxiety, depressive behaviour and anger are reactions usually observed. For inpatients, the psychological status seems to follow the somatic status. Among patients with a kidney transplant, some authors have observed psychological problems related to the patient's impression of acquiring some of the donor's characteristics through the graft. Little is known about psychological issues following bone marrow transplantation, which requires a donor in the case of allogeneic transplantation. Thirty-nine patients (12 auto, 27 allogeneic) are included in this study. Patients had been undergoing BMT for six to eighteen months at the time of the study. All patients were in complete remission of their disease. To conduct our study, we used interviews, a rating scale of depression and anxiety, vocabulary testing, and a Rotter's scale. The patients were not suffering specifically from depression or anxiety. After BMT, they experienced both objective and subjective changes in their way of life. The majority appear to be well adapted to their new situation, and some patients have received some benefit from their experience. For a minority, the transplantation was disruptive. Psychological problems encountered in such cases were related to the personality and personal history of the patients. After an allogeneic BMT, some patients approached, by means of a complaint or a joke, the theme of transformation induced by the transplanted marrow. This suggests that psychological integration of an allogeneic marrow transplant is a complex procedure. The various components of this elaboration could be studied further by using more sophisticated means to analyse interviews.


In vivo effects of thymic hormones (Imunotim) on immunologic parameters of patients with secondary immunodeficiency diseases.

In previous papers we revealed the immuno-restorative capacity of thymic hormones both in vitro and in vivo. This paper is concerned with the investigation in a phase I trial of the effects of a thymic extract (Imunotim) prepared in the Cantacuzino Institute, Bucharest upon patients with immunodeficiencies manifest as recurrent or persistent infections resistant to classical treatments. Twenty five testings were performed on 23 patients with secondary immunodepressions: carcinomas with immunodepressions following the acute phase of the disease or cytoreductive treatments (10 cases), allergic and autoimmune conditions (3 cases) and frequent, recurrent infections with prolonged evolution. In 73.9% of cases (17/23) after administration of Imunotim per os the immunologic parameters showed a tendency to return to normal values. In patients with low pre-therapy values the following were noticed: a remarkable increase of the number of lymphocytes from 1317.2 +/- 506.40 to 1961.1 +/- 899.11 (p < 0.01), of PMN from 1401.7 +/- 444.21 to 2651.0 +/- 755.31 (p < 0.01), of the absolute number of B lymphocytes from 192.0 +/- 79.00 to 444.8 +/- 299.75 (p < 0.05); the rise in the rate and absolute number of total T lymphocytes (628.2 +/- 192.81, 1041.0 +/- 441.84, respectively; p < 0.05), T helper (from 29.84 +/- 12.75% to 44.14 +/- 18.76%, respectively from 421.6 +/- 127.29 to 1058.7 +/- 411.05; p < 0.01 respectively p < 0.02) and the rise of the absolute number of T suppressor/cytotoxic lymphocytes from 92.8 +/- 97.11 to 368.3 +/- 368.53 (p < 0.05). In some patients with lymphocytosis and adenopathies the absolute lymphocyte number and the maturation of the lymphocyte populations and subpopulations in blood showed a decline. Individual variations could be noticed even within one and the some primary affection.


Serological diagnosis in human Yersinia infections.

Serological examinations revealed Y. enterocolitica 0:3 specific antibodies in 34% of 80 children affected by a yersiniosis epidemic at titres of 1/40-1/640 and in all sporadic Yersinia enteritis cases. Similarly, agglutinins were identified for Y. enterocolitica 0:3 and 0:9 in 3.4% of 430 appendectomized children hospitalized with painful syndrome in the right iliac fossa. In 10% of 101 patients hospitalized with arthritis and Reiter's syndrome antibodies to Y. enterocolitica 0:3 (8 cases) and Y. pseudotuberculosis (3 cases) were detected at titres of 1/80-1/640. Our results stress the importance of serological examinations in diagnosing human infections with Yersinia.


[Association studies in cancerology: the example of gene L-myc polymorphism associated with the incidence of pulmonary metastases in breast cancers].

Genes, which are implicated in the pathogenesis of human tumors, may demonstrate Restriction Fragment Length Polymorphism (RFLP). Population studies of such RFLP are of potential interest for a genetic analysis of cancer susceptibility. The initial data of Krontiris et al has shown a significant increase of rare c-Ha-ras-1 alleles in individuals with tumors. Since then, other significant associations have been found between other genes RFLPs and various tumors. In this paper, we report that L-myc RFLP may prove useful in identifying breast cancer patients at high risk of developing lung metastases.


Immunologic and antiviral therapy in herpetic keratitis.

A group of 113 patients with herpetic keratitis who were administered besides the classical therapy (local and general) immunologic and antiviral treatments was studied. The following Romanian non-toxic preparations were used: "SRE Immunostimulent", "Moroxidin" chlorhydrate and "Antiherpin". The control group including 120 patients received a classical treatment and the first signs of recovery appeared after 30 days; in 32% of the patients recurrences, were recorded during the first year. The recovery of the patients included in the experimental lot (113) consisted in the disappearance of congestive phenomena after 3-4 days; recurrences occurred in 14% of the patients after 1, 2 or even 4 years, especially in those who received an incomplete treatment.


Contributions to the taxonomy and biology of Clostridium difficile.

Clostridium difficile was incriminated by Hughes and Jarvis (1987) as a cause of intestinal infections in USA in the 1980-1984 period in 45 p. 100 of cases, whereas Salmonellae only in 12 p. 100. Four strains of this organism are studied in this paper in comparison with ten strains of C. bifermentans and six of C. sordellii, because the three species share a common antigen and have other common characteristics, as well. However, spores of C. difficile swell the bacteria and those of other bacteria (C. bifermentans and C. sordellii) do not; C. difficile does not produce indole, whereas the other species produce it. We confirmed the selective capacity of the medium of George et al. (1979) using the "alcohol shock" and as selective agents cycloserine D and cefoxitin. C. difficile proved to be most susceptible to metronidazole and rifampin. Whereas the former antibiotic was considered as a cause of post-antibiotic intestinal infections by different authors, the second was not, to our knowledge. The strain 10463 has a considerable toxicity (1000 DLM/ml for the white mouse, and pathogenicity--2000--5000 DCL for the white mouse, as compared to 25 DCL of the other three strains). Using this toxin an antitoxic serum was obtained in horse, with a capacity of neutralizing the action of the toxin up to a dilution of 1 p. 1000.


[Dihydropyrimidine dehydrogenase activity in lymphocytes: predictive factor for 5-fluorouracil clearance].

We previously have shown that pharmacokinetic monitoring of 5-fluorouracil (5-FU) could significantly improve the 5-FU therapeutic index when given in continuous venous infusion (Br J Cancer 59, 287-290, 1989). However, the more rational approach would be to find individual biological factors which could predict 5-FU clearance. Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme in the catabolism of 5-FU. DPD activity was measured in 57 head and neck cancer patients receiving CDDP (100 mg/m2, day 1) plus 5-FU (1 g/m2/day x 5, day 2-day 6). DPD activity was measured in lymphocytes using a radioenzymatic assay (2.5 mM MgCl2, 250 microM NADPH, 20 microM 14C-5-FU) with separation of 14C-5-FU from 14C-5-FUH2 by HPLC coupled with a radiodetector. The average DPD activity measured in lymphocytes was 0.186 +/- 0.068 nmol/min/mg protein (range 0.058-0.357) and the average 5-FU clearance (Cl) was 2,523 +/- 684 ml/min/m2 (range 1,052-4,029). A significant linear correlation was demonstrated between DPD activity and 5-FU clearance (Cl = 1,099 + 7,580 DPD, r2 = 0.613, P < 0.0001). In patients evaluated for more than one cycle (n = 18), variations in 5-FU clearance were associated with corresponding variations in DPD activity. The individual determination of DPD activity measured in lymphocytes could be useful for identifying patients at risk for altered 5-FU pharmacokinetics and could be used to adjust the optimal 5-FU dose for each patient before starting the treatment.


Acute myelogenous leukemia with T-cell features.

From May 1985 to June 1990, 94 newly diagnosed cases of acute myelogenous leukemia (AML) were treated at the Institut Gustave-Roussy, of which four (4.3%) demonstrated mixed cell lineage. All these cases were morphologically and cytochemically considered as myelogenous leukemias according to the FAB classification. Immunophenotyping revealed in all four cases that the blast population had T-lymphoid (CD2, CD5, CD7 and cytoplasmic CD3) markers. In three of these cases, blast cells co-expressed myelogenous CD13 and CD33 markers. Cytogenetic analysis of the blast cells revealed a normal karyotype in all cases. The response to therapy has been poor. The two patients initially treated with a regimen usually used for AML did not achieve complete remission. By contrast, in three cases, complete remission was obtained with a drug combination used for lymphoblastic leukemia (in one case after failure of first line AML regimen). Only one patient remained disease-free for more than 18 months. We conclude that this form of leukemia is a distinct biological and clinical entity and may benefit form alternative therapy.


Seroepidemiological study of the circulation of influenza C virus in man.

The seroepidemiological study of the circulation of influenza C virus was achieved by testing HI antibodies in human sera collected from healthy subjects in Bucharest belonging to various age groups. The investigations were carried out over a two years period (October 1988--September 1990), using 3 influenza C virus strains: C/Taylor/1233/47, C/USSR/0303/77 and C/Moscow/1/84, the last two being antigenically similar. The analysis of the distribution of HI antibody values against the three influenza C virus strains showed that over October 1988--June 1989 the reported circulating viruses belonged to two distinct antigenic groups, one similar to the prototype C/Taylor/47 strains and the other to C/USSR/and/C/Moscow strains, whilst during the July 1989--September 1990 time interval viruses belonging solely to the C/USSR and C/Moscow antigenic group circulated. Similarly, one should note that circulation of influenza C viruses is not seasonal, it can appear anytime and even several times per year; this accounts for the relatively high and permanent antibody level in the population belonging to all age groups.


[Ezhu intravenous injection in treating infantile respiratory syncytial virus pneumonia and its therapeutical mechanism].

Ezhu Intravenous Injection (EII) in treating respiratory syncytial virus (RSV) pneumonia was studied. 45 patients in therapeutic group were treated with 0.04% EII in dose of 20 ml/kg/day for 7-14 days. 20 patients in the control group were treated with Huayu decoction, its efficacy on RSV pneumonia has been confirmed. The results showed that all the patients of both groups were cured and there were no statistic differences in the average days of clearing up the temperature, dyspnea and wheezing (P > 0.05). Experimental studies showed that the isolation rate of RSV on the 5th and 8th day in the therapeutic group were lower than that of infected and glucose control groups. The difference was significant (P < 0.001 and P < 0.0001). Pathological examination showed that on 5th day in infected and control group there were epithelial damages and bronchial lumen obstruction, peri-bronchial and periseptal edema, eosinophilic leucocytes and inflammatory infiltrations. The endothelial cells of alveolar capillaries swelled, platelet aggregations and thrombi occurred in its lumens. On 8th day the platelet aggregations diminished, but cellular infiltrations and damages of bronchial epithelium still existed. In therapeutic group on 5th day most of the alveolar capillary endothelial cells were normal with mild edema only. The platelet aggregation were much less than the infected and control groups. No peribronchial and peri-septal edema was observed. The results of both clinical and experimental studies showed that EII had good therapeutic effect on RSV pneumonia with no adverse reaction.


[Short-term effects of Angelica sinensis and nifedipine on chronic obstructive pulmonary disease in patients with pulmonary hypertension].

40 COPD cases with pulmonary hypertension (PH) in remission stage were equally divided into four groups, 10 cases in each. Group 1-4 were treated with 25% Angelica sinensis (250 ml, iv. drip, qd), nifedipine (10 mg, po, tid), both Angelica sinensis+nifedipine and blank control respectively. The study was designed to investigate the changes of hemodynamics, pulmonary function and blood gas before and after the treatments by impedance rheopneumogram, lung function examination and blood gas analysis. Mean pulmonary arterial pressure was decreased and cardiac output, PaO2 were increased significantly (P < 0.05 or P < 0.01) in group 3. The effects of group 3 appeared to be better than in other groups. The side effect of PaO2 lowering in group 2 was overcome in adding Angelica sinensis.


[Clinical and experimental study of xiao er ke cuan ling oral liquid in the treatment of infantile bronchopneumonia].

Xiao Er Ke Chuan Ling Oral Liquid (KCL) is a Chinese herbal preparation consisted of 10 herbs such as Prunus armeniacae, Scutelaria baicalensis, Lonicera japonica etc. 30 children suffering from bronchopneumonia and/or acute bronchitis were treated with KCL (treated group) and another 30 cases were treated with penicillin and aminophylline (control group). cure rate and effective rate in treated group was 26.6%, and 93.3% respectively. While in control group was 30% and 96.6% respectively. No significant differences were seen between them(P > 0.05). The pharmacodynamic experiment showed KCL had potent pharmacological action. The experiment on tracheal fragment of Guinea pig in vitro showed it caused moderately strong smooth muscle relaxation, through inhibition the effect of histamine and acetylcholine. Asthma induction experiment of Guinea pig in vivo showed KCL could significantly prolong the latent period of asthma and alleviate asthmatic symptom. Ammonium water cough induction experiment in mice showed it may apparently prolong cough latent period and reduce times of cough relapse and alleviate cough symptom. KCL had potent antipyretic effect on fever model induced by triple vaccine in rabbits. Bacteriostatic and antiviral experiment in vitro showed the drug had quite strong inhibitory effects for Streptococcus hemolyticus, Staphylococcus aureus, Flexners Dysentery bacillus, Diplococcus pneumoniae and Pseudomonas aeruginosa, and it could potently inhibit the respiratory syncytial virus. KCL is an effective drug in treating bronchopneumonia and acute bronchitis.


[Electron microscopy observation on pre- and post-treatment skin of dorsum pedis of chyluric patient treated with heat-clearing and hemostatic drugs--the lymphangial wall damage being the pathogenesis of chyluria].

Heat-clearing and hemostatic drugs were effective in treating chyluria. In order to explore the curative mechanism, a group of 10 in-patients with chyluria were chosen and treated with heat-clearing and hemostatic drugs for 45 days. Some pre- and post-treatment skin from dorsum pedis of the patients were taken off and observed under electron microscope. It showed that the walls of lymphatic capillaries were seriously damaged before treatment and rehabilitated wall after treatment. The pinocytotic vesicles were packed tightly in the endothelial cells of capillaries before treatment and their number was decreased remarkably after treatment; the mast cells were rare in number and abnormal as spindle shape before treatment and they increased in number and became round or oval in shape, the nucleus became large, the granules they contained became more in number and larger in size after treatment, lymph edema was noticed before treatment and it disappeared after treatment. The result showed that the extensive lesion of lymphangial walls is the pathogenesis of chyluria, while the rehabilitation of them is the curative mechanism. It seems that in the process of rehabilitation the mast cells act took the role of "soldier-engineer".


[Abdominal diagnosis in blood-stasis syndrome].

In order to make the abdomen diagnosis in Blood Statis Syndrome more objectively and quantitatively, some comprehensive quantitative analysis have been done by authors. The chief evidences between abdomen syndrome of Blood-stasis Symptoms and Blood-stasis Syndrome were correlated. The main parameters of abdomen diagnosis in Blood-stasis syndrome have been selected by computerized stepwise regression analysis in 152 patients with above-mentioned Syndromes. They were:Blood viscosity, erythrocyte deformation, extracorporeal thrombosis, platelet aggregation, thromboelastograph, electromyogram, etc. All of these can be used as objective parameters of abdomen diagnosis in Blood-stasis Syndrome to make the further study of it.


[Mechanism of shen qian gujing granule in the treatment of menorrhagia].

Shen Qian Gujing Granule, a Chinese herbal preparation has shown its efficacy of 87.7% in treating menorrhagia. PGE2, PGE2 alpha, TXB2 and 6-Keto-PGF1 alpha levels were measured in the endometrium and menstrual blood of both normal menstrual women and patient with menorrhagia before and after the treatment. Local TXB2 values of endometrial and menstrual blood were significantly higher in menorrhagia patients than that in normal subjects (P < 0.05). And the local PGE2 values were higher in patients accompanied with Qi Deficiency (P < 0.05) and lower in patients without Qi Deficiency (P < 0.05). After the treatment, the local TXB2, PGE2 levels normalized. It suggests that Shen Qian Gujing Granule had a biphasic regulation on local PG values which yields good results for menorrhagia. Some mechanism were discussed.


[Research on liu wei Rehmannia oral liquid against side-effect of drugs of anti-tumor chemotherapy].

Using tumor-bearing mice as experimental animals, the survival rate, hematopoietic, heart, liver, kidney and immunological functions as indexes, Liu Wei Rehmannia Oral Liquid against the side-effect of drugs of anti-tumor chemotherapy (ADM, CTX, DDP, VCR and 5FU) were observed. The results showed that the survival rate in the treated group was significantly higher than that in control, the survival days were highly different (P < 0.01); the hemopoietic functions (HB, WBC, PL) in the treated group were higher also (P < 0.05 & P < 0.01); in the functions of heart, liver and kidney the treated group could protect the above-mentioned three organs (P < 0.01); in immune functions, the oral liquid could protect the NK and T-, B-Lymphocyte transformation, being inhibited by drugs of chemotherapy. In comparing with control group NK and T lymphocyte transformation were all significantly different (P < 0.01 & P < 0.05), while only 3 chemotherapy drugs were markedly different in B lymphocyte transformation (P < 0.05 & P < 0.01). The discussion indicated that Liu Wei Rehmannia Oral Liquid is effective in protecting the functions of hematopoiesis, immunity, heart, liver and kidney during chemotherapy, which provides an objective data for the clinical application.


[Reinforcing qi and promoting blood circulation to prevent hepatic fibrosis due to bovine serum albumin immunologic injury].

44 Wistar female rats were divided randomly into 4 groups--normal control(I), case control (II), reinforcing Qi and promoting blood circulation(III) and nourishing Yin and promoting blood circulation(IV). After 4 times of bovine serum albumin (BSA) shock injection, the group III and the group IV were medicated through gastric intubation for 40 days respectively with 300% mixture of reinforcing Qi and promoting blood circulation and 300% mixture of nourishing Yin and promoting blood circulation. The results suggest the mixture of reinforcing Qi and promoting blood circulation has the function of alleviating pathological changes of liver, reducing the content of liver collagen, improving erythrocytic function of clearing away immune complexes and regulating humoral immune response.


[Effects of crude extract of earthworm on promoting blood circulation to removing stasis].

The crude extract of earthworm which has a thrombolytic effect could significantly decrease the plasma fibrinogen content and euglobulin lysis time (P < 0.01). An enzymatic preparation, containing many fibrinolytic enzymes, was prepared from the crude extract by ammonium sulphate precipitation and DEAE-cellulose chromatography. When administered in rabbits, the enzymatic preparation had an effect in hemorheology improvement. The experiment shows the enzymatic preparation could obviously lower the aggregation of platelets, decrease the viscosity of whole blood and plasma as well as the index of erythrocyte rigidity significantly (P < 0.001). All these effects demonstrated its ability in promoting blood circulation to remove stasis.


[Plant lectins and embryonic tissue factors as probes for studying the mechanisms of neural induction in amphibians].

Using various experimental techniques, we have demonstrated that animal pole ectoderm (APE) of Rana temporaria embryos at the stage of early gastrula is a good target tissue for testing the neuralizing (N) factors. In this respect R. temporaria APE is comparable with APE of some other amphibian species. We found that concanavalin A (con A), phytohemagglutinin (PHA) and embryonic brain-derived neuralizing factor (EBDNF; a factor extracted from the chick embryonic brain and partially purified) have a pronounced N-effect on the APE of R. temporaria. In order to analyse possible mechanisms of N-action of these factors, we have cultured APE explants for 3 or 18 h in the medium containing various concentrations of con A, PHA of EBDNF. All these factors could produce neuralization in 50% explants. However, the optimal concentration and time of exposure were different. This is an evidence for different mechanisms of reception and transmission of a N-signal in each particular case. It appears that the APE consists of several cell subpopulations which differ in their threshold sensitivity to the N-effect of studied agents.


[The effect of 6-methyluracil on oxidative reactions in model systems of varying degrees of complexity].

Effect of 6-methyluracyl (MU) on oxidative processes was studied on model systems of different degrees of complexity (methyloleate model, mitochondria isolated from rat liver, surviving slices of mammalian muscle tissues). On two former models it was shown that MU exerted antioxidant (AO) properties inferior to those of such antioxidants as ionol and K(+)-phenosan. The AO efficiency depended on the intensity of peroxide processes. The AO activity of MU was most fully realized on the model of isolated surviving slices of muscle tissue. The totality of data obtained suggests that participation of the substance in regulation of lipid peroxidation in the organism underlies its therapeutic activity in pathological states.


[The interorgan distribution of tritium-labelled para-aminobenzoic acid in the body of rats after its subcutaneous injection].

To study the pharmacokinetics of para-aminobenzoic acid (PABA), rats were subcutaneously injected with 3H-PABA (1 mkg/g body weight) at the stage (P7) when stimulation of outer segments of the photoreceptor cell morphogenesis is possible (Stroeva et al., 1990). The maximum labelling assayed for radioactivity by scintillation counting was observed in the liver 3 h after the injection while, in the blood the labelling was 3 times lower and, in the retina, one order lower than in the liver and blood. The label was detected in all the three types of tissue in 24 and 48 hours, being reduced by an order in comparison with each previous term of measurement. Biological structures binding 3H-PABA were not determined in the present study.


Localization and function of dopamine in the adult vertebrate retina.

Dopamine (DA) has satisfied many of the criteria for being a major neurochemical in vertebrate retinae. It is synthesized in amacrine and/or interplexiform cells (depending on species) and released upon membrane depolarization in a calcium-dependent way. Strong evidence suggests that it is normally released within the retina during light adaptation, although flickering and not so much steady light stimuli have been found to be most effective in inducing endogenous dopamine release. DA action is not restricted to those neurones which appear to be in "direct" contact with pre-synaptic dopaminergic terminals. Neurones that are several microns away from such terminals can also be affected, presumably by short diffusion of the chemical. DA thus affects the activity of many cell types in the retina. In photoreceptors, it induces retinomotor movements, but inhibits disc shedding acting via D2 receptors, without significantly altering their electrophysiological responses. DA has two main effects upon horizontal cells: it uncouples their gap junctions and, independently, enhances the efficacy of their photoreceptor inputs, both effects involving D1 receptors. In the amphibian retina, where horizontal cells receive mixed rod and cone inputs, DA alters their balance in favour of the cone input, thus mimicking light adaptation. Light-evoked DA release also appears to be responsible for potentiating the horizontal cell-->cone negative feed-back pathway responsible for generation of multi-phasic, chromatic S-potentials. However, there is little information concerning action of DA upon bipolar and amacrine cells. DA effects upon ganglion cells have been investigated in mammalian (cat and rabbit) retinae. The results suggest that there are both synaptic and non-synaptic D1 and D2 receptors on all physiological types of ganglion cell tested. Although the available data cannot readily be integrated, the balance of evidence suggests that dopaminergic neurones are involved in the light/dark adaptation process in the mammalian retina. Studies of the DA system in vertebrate retinae have contributed greatly to our understanding of its role in vision as well as DA neurobiology generally in the central nervous system. For example, the effect of DA in uncoupling horizontal cells is one of the earliest demonstrations of the uncoupling of electrotonic junctions by a neurally released chemical. The many other, diverse actions of DA in the retina reviewed here are also likely to become model modes of neurochemical action in the nervous system.(ABSTRACT TRUNCATED AT 400 WORDS)


[Histological variants of the hepatoid glands in representative Canidae and Cavicornia: the structural and histochemical characteristics].

Both the original material and the data from literature collected in two previous papers, show a considerable diversity of structure and secretion of the glands which constitute the hepatoid gland type. According to the body of available histological (including morphometrical) and histochemical data, the authors distinguish: a) features characteristic of the hepatoid gland type only which do not appear in other types of skin glands; b) seven histological varieties within the hepatoid gland type. These seven varieties can be united, according to the laws of their structure and function, into two groups (1-3 and 5-7), with an intermediate variety (4) between them. The first group includes hepatoid glands of the "classical" structure which has been previously detaily exampled by circumanal glands of the dog: these glands possess cysts, produce a purely protein secretion; the secretion type is merocrine. The second group comprises mero-holocrine hepatoid glands which are characterized by the absence of cysts, broader excretion ducts, and a capability to produce, besides protein, considerable amounts of melanin and/or hydrophobic lipids.


[The principles of left-right hemispheric interactions of pharmacological preparations exemplified by Brinaldix].

The effect of 4-chlor-N-(cys-2,6-dimethyl peridine)-3-sulfomoin-benzamide as an anti-hypertensive drug on left-to-right hemisphere interactions was studied on experimental animals. Its effect on hemispheres was shown to be asymmetrical. The most effective action of the drug was demonstrated on the left hemisphere of rats differing in their trends of interhemispheric interactions.


[The morphogenesis of the human brain on the 27th-35th day of development with disordered neurulation].

Human embryos with anomalies of brain and spinal cord were studied on 27-35th day of the development. It was established that developmental anomalies were associated with the disturbed formation of neural tube. The reconstruction of three-dimensional brain arrangement of embryos has shown the presence of different variants of nonclosed medullar tori. In mild disturbance of neurulation, there is an acceleration of organogenesis and morphological differentiation induced by the shackening of normal mechanical tensions and relaxation of neuroepithelial layers. In presence of substantial parts of open neural tube, the development of brain and ectodermal derivatives becomes disturbed to be a possible cause of anencephaly and hypotelorism. The type of disturbed neurulation inducing developmental anomalies of nervous system is suggested to be a factor which determines forms of secondary craniofacial and cranial pathology.


Effect of choline esters on the decarbamylation of dimethylcarbamyl-acetylcholinesterase.

Acetylcholine and butyrylcholine exhibited the dose-dependent decarbamylation up to 0.2 mM, although at higher concentrations the decarbamylation degree declined. In combination with choline, butyrylcholine potentiated the choline-catalyzed decarbamylation by 30-100%, and was found to be more effective than acetylcholine in enhancing the decarbamylation. In kinetic analysis, it was observed that Ka value of choline was not remarkably altered by butyrylcholine whereas the maximum rate for decarbamylation was enhanced significantly in the presence of butyrylcholine, suggesting that butyrylcholine may affect the decarbamylation by interacting with the peripheral sites, different from the central active site which choline is known to interact with. In support of the suggestion, butyrylcholine was observed to compete with gallamine, a well known peripheral activator, and the effect of butyrylcholine was enhanced by three times at low ionic strength. In addition, acetylcholinesterase from mouse brain or bovine erythrocyte seemed to differ from electric eel enzyme in the interaction with butyrylcholine.


[The effect of isomeric alkyl (C19--C25) methoxybenzoquinones on mitochondrial respiration].

The influence of 2-methoxy-6-alkyl(C19-C25)benzoquinones-1,4 and 4-methoxy-6-alkyl(C19-C25)benzoquinones-1,2 on the respiration of isolated rat liver mitochondria has been studied. As a concentration of each of the quinones increased gradually from zero, the rate of the respiration using NAD-dependent substrates firstly increased but then decreased, 1,2-quinones being the more potent inhibitors. Concurrently, the respiratory control of mitochondria was lowered, indicating uncoupling effect of these quinones. With succinate the latter exhibited moderately accelerating effect. Uncoupled by 2,4-dinitrophenol, the respiration was stimulated by the both preparations in the presence of succinate but was suppressed when NAD-dependent substrates were used. The observed inhibitory effects have been assumed to be caused by a competition between the quinones and endogenous co-enzyme Q for the active site of NADH-dehydrogenase complex of the respiratory chain.


The mode of interaction of amiloride and some of its analogues with the adenosine A1 receptor.

Amiloride, a potassium sparing diuretic, inhibits adenosine A1 receptor-radioligand binding in calf and rat brain membranes in the low micromolar range. The drug interacted with the A1 receptor in a manner different from classical A1 ligands, but structure-activity relationship studies indicated that this inhibitory effect is not related to the ion transport inhibiting properties of amiloride (Garritsen et al., 1990a,b) In the present study, the question is addressed how amiloride interacts with the adenosine A1 receptor. Amiloride and two of its analogues, in concentrations equivalent to their Ki values in displacement studies, decrease the affinity of the A1 antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine, but not the maximal binding capacity of the radioligand. Furthermore, the dissociation rate of the receptor-ligand complex is unaltered in the presence of amiloride or its analogues in a concentration exceeding the Ki value 10-fold. These characteristics argue for a purely competitive mode of interaction. The functional consequences of the interaction between amiloride analogues and the A1 receptor were investigated at the level of cyclic adenosine 3',5'-monophosphate (cAMP) formation. The amiloride analogue 5-(N-butyl-N-methyl) amiloride (MBA) reversed A1-receptor mediated inhibition of forskolin-stimulated cAMP formation in rat fat cell membranes. In this model, the antagonist potency of MBA is ca 5 microM. This value is in fair agreement with a Ki value of 3.5 microM in binding assays under similar conditions. In conclusion, amiloride inhibits A1 receptor binding in an apparently competitive manner. This suggests that the binding sites of amiloride and the classic A1 receptor ligands may at least partially overlap.(ABSTRACT TRUNCATED AT 250 WORDS)


[The effect of mexidol on the dynamics of the normalization of the neurochemical and behavioral parameters of rats after deprivation of the paradoxical sleep phase].

Using cytophotometric method it was shown that intraperitoneal injection of mexidol in the dose of 50 mg/kg resulted in a significant accumulation of proteins and RNA in cells of dorsal raphe nucleus (DRN) and locus coeruleus (LC) 6 hs following the injection. In rats deprived of paradoxical sleep for 24 hours, the effect of mexidol prevented the decrease in DRN proteins and RNA and even increased them, and prevented their decrease in LC. In the postdeprivation period, the beneficial effect of mexidol manifested itself in the acceleration of normalizing the levels of proteins and RNA in DRN and LC. The antistress effect of mexidol manifested itself also in the acceleration of normalizing the rat memory in the restoration period following the deprivation of paradoxical sleep. The normalization of metabolism and functional activity in serotonergic DRN is suggested to determine to a considerable extent the functional state of cerebral structures participating in the control of memory as well.


Measurement of nerve growth factor-like immunoreactivity in human brain using an anti-mouse-NGF enzyme immunoassay.

Nerve growth factor (NGF) like immunoreactivity, expressed as mouse 2.5S nerve growth factor equivalents, was evaluated in three structures of the human brain post-mortem using a commercially available enzyme immunoassay. Regional differences in NGF-like immunoreactivity were observed. Highest levels were found in hippocampus (148 pg/g) compared to putamen (76 pg/g) and frontal cortex (34 pg/g). In addition, these results suggest differences in the distribution of brain NGF between human and rodent, where relatively high levels of NGF are found in the cortex.


Further studies on the relationship between tyrosine supply and catecholamine production in cultured adrenal chromaffin cells.

To elucidate a possible role of tyrosine supply as a factor modulating catecholamine biosynthesis in the adrenergic cell, the transport of [14C]tyrosine into cultured bovine adrenal chromaffin cells was first examined, and the relationship between [14C]tyrosine transport and [14C]catecholamine formation was then investigated. Under the conditions which were routinely employed to determine the rate of catecholamine biosynthesis, tyrosine was taken up into the cells in a manner independent of extracellular Na+ and Ca2+, and this uptake was also insensitive to ouabain and various metabolic inhibitors. The stimulation of these cells with high K+ and other secretagogues caused no significant alteration in the uptake. While, tyrosine transport was markedly inhibited by tyrosine analogues and other L-aromatic amino acids, and this inhibition was accompanied by the reduction of [14C]catecholamine formation. In contrast, tyrosine transport was markedly enhanced by flavone, and this enhancement was also accompanied by the augmentation of catecholamine production under the same experimental conditions. These results seem to indicate that the transport of tyrosine into the cells may be closely related to catecholamine formation within the cells, thus providing an evidence for a possible role of tyrosine supply as one of the factors affecting catecholamine production in the adrenal chromaffin cell.


Two polypeptides from Dendroaspis angusticeps venom selectively inhibit the binding of central muscarinic cholinergic receptor ligands.

Two new polypeptides were isolated and purified from the venom of the snake Dendroaspis angusticeps, which also contains other neuroactive peptides such as Dendrotoxins and Fasciculins. The amino acid composition of the peptides was determined and the first 10 amino acids from the MTX2 N-terminal fragment were sequenced. The so-called muscarinic toxins (MTX1 and MTX2) have been shown to inhibit the specific binding of [3H]QNB (0.15 nM), [3H]PZ (2.5 nM) and [3H]oxoM (2 nM) to bovine cerebral cortex membranes by 60, 88 and 82% respectively. In contrast, they caused only a 30% blockade of the [3H]QNB specific binding to similar membrane preparations from the brainstem. The Hill number for the [3H]PZ binding inhibition by the putative muscarinic toxin MTX2 was 0.95 suggesting homogeneity in the behaviour of the sites involved. The data from [3H]oxoM binding gave a Hill number of 0.83. The decreases in the specific binding involved increases in KD for the three different ligands (8-fold for [3H]QNB, 4-fold for [3H]PZ and 3.5-fold for [3H]oxoM) without significant changes in Bmax, except for a slight decrease in the [3H]oxoM binding sites (-19%); such results suggest that there may be a competitive inhibition between the MTXs and these ligands. The Ki for MTX2/[3H]PZ was 22.58 +/- 3.52 nM; for MTX2/[3H]oxoM, 144.9 +/- 21.07 nM and for MTX2/[3H]QNB, 134.98 +/- 18.35 nM. The labelling of MTX2 with 125I allowed direct demonstration of specific and saturable binding to bovine cerebral cortex synaptosomal membranes. In conclusion, the results reported in this study strongly support the hypotheses that the two polypeptides isolated from D. angusticeps venom selectively inhibit specific ligand binding to central muscarinic receptors, in a competitive manner at least for the antagonist [3H]PZ and that the MTX2 specifically binds to a central site that is suggested to be a muscarinic receptor of the M1 subtype.


Interaction of sialosyl cholesterol with the cell surface of rat astrocytes and its biological activities.

The interaction between sialosyl cholesterol (alpha- or beta-D-N-acetyl neuraminyl cholesterol, alpha- or beta-SC) and the plasma membrane of astrocytes was investigated by the use of 14C-labeled alpha- or beta-SC. Both alpha- and beta-SC were dose-dependently and time-dependently bound to rat astrocytes. The Scatchard plot analyses showed that rat astrocytes bound apparently 9.69 x 10(9) molecules of both alpha-SC/cell (apparent Kd = 2.29 x 10(-5) M) and beta-SC/cell (apparent Kd = 5.39 x 10(-5) M) at 37 degrees C. Both the binding of alpha-SC to astrocytes and the subsequent inhibition of DNA synthesis were decreased at the low temperature (4 degrees C), and also suppressed by serum proteins including albumin. One molecule of bovine serum albumin (BSA) bound 2.3 molecules of alpha-SC with the slightly lower Kd-value (8.03 x 10(-6) M) than that for the binding site on astrocytes. BSA not only suppressed the alpha-SC-binding to astrocytes but also increased its release from the cells to the culture media. Gangliosides such as GM1 and GM3 unaffected the alpha-SC-binding, promoted the small release of alpha-SC from the cell surface, and inhibited the morphological changes of astrocytes induced by alpha-SC. The mechanism of alpha-SC-binding to cultured astrocytes with reference to the effects of serum or gangliosides is discussed.


Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins.

Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [35S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS-PAGE of the treated cells showed incorporation of label into a broad 97-121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97-121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5'-O-(3-thiotriphosphate). The 97-121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5'-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteolgycans.(ABSTRACT TRUNCATED AT 250 WORDS)


Inorganic thiophosphate effects on chromaffin cell structure and function.

The effect was determined of replacing medium inorganic phosphate with thiophosphate on the structure and function of cultured bovine chromaffin cells. Cell cultures were incubated in normal medium containing fetal bovine serum, phosphate free medium or similar medium supplemented with inorganic phosphate or thiophosphate. In contrast to the other media, cells cultured with thiophosphate medium for 3-4 days showed seriously compromised structure and functions. The cells lost 75% of their catecholamine content and their ability to secrete remaining catecholamines in response to nicotine stimulation. Radiolabelled thiophosphate was rapidly taken up by the cells and, in long-term experiments, was incorporated largely into a 97-121 kDa protein band on SDS-PAGE. Additional minor bands were found to a lesser, variable extent. Transmission electron micrographs of cells treated with thiophosphate showed extensive depletion of chromaffin vesicles and disruption of mitochondria, suggesting that the functional damage noted with these cells could be associated with damage to mitochondria. Analysis of general cell metabolic activity by conversion of the dye (3-[3,4-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide) to its formazan derivative indicated increased metabolic activity at early stages of exposure to thiophosphate followed by a decline with continued exposure, supporting the argument for an overall depression of cell metabolism. Uptake of the dye neutral red, which is avidly accumulated by chromaffin cells, was also reduced for cells exposed to thiophosphate. The data suggest that thiophosphate enters chromaffin cells and disrupts energy dependent cell functions, including catecholamine storage and secretion.


Role of histidine residues in the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor.

This paper studies the effect of histidine chemical modification of the membrane-bound acetylcholine receptor from Discopyge tschudii on its specific alpha-bungarotoxin binding. The acylating reagent ethoxyformic anhydride (diethyl pyrocarbonate, DEP), was used. DEP-treatment induces a loss of binding capacity, time and DEP-concentration dependent. After a 30 min period of derivatization with 2 mM final DEP-concentration, at pH 7.4, the decrease reaches 70%; the loss of binding capacity is faster at pH 7.4 than at pH 6.0, as expected, since the amount of unprotonated species is higher under the first condition. Moreover, when ethoxyformylation is carried out at different pH values, the most important neurotoxin binding decrease occurs between pH 6.0 and 8.0. Furthermore, ethoxyformylation reversion restores such capacity. Consistent with the modification of a binding site, the ethoxyformylation does not bear on the affinity but reduces the number of receptors. Ethoxyformylation in the presence of carbamylcholine shows some ligand protective effect. These results, as a whole, strongly indicate a relevant role for histidine residues at the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor.


Influence of the tumor mass on the valine rate constants and on valine incorporation into proteins in an experimental brain tumor model.

Quantitative autoradiography was used to estimate regional transfer coefficients for valine incorporation and the rate of valine (exogenous and total) incorporation into proteins in an implanted brain-tumor model (AA ascites tumor). Special attention was paid to the evaluation of the tumor mass influence on the transfer coefficients and the rate of incorporation. The size of the tumors used in this study ranged from 2 to 5 mm in diameter. Nine groups of two to three animals each were used to determine the transfer coefficient. The transfer coefficients for movement of the label between different compartments were significantly greater in the tumor than in the normal brain. There is no tumor mass effect on the transfer coefficients or the rate of valine incorporation into proteins in surrounding or remote brain structures. The ratio between specific radioactivities of the free value in tissue and plasma was also measured. Results indicate that approximately the same fraction of the total valine is recycled in cortex as in the tumor tissue. The mean rates of exogenous valine incorporation into proteins (nmol g-1 min-1) is about one order of magnitude greater in the tumor than in the contralateral parietal cortex.


Purification and partial characterization of K+ channel blockers from the venom of Dendroaspis angusticeps.

Two polypeptides (designated DTX-A and DTX-B) were purified from crude snake venom of Dendroaspis angusticeps using gel filtration, cation exchange column chromatography and cation exchange high performance liquid chromatography, and their blocking actions of K+ channels were investigated in rat brain synaptosomes. Both DTX-A and DTX-B inhibited the voltage-dependent 42K efflux from the synaptosomes. DTX-A blocked 42K efflux of both the rapidly inactivating phase (component T) and the slowly inactivating phase (component S). The inhibitory effect of DTX-A on component T was pronounced compared with that on component S. However, DTX-B selectively blocked 42K efflux of component S. The molecular weights of DTX-A and DTX-B were estimated to be ca 10,000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition of these toxins is different from that of polypeptide purified from the venom of D. angusticeps (alpha-, beta-, gamma- and delta-DTX). These results suggest that DTX-A and DTX-B are new polypeptides which block voltage-dependent K+ channels selectively, and that they are useful tools for investigating the K+ channel.


Dopamine receptors in human foetal brains: characterization, regulation and ontogeny of [3H]spiperone binding sites in striatum.

Eighteen corpora striata from normal human foetal brains ranging in gestational age from 16 to 40 weeks and five from post natal brains ranging from 23 days to 42 years were analysed for the ontogeny of dopamine receptors using [3H]spiperone as the ligand and 10 mM dopamine hydrochloride was used in blanks. Spiperone binding sites were characterized in a 40-week-old foetal brain to be dopamine receptors by the following criteria: (1) It was localized in a crude mitochondrial pellet that included synaptosomes; (2) binding was saturable at 0.8 nM concentration; (3) dopaminergic antagonists spiperone, haloperidol, pimozide, trifluperazine and chlorpromazine competed for the binding with IC50 values in the range of 0.3-14 nM while agonists--apomorphine and dopamine gave IC50 values of 2.5 and 10 microM, respectively suggesting a D2 type receptor. Epinephrine and norepinephrine inhibited the binding much less efficiently while mianserin at 10 microM and serotonin at 1 mM concentration did not inhibit the binding. Bimolecular association and dissociation rate constants for the reversible binding were 5.7 x 10(8) M-1 min-1 and 5.0 x 10(-2) min-1, respectively. Equilibrium dissociation constant was 87 pM and the KD obtained by saturation binding was 73 pM. During the foetal age 16 to 40 weeks, the receptor concentration remained in the range of 38-60 fmol/mg protein or 570-1080 fmol/g striatum but it increased two-fold postnatally reaching a maximum at 5 years.(ABSTRACT TRUNCATED AT 250 WORDS)


Preparation of noradrenaline-storing organelles from bovine sympathetic ganglia: biochemical and morphological evaluation of partly purified large dense cored vesicles.

Large dense cored vesicles from bovine sympathetic ganglia were isolated and partly purified. Biochemical and morphological evaluation of the present vesicle-preparation revealed that it represents a convenient fraction for the characterization of perikaryal noradrenergic vesicles. Homogenates of bovine stellate ganglia were subjected to differential centrifugation and D2O-sucrose density gradient centrifugation. Biochemical evaluation of gradient fractions was performed by measuring marker enzyme activities reflecting subcellular contamination, while morphological evaluation was performed by electron microscopic analysis of the isolated fractions. Both techniques revealed that the vesicle-preparation was, at first, still considerably contaminated by mitochondria and lysosomes. An improved purification could be achieved by subjecting this fraction to an additional centrifugation under iso-osmotic conditions, also applied for the preparation of highly purified splenic nerve vesicles. The resulting vesicle-fraction was almost completely free of contaminating enzyme activities and consisted merely of large dense cored vesicles as revealed by electron microscopic observations (50-70% purity). Neuropeptide Y and chromogranin A were enriched more than 50 times as compared to the total homogenate. Although the purity of these vesicles was still not satisfactory for direct chemical analysis, this vesicle-preparation seemed very well suited for immunological characterization of perikaryal large dense cored vesicles.


Modulation of cerebral acetylcholine metabolism by the dorsal diencephalic conduction system in the rat.

We investigated the effects of interruption of the impulse flow in the habenulopeduncular pathways by local infusion of tetrodotoxin on the acetylcholine and choline content in selected dopamine rich regions in the forebrain and midbrain in rats. The tetrodotoxin infusion caused a marked increase in acetylcholine content in the medial frontal cortex, striatum and ventral tegmental area+interpenduncular nucleus, but not in the limbic area or the substantia nigra, whereas choline content was reduced only in both the striatum and ventral tegmental area+interpeduncular nucleus. There was an increase in 3,4-dihydroxyphenylacetic acid content in the striatum after the manipulation. These findings suggest that the dorsal diencephalic conduction system may be involved in the integration of the activity of cholinergic neurons in the forebrain and midbrain regions and striatal dopanine neurons may play a role in the modulation of cholinergic neurons.


3-mercaptopropionic acid administration increases the affinity of [3H]quinuclidinyl benzilate binding to membranes of the striatum and cerebellum.

It is known that quinuclidinyl benzilate (QNB) binds specifically and with high affinity to the cholinergic muscarinic receptor and that behaves as a potent antagonist of this receptor. We have analysed L-[3H]QNB binding to rat CNS membranes after the administration of the convulsant 3-mercaptopropionic acid (MP) (150 mg.kg-1, i.p.). The studies were done in rats killed at two stages: during and after seizures. No changes in [3H]QNB binding to hippocampus and cerebral cortex membranes were found. [3H]QNB binding increased about 40 and 80% in striatum and cerebellum membranes, respectively. The changes were observed both in seizure and postseizures states. The study was extended to the assay of [3H]QNB binding kinetic constants in the anatomical areas modified by the convulsant. The analysis of the saturation curves indicated an increase in the binding affinity but no change in the number of binding sites. Hill number values were near the unit suggesting a non-cooperative interaction between the ligand and the receptor, and the labelling of a homogeneous population of receptor sites. The results suggest the participation of some cholinergic pathways in the development and maintenance of MP-induced seizures.


Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast.

The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.


The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc-dependent binding to an anionic surface.

The histidine-glycine-rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin-forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) C11C1 to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 microM and 27 microM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M(r) was 7.3 kDa (range 6.2-8.1 kDa). Based on N-terminal sequencing, this polypeptide (1(2)), which extends from the histidine residue 459 to a lysine at position 505, 509, 511, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 microM. The synthetic peptide HGLGHGH representing the N-terminal of the 1(2)) fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459-475) included within the 1(2) fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 microM in the absence and presence of added Zn2+ (30 microM). The specific binding of 125I-HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 microM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of 125I-HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+. In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 microM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the 1(2) fragment was found to inhibit 50% of the 125I-HK binding to kaolin at a concentration of 380 microM. These results suggest that residues contained within the 1(2) fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.


Primary structure of a protein isolated from reef shark (Carcharhinus springeri) cartilage that is similar to the mammalian C-type lectin homolog, tetranectin.

During the course of characterization of low molecular weight proteins in cartilage, we have isolated a protein from reef shark (Carcharhinus springeri) cartilage that bears a striking resemblance to the tetranectin monomer originally described by Clemmensen et al. (1986, Eur. J. Biochem. 156, 327-333). The protein was isolated by extraction of neural arch cartilage with 4 M guanidine hydrochloride, dialysis of the extract to bring the guanidine to 0.4 M (reassociating proteoglycan aggregates), followed by cesium chloride density gradient removal of the proteoglycans. The amino acid sequence had 166 amino acids and a calculated molecular weight of 18,430. The shark protein was 45% identical to human tetranectin, indicating that it was in the family of mammalian C-type lectins and that it was likely to be a shark analog of human tetranectin. The function of tetranectin is unknown; it was originally isolated by virtue of its affinity for the kringle-4 domain of plasminogen. Sequence comparison of human tetranectin and the shark-derived protein gives clues to potentially important regions of the molecule.


Reduction of thioredoxin significantly decreases its partial specific volume and adiabatic compressibility.

The partial specific volume and adiabatic compressibility were determined at several temperatures for oxidized and reduced Escherichia coli thioredoxin. Oxidized thioredoxin had a partial specific volume of 0.785-0.809 mL/g at the observed upper limit for all proteins whereas the partial specific volume of reduced thioredoxin was 0.745-0.755 mL/g, a value in the range found for a majority of proteins. The adiabatic compressibility of oxidized thioredoxin was also much larger (9.8-18 x 10(-12) cm2 dyne-1) than that of the reduced protein (3.8-7.3 x 10(-12)). Apart from the region immediately around the small disulfide loop, the structures of the oxidized (X-ray, crystal) and reduced protein (nuclear magnetic resonance, solution) are reported to be very similar. It would appear that alterations in the solvent layer in contact with the protein surface must play a major role in producing these large changes in the apparent specific volumes and compressibilities in this system. Some activities of thioredoxin require the reduced structure but are not electron transfer reactions. The large changes in physical parameters reported here suggest the possibility of a reversible metabolic control function for the SS bond.


The kinemage: a tool for scientific communication.

A "kinemage" (kinetic image) is a scientific illustration presented as an interactive computer display. Operations on the displayed kinemage respond within a fraction of a second: the entire image can be rotated in real time, parts of the display can be turned on or off, points can be identified by selecting them, and the change between different forms can be animated. A kinemage is prepared and specified by the author(s) of a journal article, in order to better communicate ideas that depend on three-dimensional information. The kinemages are distributed as plain text files of commented display lists and accompanying explanations. They are viewed and explored in an open-ended way by the reader using a simple graphics program, such as the one described here (called MAGE), which presently runs on Macintosh computers. A utility (called PREKIN) helps authors prepare the kinemages. Kinemages are being implemented under the auspices of the Innovative Technology Fund.


The importance of surface loops for stabilizing an eightfold beta alpha barrel protein.

An important step in understanding how a protein folds is to determine those regions of the sequence that are critical to both its stability and its folding pathway. We chose phosphoribosyl anthranilate isomerase from Escherichia coli, which is a monomeric representative of the (beta alpha)8 barrel family of proteins, to construct a variant that carries an internal tandem duplication of the fifth beta alpha module. This (beta alpha)9 variant was enzymically active and therefore must have a wild-type (beta alpha)8 core. It had a choice a priori to fold to three different folding frames, which are distinguished by carrying the duplicated segment as an insert into one out of three different loops. Steady-state kinetic constants, the fluorescence properties of a crucial tryptophan residue, and limited proteolysis showed that the stable (beta alpha)9 variant carries the insertion between beta-strand 5 and alpha-helix 5. This preference can be explained by the important role of loops between alpha helices and beta strands in stabilizing the structure of the enzyme.


Structure of a stabilizing disulfide bridge mutant that closes the active-site cleft of T4 lysozyme.

The engineered disulfide bridge between residues 21 and 142 of phage T4 lysozyme spans the active-site cleft and can be used as a switch to control the activity of the enzyme (Matsumura, M. & Matthews, B.W., 1989, Science 243, 792-794). In the oxidized form the disulfide increases the melting temperature of the protein by 11 degrees C at pH 2. The crystal structure of this mutant lysozyme has been determined in both the reduced and oxidized forms. In the reduced form, the crystal structure of the mutant is shown to be extremely similar to that of wild type. In the oxidized form, however, the formation of the disulfide bridge causes the alpha-carbons of Cys 21 and Cys 142, on opposite sides of the active-site cleft, to move toward each other by 2.5 A. In association with this movement, the amino-terminal domain of the protein undergoes a rigid-body rotation of 5.1 degrees relative to the carboxy-terminal domain. This rotation occurs about an axis passing through the junction of the amino-terminal and carboxy-terminal domains and is also close to the axis that best fits the apparent thermal motion of the amino-terminal domain seen previously in crystals of wild-type lysozyme. Even though the engineered Cys 21-Cys 142 disulfide links together the amino-terminal and carboxy-terminal domains of T4 lysozyme, it does not reduce the apparent mobility of the one domain relative to the other. The pronounced "hinge-bending" mobility of the amino-terminal domain that is suggested by the crystallographic thermal parameters of wild-type lysozyme persists in the oxidized (and reduced) mutant structures. In the immediate vicinity of the introduced disulfide bridge the mutant structure is more mobile (or disordered) than wild type, so much so that the exact conformation of Cys 21 remains obscure. As with the previously described disulfide bridge between residues 9 and 164 of T4 lysozyme (Pjura, P.E., Matsumura, M., Wozniak, J.A., & Matthews, B.W., 1990, Biochemistry 29, 2592-2598), the engineered cross-link substantially enhances the stability of the protein without making the folded structure more rigid.


Carboxylic acid-modified polyethylene: a novel support for the covalent immobilization of polypeptides for C-terminal sequencing.

We have developed a method for the covalent immobilization of peptides, for the purpose of C-terminal sequencing, to a novel solid support, carboxylic acid-modified polyethylene (PE-COOH) film. The peptides are attached by coupling the N-terminal amino group to the activated carboxyl groups of the film. Reagents for carboxyl group activation, including 1,3-dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,3-diisopropylcarbodiimide (DICD) were compared. The best yields were obtained with DCC for a variety of tested peptides and averaged approximately 50%. The covalent attachment at pH 6.7 of peptides was shown to occur predominantly thorough the alpha-amino group for the peptide, SIGSLAK, which after attachment to the PE-COOH support permitted the C-terminal lysine residue to be sequenced in good yield, indicating that the epsilon-amino group of lysine is not covalently attached. This support offers a number of advantages over other solid supports, such as silica and polyvinylidene difluoride, for C-terminal sequencing including (1) stability to base and the high temperatures (65 degrees C) employed for C-terminal sequencing, (2) wettability with both aqueous and organic solvents, (3) a high capacity (1.6 nmol/mm2) for covalent coupling of polypeptides, and (4) easy divisibility into 1 x 5-mm pieces for use in our continuous flow reactor (CFR), which is also used for automated N-terminal sequencing (Shively, J.E., Miller, P., & Ronk, M., 1987, Anal. Biochem. 163, 517-529). Automated C-terminal sequencing on these supports is described in the companion paper (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80).


Automated carboxy-terminal sequence analysis of peptides.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-terminal sequence analysis of peptides.


The three-dimensional structure of the first EGF-like module of human factor IX: comparison with EGF and TGF-alpha.

The three-dimensional structure of the first epidermal growth factor (EGF)-like module from human factor IX has been determined in solution using two-dimensional nuclear magnetic resonance (in the absence of calcium and at pH 4.5). The structure was found to resemble closely that of EGF and the homologous transforming growth factor-alpha (TGF-alpha). Residues 60-65 form an antiparallel beta-sheet with residues 68-73. In the C-terminal subdomain a type II beta-turn is found between residues 74 and 77 and a five-residue turn is found between residues 79 and 83. Glu 78 and Leu 84 pair in an antiparallel beta-sheet conformation. In the N-terminal region a loop is found between residues 50 and 55 such that the side chains of both are positioned above the face of the beta-sheet. Residues 56-60 form a turn that leads into the first strand of the beta-sheet. Whereas the global fold closely resembles that of EGF, the N-terminal residues of the module (46-49) do not form a beta-strand but are ill-defined in the structure, probably due to the local flexibility of this region. The structure is discussed with reference to recent site-directed mutagenesis data, which have identified certain conserved residues as ligands for calcium.


Internal water molecules and H-bonding in biological macromolecules: a review of structural features with functional implications.

Conserved structural patterns of internal water molecules and/or H-bond chains were observed and are here correlated in this review, which then describes two functional properties: equilibration of hydrostatic pressure and proton transport. Available evidence in support of these hypotheses is presented, together with suggested experiments to test them. High-resolution crystal structures of a variety of proteins were studied with interactive computer graphics. Conserved H-bonding linkages may be used as a paradigm for a rationalization of proton transport in membranes. The concept of the "proton wire," which links buried active-site amino acids with the surface of the protein raises the more general question of the functional role of the various molecular components.


A detailed consideration of a principal domain of vertebrate fibrinogen and its relatives.

Vertebrate fibrinogen is a complex multidomained protein, the structure of which has been inferred mainly from electron microscopy and amino acid sequence studies. Among its most prominent features are two terminal globules, moieties that are mostly composed of the carboxyl-terminal two-thirds of the beta and gamma chains. Sequences homologous to the latter segments are found in several other animal proteins, always as the carboxyl-terminal contributions. An alignment of 15 amino acid sequences from various fibrinogens and related proteins has been used to make judgments about secondary structure. The nature of amino acids at each position in the alignment was used to distinguish alpha helices and beta structure on the one hand from loops and turns on the other, and the resulting assignments compared with predictions of secondary structure by other methods. Additionally, constraints imposed by the locations of cystines, carbohydrate attachment residues, and proteinase-sensitive points provided further insights into the general organization of the postulated secondary structures. Other ancillary data, including the effects of bound calcium and the locations of labeled or variant residues, were also considered. An intriguing similarity to a portion of the recently reported structure of a calcium-dependent lectin is noted.


Structure of the complex between trypanosomal triosephosphate isomerase and N-hydroxy-4-phosphono-butanamide: binding at the active site despite an "open" flexible loop conformation.

The structure of triosephosphate isomerase from Trypanosoma brucei complexed with the competitive inhibitor N-hydroxy-4-phosphono-butanamide was determined by X-ray crystallography to a resolution of 2.84 A. Full occupancy binding of the inhibitor is observed only at one of the active sites of the homodimeric enzyme where the flexible loop is locked in a completely open conformation by crystal contacts. There is evidence that the inhibitor also binds to the second active site of the enzyme, but with low occupancy. The hydroxamyl group of the inhibitor forms hydrogen bonds to the side chains of Asn 11, Lys 13, and His 95, whereas each of its three methylene units is involved in nonpolar interactions with the side chain of the flexible loop residue Ile 172. Interactions between the hydroxamyl and the catalytic base Glu 167 are absent. The binding of this phosphonate inhibitor exhibits three unusual features: (1) the flexible loop is open, in contrast with the binding mode observed in eight other complexes between triosephosphate isomerase and various phosphate and phosphonate compounds; (2) compared with these complexes the present structure reveals a 1.5-A shift of the anion-binding site; (3) this is the first phosphonate inhibitor that is not forced by the enzyme into an eclipsed conformation about the P-CH2 bond. The results are discussed with respect to an ongoing drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.


Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines.

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.


A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I.

The troponin I peptide N alpha-acetyl TnI (104-115) amide (TnIp) represents the minimum sequence necessary for inhibition of actomyosin ATPase activity of skeletal muscle (Talbot, J.A. & Hodges, R.S. 1981, J. Biol. Chem. 256, 2798-3802; Van Eyk, J.E. & Hodges, R.S., 1988, J. Biol. Chem. 263, 1726-1732; Van Eyk, J.E., Kay, C.M., & Hodges, R.S., 1991, Biochemistry 30, 9974-9981). In this study, we have used 1H NMR spectroscopy to compare the binding of this inhibitory TnI peptide to a synthetic peptide heterodimer representing site III and site IV of the C-terminal domain of troponin C (TnC) and to calcium-saturated skeletal TnC. The residues whose 1H NMR chemical shifts are perturbed upon TnIp binding are the same in both the site III/site IV heterodimer and TnC. These residues include F102, I104, F112, I113, I121, I149, D150, F151, and F154, which are all found in the C-terminal domain hydrophobic pocket and antiparallel beta-sheet region of the synthetic site III/site IV heterodimer and of TnC. Further, the affinity of TnIp binding to the heterodimer (Kd = 192 +/- 37 microM) was found to be similar to TnIp binding to TnC (48 +/- 18 microM [Campbell, A.P., Cachia, P.J., & Sykes, B.D., 1991, Biochem. Cell Biol. 69, 674-681]). The results indicate that binding of the inhibitory region of TnI is primarily to the C-terminal domain of TnC. The results also indicate how well the synthetic peptide heterodimer mimics the C-terminal domain of TnC in structure and functional interactions.


Automated carboxy-terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on the automation of the thiocyanate chemistry using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80). A major limitation of this approach was the need to activate the C-terminus with acetic anhydride. We now describe the use of a new reagent, diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine, which combines the activation and derivatization steps to produce peptidylthiohydantoins. Previous work by Kenner et al. (Kenner, G.W., Khorana, H.G., & Stedman, R.J., 1953, Chem. Soc. J., 673-678) with this reagent demonstrated slow kinetics. Several days were required for complete reaction. We show here that the inclusion of pyridine was found to promote the formation of C-terminal thiohydantoins by DPP-ITC resulting in complete conversion of the C-terminal amino acid to a thiohydantoin in less than 1 h. Reagents such as imidazole, triazine, and tetrazole were also found to promote the reaction with DPP-ITC as effectively as pyridine. General base catalysts, such as triethylamine, do not promote the reaction, but are required to convert the C-terminal carboxylic acid to a salt prior to the reaction with DPP-ITC and pyridine. By introducing the DPP-ITC reagent and pyridine in separate steps in an automated sequencer, we observed improved sequencing yields for amino acids normally found difficult to derivatize with acetic anhydride/TMS-ITC. This was particularly true for aspartic acid, which now can be sequenced in yields comparable to most of the other amino acids. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene and proteins (200 pmol to 5 nmol) noncovalently applied to Zitex (porous Teflon). The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids tested were found to sequence in good yield except for proline, which was found not to be capable of derivatization. In spite of this limitation, the methodology should be a valuable tool for the C-terminal sequence analysis of peptides and proteins.(ABSTRACT TRUNCATED AT 400 WORDS)


Effects of calcium on recombinant bovine chromogranin A.

Bovine chromogranin A, the acidic calcium-binding protein characteristic of endocrine secretory vesicles, has been expressed in Escherichia coli using the pET3a vector system under T7 polymerase control. The expressed protein is located in the bacterial cytosol and can be purified from bacterial proteins by a heat treatment step, followed by gel filtration, anion-exchange, and reversed-phase chromatography. The purified recombinant chromogranin A has an apparent M(r) of ca. 72,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in spite of its 432-amino acid polypeptide chain, consistent with observations on natural chromogranin A. The primary structure has been confirmed by mass spectral analysis of tryptic peptides, by Edman degradation of the intact protein, and by immunoreactivity with sequence-specific antibodies. Analysis by circular dichroism spectroscopy shows pH- and concentration-dependent spectra. The spectra are Ca2(+)-dependent from 5 to 40 microM.