Effects of physiological cycles of infused melatonin on circadian rhythmicity in pigeons. The role of the hormone melatonin in the circadian system of pigeons (Columba livia) was investigated. Using an automatic infusion system, melatonin at physiological levels was delivered for 10 h each day to cannulated, pinealectomized (P-X) pigeons in constant darkness. These cyclic infusions of melatonin entrained feeding rhythms in P-X pigeons while vehicle infusions were ineffective entraining agents. When the retinae of P-X pigeons were removed (E-X), feeding rhythms were abolished in constant darkness. When cyclic melatonin infusions were delivered to these birds (E-X and P-X), feeding rhythmicity was restored whereas vehicle infusions alone did not restore rhythmicity. When melatonin infusions were terminated in E-X/P-X pigeons, feeding rhythms persisted for several days but eventually decayed. Blood melatonin levels were measured in both P-X and E-X/P-X birds infused cyclically with exogenous melatonin and were found to be within the physiological range both in level and pattern. These results strongly suggest that endogenous melatonin, released by the pineal gland and the retinae, regulates the timing of feeding rhythms by entraining other oscillators in the circadian system of the pigeon.


The influence of tactile stimulation of nipples on the milk ejection reflex in the rat. Changes of positive pressure exerted by pups on nipples during sucking were investigated using anesthetized, lactating dams. It was found that, every 50-60 s, individual pups performed bouts of pressure oscillations (3/s) of high amplitude which lasted about 10-12 s and coincided with periods of increased motor activity. During the intervals, when pups were quiet, series of low-amplitude oscillations (3/s) were also observed. Using a strain measuring method to record the activity of sucking pups, synchronization of activity of two or more pups was found to occur periodically every 25-30 s and, most frequently, 10-30 s before the reflex increase of milk pressure. In further experiments, artificial tactile stimulation was applied to the dam's nipples using the joint action of suction and positive pressure. Following a short-term (10-20 s) increase in frequency and amplitude of artificial nipple stimulation, 60%-80% of all reflexive peaks of milk pressure were elicited with a latency of 19 +/- 5 s. This suggests that there are specific conditions under which the stimulation of nipples by pups may trigger the formation of the milk ejection reflex in the rat.


Electroantennogram of the American cockroach: effect of oxygen and an electrical model. 1. With a view to clarifying the role of oxygen in the olfactory reception of insects, the intact antenna of the male American cockroach (Periplaneta americana) was studied under normal conditions and during reversible anoxia, external or tracheal, using an electroantennogram (EAG) elicited by a sex pheromone stimulation. As a first step, the anatomical and electrical characteristics of the antenna were investigated. 2. Based on the microanatomical study, a cockroach antenna was characterized by structural parameters reflecting the haemolymph and gas exchange in the antenna. Valves in the wall of a haemolymphatic vessel were discovered. 3. The resting DC background voltage (DCBV) and the EAG were continuously recorded and proved to be more strongly affected by tracheal anoxia than by anoxia from the outside. 4. A minimal electrical model of the antenna based on the Thurm-Kaissling-De Kramer equivalent circuit of a sensillum was shown to be valid to explain the origin of the DCBV and EAG as well as their changes after oxygen cutoff and resumption. 5. Two formal opposing processes, excitation and desensitization, probably related to the receptor mechanisms, have been used to interpret the kinetics of the EAG and the effect of anoxia on EAG parameters. The desensitization is thought to be more sensitive to oxygen lack than the excitation.


Programmed cell death in whole body and organ systems by low dose radiation. New whole-body and organ systems were established to detect interphase cell death in the thymus, spleen and epithelial cells of intestinal crypts by low-dose radiation. Frozen sections of the thymus, spleen and intestine as thick as 8 microns were made after X-irradiation of whole body or removed organs, and then sections were stained with 0.02% erythrosin B solution. In unirradiated controls, a few numbers of erythrosin B positive cells (dead or dying cells) were observed in the thymus, spleen and intestinal crypt as a single cell death. When X rays were given to various strains of mice as a whole body dose, clusters of erythrosin B positive cells were produced. They appear at 2 hr after irradiation and reached maximum at 4 hr, remaining at a similar level until 8 hr after irradiation. The number of erythrosin B positive cells decreased after then by the elimination of dead cells, and they were observed like a single cell death at 24 hr after irradiation. When erythrosin B positive cells were scored 4 hr after irradiation, their total number and the number of cluster increased with increasing doses of X rays in the dose range from 0.05 to 0.5 Gy. It is noted that there were large differences in the radiation susceptibility among the inbred strains of mice for the induction of interphase cell death of thymic lymphocytes: e.g., high susceptibility in C57BL/6J and AKR/J, intermediate in N4, A/J, PT and ST, low in C3H/HeJ, HT, 101/H and DBA/2J, indicating that interphase cell death is genetically programmed. Similar results were observed with some chemical mutagens. Although a large increase of erythrosin B positive cells was observed in the thymus and spleen with methylprednisolone, there was no increase in the intestinal crypt, and vice versa with bleomycin, suggesting the organ specificity for the induction of interphase cell death by chemicals. For the in vitro method, the removed thymus was irradiated on the agar plate, and then incubated on the agar plate which was placed on the grid in the medium, so that the medium comes up to the organ through the agar plate. Frozen sections were made and stained with erythrosin B solution in the same way as the in vivo method. The number of erythrosin B positive cells in the organ culture system reached maximum at 5 hr after X-irradiation, e.g. slightly later than in the whole-body system. The efficiency was about 60% in C57BL/6J mice when compared with whole-body system.


The PSI automatic metaphase finder.

The PSI automatic metaphase finder. The Genetiscanner automatic metaphase finder (AMF) instrument manufactured by Perceptive Scientific Instruments, Inc. is capable of searching a microscope slide and generating a list of the stage coordinates of the metaphases within the 20 mm x 40 mm search area in ten minutes. It can process a cassette load of sixty slides in approximately ten hours, ranking the metaphases according to six categories of quality. It uses a robot arm to load and unload slides from a 60-slide cassette onto an automated microscope stage. It searches at approximately 1.5 sq. mm per second and uses automatic focus to keep the image sharp during the search. On good quality blood slides it detects greater than 80% of the metaphases with greater than 80% ranking accuracy and less than 20% false positives. It can also search amniotic fluid, bone marrow and mammalian cell culture metaphase preparations. The pattern recognition algorithms use classifiers specifically trained for each specimen type, and for the rejection of non-metaphase material. Such units have been in continuous operation for up to two years with good performance and reliability.


A high resolution chromosome image processor for study purposes, NIRS-1000:CHROMO STUDY, and algorithm developing to classify radiation induced aberrations. Since 1989 we have promoted a project to develop an automated scoring system of radiation induced chromosome aberrations. As a first step, a high resolution image processing system for study purposes, NIRS-1000:CHROMO STUDY, has been developed. It is composed of: (1) CHROMO MARKER whose main purpose is to mark on images to make image data base, (2) CHROMO ALGO whose purpose is algorithm development, and (3) METAPHASE RANKER whose purposes are metaphase finding and ranking with a high power objective lens. However, METAPHASE RANKER is presently under development. The system utilizes a high definition video system so as to realize the best spatial resolution that is achievable with an optical microscope using an objective lens (x 100, numerical aperture 1.4). The video camera has 1024 effective scan lines to realize 0.1 microns sampling on a specimen. The system resolution achieved on the hard copy is less than 0.3 microns on a specimen. A preliminary algorithm has been developed to classify the aberrations on the system using projection information of gray level. The preliminary test results on excellent 10 metaphases show that the correct classification ratio is 92.7%, that the detection rate of the aberrations is 83.3% and that the false positive rate is 6.1%.


Automatic assessment of the quality of G-banded metaphases. A preliminary study. Assessment of the quality of cytogenetic preparations is important for 1) a general quality control of the cytogenetic laboratory and 2) for determining the quality of individual cytogenetic analyses. It is generally accepted that good preparations allow a more detailed search for structural abnormalities than poor preparations. As part of a comprehensive study on automated assessment of slide quality we have developed a simple algorithm for automated measurement of metaphase resolution. During semi-automated karyotyping with the Magiscan chromosome analysis system (Joyce-Loebl) three measurements related to resolution are automatically extracted 1) the total number of dark bands of the chromosomes of the metaphase (TB), 2) the normalized average length of the chromosomes (NL), and 3) the average "thickness" of the chromosomes (T). The algorithm TB x NL/T has been tested on 35 metaphases of various quality and compared with visual assessment of the same 35 metaphases. The results indicate that the automatic assessment of metaphase resolution is superior to the visual.


Adaptive classifiers for dicentric chromosomes. Classification of dicentric chromosomes in a practical automatic screening system comprises three stages. The first generates plausible centromere candidates from each chromosome in an automatically segmented metaphase, and uses contextual knowledge to generate distributions of "probably true" and "probably false" centromeres, thus adapting to the conditions within a particular metaphase. The second stage classifier uses these distributions to re-classify the candidates as centromeres or non-centromeres. From this classification, likely dicentrics are found by counting centromeres; a third classifier attempts to reject false positives among the likely dicentric chromosomes, by comparing the feature values of the proposed centromeres of a chromosome and rejecting chromosomes for which these values do not satisfy certain similarity criteria. The second stage classifier may be a simple box classifier, or may use a variety of parametric Bayesian methods. The performance of these alternatives has been tested both on reference data sets comprising about 600 metaphases, and on larger data sets when embedded in a practical fully automatic dicentric pre-screening system. When operating parameters were such that a similar number of true positives were found by both classifiers, the Bayesian classifier produced about half as many false positive errors as the box classifier, with the final false positive rate being in the region of one candidate dicentric chromosome in every four cells.


Chromosome analysis by image processing in a computerized environment. Clinical applications. Dealing with a routine regional cytogenetic activity, we have developed and adapted to clinical work a semi automatic karyotyping machine. Attempts for an accurate automated chromosome classification using a neural network have led to partial results. A specific adaptation to cancer cytogenetics is under development (determination of the modal number, translocations analysis with densitometric curves, automatic identification of markers). A specific program allows quantification of chromosome labelling with radioactive probes. Exchanges of digitized karyotypes are feasible with labs using automated karyotyping machines. A local network connects several karyotyping and metaphase finding stations. Guidelines for an international data bank concerning abnormal chromosome images have been elaborated. On the other hand the ISH techniques have been applied to the following topics: identification of human chromosome aberrations in amniotic and chorionic cells, chromosome studies of human gametes and embryos (including sex determination), identification of markers in cancer cells.


Automated detection of radiation-induced chromosome aberrations following fluorescence in situ hybridization. The cytogenetic detection of rare chromosome aberrations induced by ionizing radiation requires the evaluation of large numbers of cells. In this report, an image analysis program based on thresholding of grey level histograms is described for a rapid automated detection of chromosome translocations in metaphase spreads from human lymphocytes following irradiation and chromosome in situ suppression (CISS) fluorescence in situ hybridization (FISH). To classify a metaphase spread as "normal", "aberrant", or "excluded", a minimum medium time of less than two seconds was required on a general purpose Personal Computer. Using appropriately stained specimen, for the false classification rate an upper limit of about 10% was estimated. The upper limit for the rate of "excluded" cells was estimated to be about 11% (99% confidence ranges). FISH-procedures allow to score chromosome aberrations also in the interphase nucleus. To apply simple threshold algorithms for segmentation of the FISH-stained nuclear areas, it is required that the grey level contrast is sufficiently high. The preliminary results presented here on the image analysis of human lymphocyte nuclei suggest the feasibility of such an approach.


Factors that determine the in vivo dose-response relationship for stable chromosome aberrations in A-bomb survivors. An overview is given of the dose-response relationship for stable chromosome aberrations (i.e., translocations and inversions) in the peripheral blood lymphocytes of A-bomb survivors in Hiroshima. Special emphasis is placed on (i) the overdispersion of survivor cases with either unexpectedly high or low aberration frequencies relative to the estimated DS86 kerma values assigned to individual survivors, termed "cytogenetic outliers", and (ii) the correlation of chromosome aberration frequencies with other biological endpoints, such as acute radiation symptoms (severe epilation). A new molecular biological technique, known as fluorescence in situ hybridization (FISH) with composite, whole-chromosome probes to paint differentially the target chromosomes, has facilitated rapid, efficient, and extensive scoring of translocation-type chromosome aberrations in which the target chromosomes are involved. Using this methodology, the observed findings on translocation frequencies in A-bomb survivors have shown that the frequency of stable chromosome aberrations, which have persisted for years without change in frequency in irradiated persons, is indeed useful as an indicator for biological dosimetry.


A preliminary evaluation of the Edinburgh dicentric hunter. Computer programs have recently been developed to enable automatic detection of dicentric chromosomes on microscope slides. Theoretical calculations based on the probable limits of resolution of such a system suggested that about two thirds of dicentrics might be detectable. We present here some preliminary results from an ongoing evaluation of an automatic dicentric hunter using slides of lymphocyte preparations which have also been examined by eye. The data indicate an efficiency approaching 40% in dicentric detection.


Combined use of several mitogens for mitotic stimulation to human lymphocytes. Several different type of mitogens, such as phytohemagglutinin (PHA) including PHA-P, -M, -W, -C, -L and -E+L, pokeweed mitogen (PWM), Concanavalin A (Con-A), lipopolysaccharide (LPS), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) were compared their mitotic stimulating effects to human lymphocytes in single use and also combined use of these mitogens. In the tests with single use of each mitogen, PHA was confirmed to be the most superior mitogen. Of several different types of PHA, PHA-C, -L and -E+L were not appropriate to stimulate human lymphocytes, though PHA-P, -M and -W induced sufficiently mitoses. The most appropriate concentration of PWM was 50 micrograms/ml, but the highest mitotic index reached at 5 days after the initiation of culture. Con-A did not induce sufficient mitoses, and LPS, WGA and SBA were inadequate to stimulate human lymphocytes in the single use. In the combined use of two mitogens, there was no reliable combination to enhance mitotic stimulation. When three different mitogens were used simultaneously, only one combination with PHA-M, PWM and SBA effectively enhanced the mitotic index when compared to the single use. In the confirmatory experiment for this combination carried out on 5 different donors, the enhanced effect to mitotic index was recognized in two out of three donors with low response to PHA, but the low level of mitotic indices was seen in remaining 2 donors with high response to PHA. This finding suggests that this combination may overcome a disadvantage in some of the low responders to PHA.


Robot system for preparing lymphocyte chromosome. Towards the automatization of the scoring of chromosome aberrations in radiation dosimetry with the emphasis on the improvement of biological preparations, the conventional culture and harvesting method was modified. Based on this modified method, a culture and harvest robotic system (CHROSY) for system (CHROSY) for preparing lymphocyte chromosome was developed. The targeted points of the modification are as in the following. 1. Starting culture with purified lymphocytes in a fixed cell number. 2. Avoiding the loss of cells in changing the liquids following centrifugalization. 3. Keeping the quantity of the liquids to be applied to the treatments of cells fixed. 4. Building a system even a beginner can handle. System features are as follows. 1. Operation system: Handling robot having 5 degrees of freedom; a rotator incubator with an automatic sliding door; units for setting and removing pipette tips; a centrifuge equipped with a position adjuster and an automatic sliding door; two aluminum block baths; two nozzles as pipettes and aspirators connected to air pumps; a capping unit with a nozzle for CO2 gas; a compressor; and an air manipulated syringe. 2. Control system: NEC PC-9801RX21 with CRT; and program written in Basic and Assembly languages on MS-DOS. It took this system 2 hours and 25 minutes to harvest 2 cultures. A fairly good chromosome slide was made from the sample harvested by CHROSY automatically.


Development of an instrument for chromosome slide preparation. For cytogenetic analysis of chromosome abnormalities, we have developed an instrument which can produce semi-automatically chromosome slides for microscopic observation. This instrument has automatic slide supplier and slide transfer system. Chromosome slides were obtained by the steam dry method; slides were sequentially exposed to different atmosphere controlled strictly in respect of temperature and humidity. Slides with well-spread chromosomes were obtained constantly in quality and rapidly in speed of 5 slides per minute. Precise controls of room temperature and humidity were attained by the use of 26 thermomodules. Accuracy of the controls was excellently high, showing a fluctuation of +/- 0.5 degrees C in temperature and +/- 3% in humidity for any setting in the use range. This instrument was proved to be very useful for rapid obtaining of a large number of chromosome slides with highly qualified metaphase spreads. This instrument will be utilized to assist the automation analysis system of chromosome abnormalities. Mechanism of chromosome spreading, problems in actual use, and future improvements were discussed.


A neural network chromosome classifier.

A neural network chromosome classifier. We present a chromosome classifier for automated karyotyping of banded chromosomes which uses a multi-layer perception neural network. Two network configurations have been investigated. The resulting classifiers have been trained and tested on data from three different data sets covering a range of data quality. Classification results compare very favourably with those obtained using a highly optimised parametric classifier.


Biomedical rationale for cytogenetic dosimetry. The purpose of this presentation is to initiate the discussions of automated analysis of radiation-induced chromosome aberrations with a consideration of the possible biomedical benefits to be achieved by its successful accomplishment. Biological indicators of radiation effect are considered. The biomedical indications for and usefulness of cytogenetic dosimetry are described, based on past experiences and potential applications of the developing technology. The implications of this methodology for both individual and population risk assessment are discussed. Both past experiences and potential applications are considered, with examples, including clinically significant radiation accidents involving radiation workers and members of the public; public health applications in the screening and monitoring of actually and potentially exposed population groups; occupational surveillance of special worker subsets and of the general radiation worker population; and scientific usage in epidemiologic studies and laboratory investigations.


Cytogenetic biomonitoring of human radiation exposures: possibilities, problems and pitfalls. The use of chromosome aberration analysis has progressed and is now generally recognized to provide a useful means for the assessment of dose and possible health consequences in human exposures to relatively high doses of ionizing radiations. The automated analysis of radiation-induced chromosome aberrations will facilitate the better understanding of the health effects of radiations on human populations, particularly of those at low levels. However, the reaction kinetics of chromosome aberrations in the low dose or low dose-rate exposures are not well delineated. In this paper, possible reaction kinetics of chromosome aberrations, particularly those in human lymphocyte, in low dose-rate exposures have been discussed based on the cytogenetic data on the Thorotrast patients, persons involved in the protracted occupational exposures and modulation of chromosome aberrations by radioadaptive response. In the protracted low dose-rate exposures, the levels of chromosome aberrations, whether they are stable- or unstable-type aberrations, are not in a simple function of the total accumulated dose due mainly to the lymphocyte kinetics and modulation by cellular stress response. If this formalism is realistic, the dissociation between levels of chromosome aberrations and cancer risks may be large for the low level protracted exposures.


Recent advances in fluorescence in situ hybridization. Fluorescence in situ hybridization (FISH) procedures that directly couple molecular and cytological information allow precise visualization of DNA sequences on metaphase chromosomes and interphase nuclei. These techniques can be used to identify chromosomes, detect chromosomal aberrations, and analyze linear and spatial genome organization. FISH procedures are also used to clinical fields for diagnosis of disease-related chromosome changes and tumor biology.


Development of a biological dosimeter for translocation scoring based on two-color fluorescence in situ hybridization of chromosome subsets. Recently fluorescence in situ hybridization protocols have been developed which allow the painting of individual chromosomes using DNA-libraries from sorted human chromosomes. This approach has the particular advantage that radiation induced chromosome translocations can be easily detected, if chromosomes of distinctly different colors take part in the translocation event. To enhance the sensitivity of this approach two metaphase chromosome subsets A and B (A: chromosomes 1, 2, 4, 8, 16; B: 3, 5, 9, 10, 13) were simultaneously painted in green and red color. Counterstaining of the chromosomes with DAPI resulted in a third subset which exhibited blue fluorescence only. Green-red, green-blue and red-blue translocation chromosomes could be easily detected after irradiation of lymphocyte cultures with 137Cs-gamma-rays. Analyses of painted chromosomes can be combined with conventional GTG-banding analyses. This new biological dosimeter should become useful to monitor both long term effects of single irradiation events and the cumulative effects of multiple or chronic irradiation exposures. In contrast to translocation scoring based on the analysis of banded chromosomes, this new approach has the particular advantage that a rapid, automated scoring of translocations can now be envisaged.


A quantitative analysis of radiation-induced chromosome aberrations with a fluorescent digital image microscope. Using a synthetic, alpha satellite consensus DNA unit as a probe, we could show dicentrics as well as acentric fragmented chromosomes in metaphases of gamma-irradiated lymphocyte cells and that the number of appearance of dicentrics or acentric fragments seemed to be proportional to radiation doses. To make such examination with a large number of chromosomes of metaphases, a quantitative fluorescence measurement was performed using a fluorescent microscope digital image analysis system. The relative amounts of fluoresceinated probe hybridized to alpha satellite DNA varied with chromosomes to a certain extent (1-4% of total probe-fluorescence of one metaphase). However, we could score dicentrics and acentric fragments as dots with extraordinarily higher or low percentage in plots of relative amount of probe-fluorescence on metaphase chromosome in gamma-irradiated cells. The number of appearance of acentric fragments were proportional to radiation doses of 1, 2 and 4 Gy. In the case of dicentrics, the number of appearance seemed to be proportional to square doses. Further, to reduce the variation of relative amounts of probe-fluorescence, we tried to make probe-fluorescence reflect the content of alpha satellite DNA on each chromosome more exactly, using a DNA probe amplified by polymerase chain reaction.


Radiation-induced chromosome damage in G1 phase cells as breaks in premature chromosome condensation (PCC) and its biological meaning. We studied radiation-induced chromosome fragmentation in the G1-phase of Syrian golden hamster embryo (SHE) cells by premature chromosome condensation (PCC). SHE cells were irradiated with 14N ions (95 MeV) and 4He ions (22 MeV) generated by the cyclotron and 137Cs gamma rays. The number of chromosome breaks in PCC (PCC break) induced by 14N ions was higher than in those by the others. High linear energy transfer (LET) radiation (heavy ions) is more effective in the induction of PCC breaks than low LET radiation (gamma-rays). 530 kev/microns 14N induced PCC 2.4 times more than 137Cs gamma rays. There is an inverse relationship between the number of chromatid deletions and the incidence of mutation or transformation. There is, however, a good correlation between the incidence of PCC breaks and frequencies of mutation and/or morphological transformation. Although over 95% of the PCC breaks induced by gamma-rays were rejoined within 8 hours of post-irradiation incubation, only 35 to 45% of PCC breaks induced by heavy ions were rejoined. These results suggest that there is a qualitative difference in the initial chromosome damage caused by heavy ions and gamma rays.


Resolution and characterization of polymorphic DNA by SSCP and chemical cleavage methodologies. A variety of techniques have been developed to detect single-base changes for the two different purposes. One is the detection of mutational events without phenotypic selection, and another is the rapid and conventional identification of mutations such as the specific base changes related to activation of oncogene, genetic diseases, etc. In this study, the utility of the two methods, single strand conformation polymorphism (SSCP) and chemical cleavage, was explored using 13 E. coli lacI- mutations cloned onto M13 phase. The 167 base region encompassing mutations was amplified by PCR as dsDNA. Following denaturation, these PCR products were analyzed by non-denaturing polyacrylamide gel electrophoresis (SSCP) and the separation of the ssDNA fragment carrying the altered sequence from the original sequence was found to be dependent on the location and type of the change. Hetero duplexes of changed/original sequences were also prepared by hybridization of the above PCR products. Mismatched C and T bases were modified by hydroxylamine and osmium tetroxide, respectively, and subsequently treated with piperidine to analyze the cleaved DNA fragments on a polyacrylamide gel (Chemical Cleavage). The cleavage efficiency was also found to be influenced by the type of mismatch and its surrounding sequence. Such observed characteristics should contribute to a better appreciation for these types of mutational systems, which in turn should lead to insight into the mechanisms of mutagenesis.


Species- and subtype-specific recognition by antibody WF6 of a sequence segment forming an alpha-bungarotoxin binding site on the nicotinic acetylcholine receptor alpha subunit. The monoclonal antibody WF6 competes with acetylcholine and alpha-bungarotoxin (alpha-BGT) for binding to the Torpedo nicotinic acetylcholine receptor (nAChR) alpha 1 subunit. Using synthetic peptides corresponding to the complete Torpedo nAChR alpha 1 subunit, we previously mapped a continuous epitope recognized by WF6, and the prototope for alpha-BGT, to the sequence segment alpha 1(181-200). Single amino acid substitution analogs have been used as an initial approach to determine the critical amino acids for WF6 and alpha-BGT binding. In the present study, we continue our analysis of the structural features of the WF6 epitope by comparing its cross-reactivity with synthetic peptides corresponding to the alpha 1 subunits from the muscle nAChRs of different species, the rat brain alpha 2, alpha 3, alpha 4 and alpha 5 nAChR subtypes, and the chick brain alpha-BGT binding protein subunits, alpha BGTBP alpha 1 and alpha BGTBP alpha 2. Our results indicate that WF6 is able to cross-react with the muscle alpha 1 subunits of different species by virtue of conservation of several critical amino acid residues between positions 190-198 of the alpha 1 subunit. These studies further define the essential structural features of the sequence segment alpha 1(181-200) required to form the epitope for WF6.


Scintillation proximity assay to study the interaction of epidermal growth factor with its receptor. Scintillation Proximity Assay (SPA), which does not require the physical separation of receptor bound and free ligand, was applied to study the interaction of Epidermal Growth Factor (EGF) with its receptor (EGFR) in membrane preparations from human placenta. Fluomicrospheres to which the monoclonal anti-EGFR antibody R1 was coupled, were used. Kinetic binding data of the association of 125I-labeled EGF binding to the receptor at 20 degrees C could be fitted according to a double exponential model, which is consistent with the presence of fast and slow associating EGF binding sites. Dissociation kinetics revealed that perturbation of equilibrium conditions rapidly occurs upon washing. Multiple point Scatchard analysis of equilibrium 125I-labeled EGF binding data revealed curvilinearity, indicating the presence of both high and low affinity EGF binding sites. We conclude that SPA is an interesting new tool in the exploration of the interaction of ligands with their receptors, which allows detailed ligand-receptor studies under precise in situ conditions.


Early amniocentesis.

Early amniocentesis. Early amniocentesis (EA) refers to any genetic amniocentesis performed prior to 15 weeks, 0 days' gestation. Because of technical problems, most practitioners stopped doing amniocenteses prior to 15 weeks' gestation in the late 1970s. The development of ultrasound-monitored needle insertion has allowed improved success and safety of sampling at earlier gestations, and improvements in laboratory techniques have allowed a high degree of success in culturing and processing low-volume samples. We present information on 1,805 early amniocenteses, of which 35% were performed prior to 14 weeks, 0 days' gestation. Our data indicate that with operator experience the success rate of the procedure can be similar to that seen with routine amniocentesis and that laboratory experience is not different from that with routine amniocentesis (RA) as measured by culture success, time to completion and accuracy. Pregnancy outcome, as measured by the gestational age at delivery, mode of delivery, birth weight and Apgar score, is indistinguishable between RA and EA patients. Although a randomized study is needed to accurately evaluate the fetal loss rate, the rate of birth defects and incidence of neonatal complications associated with EA procedures and examination of the crude rates for the above three parameters show that they compare favorably to the rates reported for patients who have undergone chorionic villus sampling or RA.


Fetal cells in the maternal circulation. The analysis of fetal cells from the maternal circulation would be the least invasive method of prenatal diagnosis. Potential fetal cell types to enter the maternal circulation are lymphocytes, trophoblast cells and nucleated erythrocytes. With conventional methods, such as cytology and interphase or metaphase cytogenetics, the ratio of fetal to maternal cells was overestimated in the past. Currently most groups use polymerase chain reaction-based Y-sequence analysis for the detection of fetal cells in pregnancies with male fetuses, either with or without prior enrichment of fetal cells. For fetal cell separation, fluorescence-activated cell sorting and immunomagnetic beads have been applied, and recently our group has used discontinuous density gradient centrifugation for this purpose. We have shown that the transferrin receptor antigen alone is not sufficient for enrichment of fetal nucleated erythrocytes. Despite some initial promising results with fluorescence in situ hybridization, the reproducibility and reliability of the techniques are still limited, mainly due to the lack of very specific cell markers and the very low and variable concentrations of fetal cells among numerous maternal cells.


Ultrasound detection of genetic anomalies. The prenatal detection of genetic diseases has improved remarkably. This review highlights several diagnoses that are possible to identify by ultrasound examination of the fetus, including cystic fibrosis, skeletal dysplasias, kidney diseases, cardiac disease and central nervous system disorders. Ultrasound clues to the possible existence of several syndromes and aneuploidy are also outlined.


Preimplantation genetic analysis.

Preimplantation genetic analysis. Advanced reproductive technologies have afforded us access to human preembryos, allowing the possibility of preimplantation genetic diagnosis of certain genetic diseases. This paper thoroughly reviews both the micro-manipulation and molecular biologic aspects of this new and exciting branch of medicine. In addition, some of the moral and ethical considerations relating to preembryo genetic diagnosis are discussed.


Molecular diagnosis of genetic disease.

Molecular diagnosis of genetic disease. DNA-based testing is becoming possible for an increasing number of hereditary diseases as the responsible genes are mapped to individual chromosomes and then isolated and characterized. The strategy for each test depends on the heterogeneity of mutations commonly causing the disease, the distribution of the mutations in the population and the frequency of new mutations. In many cases, interpretation of the test result requires a comparison with relatives known to carry the abnormal gene. Sickle cell anemia is an example of a recessive mutation with a strong ethnic association and little genetic heterogeneity or new mutation. Cystic fibrosis also has a strong ethnic association and a low frequency of new mutation but greater heterogeneity. Fragile X affects all ethnic groups and often appears sporadic, but all cases have the same type of mutation, and a premutation can be found in all affected families. In the near future, the extreme sensitivity of DNA analysis will allow testing for these and similar diseases to be performed in vitro on fertilized embryos before implantation.


Third-trimester uterine rupture after prostaglandin E2 use for labor induction. Prostaglandin E2 is a powerful oxytoxic agent that reliably initiates labor, even in the presence of an unripened cervix. The very low incidence of obstetric and neonatal side effects contributes to its universal use. Only nine cases of uterine rupture during the third trimester of pregnancy after application of various prostaglandin E2 preparations have been reported in English. Although uterine rupture after prostaglandin administration is a very rare complication, no prostaglandin compound seems to be exempt from it.


Vaginal birth after cesarean section in rural Zaire. Changing concepts. The safety of trials of labor after cesarean section has yet to be established in developing countries. Centre Médical Evangélique is a referral hospital in rural northeastern Zaire. From August 1, 1989, to January 15, 1990, 33 women with previous cesarean deliveries were offered a trial of labor. Of them, 22 (67%) had successful vaginal deliveries. The indications for the previous cesarean section did not influence the outcome of the trial of labor. There was a high rate of immediate maternal morbidity but no significant long-term morbidity. The rate of uterine dehiscence was 9.1%. The perinatal death rate, 60.1/1,000, was similar to the overall rate for the institution. Trials of labor after cesarean section are a valid therapeutic option in developing countries.


Eclampsia complicating a pregnancy with neurofibromatosis. A case report. Neurofibromatosis occurs in approximately 1 in 3,000 births and is known to be associated with the development and/or worsening of hypertension in pregnancy. A woman was treated for eclampsia with neurofibromatosis and had a complicated course.


Neisseria sicca endocarditis complicating pregnancy. A case report. Neisseria sicca, a commensal organism of the oropharynx, has rarely been implicated as a pathogen responsible for causing bacterial endocarditis. A pregnant woman developed N sicca endocarditis at 32 weeks' gestation. Although there was an initial delay in diagnosis and therapy, the maternal and fetal outcomes were excellent.


Cervical pregnancy. A case report.

Cervical pregnancy. A case report. A cervical pregnancy was treated successfully with systemic methotrexate and folic acid. Serial beta-human chorionic gonadotropin levels, color Doppler velocimetry and hysteroscopy were used to monitor therapy. In view of the substantial morbidity associated with this form of ectopic pregnancy, medical management is an option for treatment.


Mapping the binding site of tissue kallikrein: preparation and testing of all possible substrate analog inhibitors homologous with the sequence of kininogen between Ser386 and Gln392. Programs aimed at converting peptide inhibitors of proteolytic enzymes into more traditional drug structures require an understanding of the role played by the individual amino acid residues in the inhibitor. To this end, all possible substrate analogues occurring within the sequence Ser386-Pro-Phe-Arg-Ser-Val-Gln392 from bovine kininogen were synthesized and tested as inhibitors of tissue kallikrein (EC 3.4.21.35, beta-PPK). Of the 21 sequences which can be formed from the heptapeptide, 11 have inhibitory constants which could be measured in the chromogenic assay employed in these studies. No dipeptide and only one tripeptide, Ac-Phe-Arg-Ser-NH2 (Ki = 718 microM), measurably inhibits the enzyme. All longer peptides inhibit beta-PPK. The heptapeptide Ac-Ser-Pro-Phe-Arg-Ser-Val-Gln-NH2 is the most effective inhibitor in this series (Ki = 101 microM). Each amino acid residue in the sequence appears to alter binding in a relatively independent manner. The N-terminal seryl residue (P4) and the prolyl residue (P3) slightly improve the Ki of the various inhibitors. The phenylalanyl residue at P2 appears to have a more pronounced effect on Ki. The arginyl residue at P1 and the seryl residue at P1' appear to be the most important residues in the inhibitory sequence. They contribute approximately one-third and one-fourth of the binding energy to the interaction between the substrate analogues and beta-PPK, respectively. The valyl residue at P2', and the C-terminal glutaminyl residue improve Ki of each of the peptides tested. Almost 80% of the binding energy of the substrate analogue inhibitors comes from the core sequence Phe-Arg-Ser which occurs between P2 and P1'. Molecular models developed from the Chen-Bode coordinates of the aprotinin-beta-PPK complex have been used to interpret the results of these studies.


Synthesis and pharmacological evaluation of enantiomerically pure 4-deoxy-4-fluoromuscarines. Four isomers of [(4-fluoro-5-methyl-tetrahydrofuran-2-yl)methyl]trimethylammonium iodide (4-deoxy-4-fluoro-muscarines) were prepared in enantiomerically and diastereomerically pure form from (S)-(-)-methyl 4-methylphenyl sulfoxide, ethyl fluoroacetate, and allyl bromide. Their absolute configurations were assigned by 1H NMR analyses. The four optically pure compounds were tested in vitro on guinea pig and their muscarinic potency was evaluated at M3 (ileum and bladder) and M2 (heart) muscarinic receptor subtypes. Compound 1a, the most potent isomer of the series, was also tested in vivo on pithed rat and its muscarinic activity at the M1 receptor subtype was compared with that of muscarine. Moreover, affinity and relative efficacy were calculated in vitro for this compound at M2 (heart force and rate) and M3 (ileum and bladder) receptors in order to investigate muscarinic receptor heterogeneity. The 4-deoxy-4-fluoromuscarines display a similar trend of potency as the corresponding muscarines and compound 1a shows differences in the affinity constants among the studied tissues. Replacement of a hydroxyl group for a fluorine atom in the 4 position of muscarine produces 1 order of magnitude increase in affinity for cardiac M2 muscarinic receptors controlling rate, while the affinity at cardiac M2 muscarinic receptors controlling force is unchanged, opening the possibility of a further classification of cardiac muscarinic receptors.


1,4-Dihydropyridines as antagonists of platelet activating factor. 1. Synthesis and structure-activity relationships of 2-(4-heterocyclyl)phenyl derivatives. A novel class of 2-(4-heterocyclylphenyl)-1,4-dihydropyridines (2-38) possessing antagonist activity against platelet activating factor (PAF) was prepared by the Hantzsch synthesis from a variety of ethyl 4'-heterocyclic-substituted benzoylacetates, aryl or heteroaryl aldehydes, and substituted 3-aminocrotonamides or 3-aminocrotonate esters. Structure-activity relationships were evaluated where PAF antagonist activity was measured in vitro by determining the concentration of compound (IC50) required to inhibit the PAF-induced aggregation of rabbit washed platelets, and in vivo by determining the oral dose (ED50) which protected mice from a lethal injection of PAF. The nature of the substituent at the dihydropyridine 2-position was found to be important for both in vitro and in vivo activity, whereas there was greater flexibility for structural variation at the 4- and 5-positions. The most potent compound was 4-(2-chlorophenyl)-1,4-dihydro-3-(ethoxycarbonyl)-6-methyl-2-[4-(2- methylimidazo[4,5-c]pyrid-1-yl)phenyl]-5-[N-(2- pyridyl)carbamoyl]pyridine (17, UK-74,505), IC50 = 4.3 nM, ED50 = 0.26 mg/kg po, which was found to be approximately 33 times more potent in vitro (rabbit platelet aggregation) and about 8 times more potent in vivo (murine lethality) than WEB2086. Compound 17 also exhibited a long duration of action in the dog (inhibition of PAF-induced whole blood aggregation ex vivo was maintained for greater than 24 h following a single oral dose of 75 micrograms/kg) and was highly selective as a PAF antagonist, showing only weak affinity (IC50 = 6600 nM) for the [3H]nitrendipine binding site. As a result of its high oral potency, selectivity, and duration of action, UK-74,505 has been selected for clinical evaluation.


Syntheses and affinities of novel organometallic-labeled estradiol derivatives: a structure-affinity relationship. Two series of novel estradiol derivatives, including cationic species, labeled with organometallic fragments Cr(CO)3, Cp*Ru+, or Cp*Rh2+ [Cp* = eta 5-C5(CH3)5] either in the 17 alpha-position or on the A-ring were synthesized, and their relative binding affinities (RBA) for the estradiol receptor were determined. The Ru(II) and the Rh(III) cationic derivatives were obtained as stable salts with the following counter anions (BF4-, PF6-, CF3SO3-). The satisfactory RBA values obtained for most complexes belonging to the 17 alpha series confirm that this position tolerates the presence of bulky neutral species. For instance, complex 4, in which the organometallic fragment Cr(CO)3 was attached to the phenyl ring of the 17 alpha-phenylethynyl fragment, exhibited an RBA value of 24%, very similar to that of the uncomplexed estrogen derivative 3. Surprisingly, the analogous cationic species 6 had no affinity for the estradiol receptor. This unprecedented result shows that the hormone binding site of the estrogen receptor does not tolerate the presence of a positive charge in the 17 alpha-position of the steroid. On the other hand, the alpha-face of the A-ring of estradiol did tolerate positively charged organometallic fragments bearing bulky substituents although the RBA value tended to decrease with increasing charge. The counterion in these cationic derivatives also affected binding affinity. For instance, the Ru(II) species 7a containing an CF3SO3- ion exhibited a reasonable RBA value (5.8%) compared to analogous species 13 with a PF6- ion (RBA of only 0.1%). Moreover, the triflate counteranion preserved the phenolic form of the A-ring of the estrogen derivative whereas the PF6- derivative was unstable and rapidly converted into the dienonylic form in buffer. The compared RBAs of the neutral and cationic species illustrate the preferences of the receptor hormone binding site in accepting or rejecting species of hydrophobic or hydrophilic character.


Synthetic nonpsychotropic cannabinoids with potent antiinflammatory, analgesic, and leukocyte antiadhesion activities. Two strategies for the design of therapeutically useful cannabinoids have been combined to produce compounds with greatly increased antiinflammatory activity and with a low potential for adverse side effects. Enantiomeric cannabinoids with a carboxylic acid group at position 7 and with an elongated and branched alkyl sidechain at position 5' have been synthesized and tested for antiinflammatory activity. They were effective when given orally at doses of 10 micrograms/kg in reducing paw edema in mice that had been induced by either arachidonic acid or platelet activating factor. Leukocyte adhesion to culture dishes was also reduced in peritoneal cells from mice in which the cannabinoids were orally administered in the same dose range as for the paw edema tests. Antinociception could be observed in the mouse hot plate assay; however, little stereochemical preference was seen in contrast to the above tests where the 3R,4R compounds are more active than the 3S,4S enantiomers. Finally, in agreement with earlier reports on the naturally occurring pentyl side chain acids, the synthetic acids showed little activity in producing catalepsy in the mouse, suggesting that they would be nonpsychotropic in humans.


Synthesis and cholinergic properties of N-aryl-2-[[[5-[(dimethylamino)methyl]-2-furanyl]methyl]thio]ethylamino analogs of ranitidine. A series of N-aryl-2-[[[5-[(dimethylamino)methyl]-2- furanyl]methyl]thio]ethylamino analogs of the H2-antagonist, ranitidine, was synthesized and the abilities of the compounds to alleviate the cholinergic deficit characteristic of Alzheimer's disease evaluated. The compounds were initially tested for their ability to inhibit human erythrocyte acetylcholinesterase activity in vitro. Selected compounds were further evaluated for butyrylcholinesterase inhibition, M1 and M2 cholinergic receptor binding, potentiation of ileal contractions, and the ability to elevate brain acetylcholine levels in mice. The analogs were compared to tetrahydroaminoacridine and to a recently reported series of bis-[[(dimethylamino)methyl]furans]. The N-aryl-2-[[[5-[(dimethylamino)methyl]-2- furanyl]methyl]thio]ethylamine derivatives were generally comparable to tetrahydroaminoacridine and the bis[[(dimethylamino)methyl]furans] in acetylcholinesterase inhibition, M1/M2 receptor binding, and the potentiation of ileal contractions, while being more potent inhibitors of acetylcholinesterase than butyrylcholinesterase. The 4-nitro-3-pyridazinyl analog, 26, was notable in demonstrating a potent and selective binding to the M2 receptor, with an M2 IC50/M1 IC50 of 0.060. Compounds in which the substituents on the dinitro-N-aryl moiety were relatively small were the best at inhibiting acetylcholinesterase in vitro. The N-aryl-2-[[[5-[(dimethylamino)methyl]-2- furanyl]methyl]thio]ethylamines in general, and those with small N-aryl substituents in particular, were superior to the bis[[(dimethylamino)methyl]furans] in elevating brain ACh levels in mice, probably due to enhanced distribution into the CNS. The 1,5-difluoro-2,4-dinitrophenyl analog, 8, resulted in the largest elevation in brain acetylcholine levels, affording a 53% increase at 88 mg/kg.


Novel 1-(pyridylphenyl)-1-phenyl-2-imidazolylethanols with topical antiinflammatory activity. The synthesis, biological evaluation, and structure-activity relationships of a series of 1-(pyridylphenyl)-1-phenyl-2-imidazolylethanols are described. These compounds show potent dose-dependent topical antiinflammatory activity in murine models of skin inflammation. This effect is likely due to inhibition of cytochrome P450 and consequent reduction in levels of 12R-HETE in the skin. These compounds were examined for their ability to inhibit the oxidative metabolism of arachidonic acid; they specifically inhibit the formation of prostacyclins in mouse macrophages. To study the effects of structure on the in vivo activity, three general features of the molecules were varied: the position of attachment of the pyridine nucleus (A), the second aromatic residue (B), and the nitrogen base on the ethanol chain (C). 1-[4-(4-Pyridyl)phenyl]-1-(4-fluorophenyl)-2- imidazolylethanol (2a, DuP 983) shows a very attractive profile of antiinflammatory activity and has been selected for clinical evaluation as a topical antiinflammatory agent.


S-(5'-deoxy-5'-adenosyl)-1-ammonio-4-(methylsulfonio)-2-cyclopentene: A potent, enzyme-activated irreversible inhibitor of S-adenosylmethionine decarboxylase. The compound S-(5'-deoxy-5'-adenosyl)-1-ammonio-4-(methylsulfonio)-2- cyclopentene (AdoMac) was prepared and evaluated as an irreversible inhibitor of S-adenosylmethionine decarboxylase (AdoMet-DC). AdoMac was shown to inhibit AdoMet-DC in a time-dependent manner with a KI of 18.3 microM and a kinact of 0.133 min-1. In addition, AdoMet-DC activity could not be restored following extensive dialysis of the enzyme-inhibitor complex, and the enzyme was protected from irreversible inactivation by the known competitive inhibitor methylglyoxal bis(guanylhydrazone). HPLC analysis of the enzymatic reaction products revealed a time-dependent decrease in the peak coeluting with AdoMac, and a corresponding increase in the peak coeluting with (methylthio)adenosine (MTA), a byproduct of the irreversible binding of AdoMac to the enzyme. Thus, AdoMac appears to function as an enzyme-activated, irreversible inhibitor of AdoMet-DC.


Application of neural networks: quantitative structure-activity relationships of the derivatives of 2,4-diamino-5-(substituted-benzyl)pyrimidines as DHFR inhibitors. A comparative study of quantitative structure-activity relationships involving diaminopyrimidines as DHFR inhibitors using regression analysis and the neural-network approach suggests that the neural network can outperform traditional methods. The technique permits the highlighting the functional form of those parameters which have an influence on the biological activity.


Hypoxia-selective antitumor agents. 5. Synthesis of water-soluble nitroaniline mustards with selective cytotoxicity for hypoxic mammalian cells. Nitroaniline mustards have potential as hypoxia-selective cytotoxic agents, with reductive metabolism activating the nitrogen mustard by converting the electron-withdrawing nitro group to an electron-donating hydroxylamine or amine. However, the parent compounds have poor aqueous solubility, and their potencies are limited by low reduction potentials (E1/2 ca. -600 mV versus the normal hydrogen electrode) and corresponding slow rates of nitro reduction. To address these limitations, a series of 4-nitroaniline mustards bearing hydrophilic side chains attached via an electron-withdrawing carboxamide group was prepared and evaluated for hypoxia-selective cytotoxicity against Chinese hamster cell lines. The N-[(N,N-dimethylamino)ethyl]carboxamide derivatives proved to have excellent aqueous solubility and improved cytotoxic potency, but their reduction potentials, while higher than the non-carboxamide compounds, were still low and little selectivity for hypoxic cells were observed. A series of carboxamides of 2,4-dinitroaniline mustard was also prepared. These compounds had reduction potentials in the desired range (E1/2 ca. -450 mV by cyclic voltammetry) and were more toxic to hypoxic than aerobic UV4 cells. The most selective compounds were 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (20, SN 23862) and its water-soluble N-[(N,N-dimethylamino)ethyl]carboxamide analogue. These showed selectivities of 60- to 70-fold for hypoxic UV4 cells. The selectivity of 20 was much superior to that of its aziridine analogue (23, CB 1954), which was only 3.6-fold more toxic to hypoxic than oxic cells in the same system. Compound 20 is a much less efficient substrate than CB 1954 for the major aerobic nitroreductase from rat Walker tumor cells, NAD(P)H:quinone oxidoreductase (DT diaphorase). Lack of aerobic bioactivation of 20 by DT diaphorases may be responsible for its higher hypoxic selectivity than that of 23.


Synthesis and antiviral evaluation of N-carboxamidine-substituted analogues of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride. Ten, hitherto unreported, analogues of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (2a, ribamidine) and methyl carboximidate 5 have been synthesized. These include the N-cyano (2b), N-alkyl (2c-e), N-amino acid (2f-h), N,N'-disubstituted (6, 7a,b), and the N-methylated carboxamide (1f) analogues of ribavirin. In addition, a new facile synthesis of carboxamidine 2a was also developed. All compounds were evaluated for biological activity against the following RNA viruses: Punta Toro (PT) and sandfly fever (SF) viruses (bunyaviruses); Japanese encephalitis (JE), yellow fever (YF), and dengue-4 viruses (flaviviruses); parainfluenza type 3 (PIV3), respiratory syncytial virus (RSV), and measles viruses (paramyxoviruses); influenza A and influenza B viruses (orthomyxoviruses); Venezuelan equine encephalomyelitis virus (VEE, alphavirus); human immunodeficiency virus type-1 (HIV-1, lentivirus); the DNA-containing vaccinia (VV) virus (poxvirus); and adeno type 5 (Ad5) viruses. All of the compounds except for 2b and 7a,b exhibited activity against the bunyaviruses such as that observed with 2a; however, higher IC50 values were generally observed. Glycine analogue 2f showed activity in PT-virus-infected mice in terms of increased survivors and decreased markers of viral pathogenicity. Carboxamidine 2a, carboximidate 5, and dimethyl amidine 6 exhibited activity against dengue type-4 virus. Monomethyl amidine 2c demonstrated activity against RSV, PIV3, and, to a lesser extent, influenza A and B. Activity of 2c generally required higher IC50 values than unsubstituted 2a. The latter exhibited hitherto unreported activity against RSV; therapeutic indices for 2a against RSV and PIV3 were greater than 64 and greater than 21. No substantial in vitro activity was observed for any of the compounds tested against Ad5, measles, JE, YF, VEE, or HIV-1. In addition, evidence is presented which argues in favor of a distinct antiviral mechanism of action for carboxamidines, e.g. 6, in contrast to a role as a carboxamide precursor.


Histamine H2-receptor agonists. Synthesis, in vitro pharmacology, and qualitative structure-activity relationships of substituted 4- and 5-(2-aminoethyl)thiazoles. It is well known that both histamine and dimaprit show moderate histamine H2-receptor agonistic activities on the guinea pig right atrium. Quantum chemical calculations on these two compounds showed similarities in electron distributions and molecular electrostatic potentials (MEP's), which could be extended to rigid analogues [2-amino-5-(2-aminoethyl)thiazoles] of the latter structure. On the base of these results a series of substituted 4- and 5-(2-aminoethyl)thiazoles was synthesized applying small alkyl substitution variations as reported for histamine. 2-Amino-5-(2-aminoethyl)-4-methylthiazole (Amthamine) proved to be the most potent full histamine H2-receptor agonist on the guinea pig right atrium, being with a pD2 value of 6.21 slightly more potent than histamine. This compound shows no affinity for H1-receptors and is a full but weak agonist on the histamine H3-receptor with a pD2 value of 4.70, thus showing a marked specificity for histamine H2-receptors. In the 5-(2-aminoethyl)thiazole series the presence of a 2-amino substituent proved to be not essential for stimulation of the histamine H2-receptor, leading to the important conclusion that in contrast to histamine, for this series, acceptance of a proton by the thiazole nucleus of the agonist from the active site of the receptor is sufficient for the stimulation of the histamine H2-receptor.


Synthesis, characterization, and Ca2+ antagonistic activity of diltiazem metabolites. Diltiazem is a calcium antagonist widely used in the treatment of angina and hypertension. The contributions of metabolites of diltiazem to the vasorelaxant effects of diltiazem were investigated. The synthesis and spectroscopic characterization of eight major cis-diltiazem metabolites are described. Three of the compounds--N, O-didemethylated metabolite (21), O-demethylated metabolite (22), and diltiazem N-oxide (27)--have been recently reported and have not previously been synthesized. The identities of all eight synthetic metabolites have been verified with samples obtained from human urine using combined LC-MS/MS. The Ca2+ antagonistic activities of diltiazem and its metabolites (except 27) were studied on hamster aorta preparations depolarized with KCl. The order of potencies (IC50 +/- SE, microM) is as follows: diltiazem (0.98 +/- 0.47) greater than 17 (2.46 +/- 0.38) greater than or equal to 23 (3.27 +/- 1.02) greater than 26 (20.2 +/- 10.5) greater than 22 (40.4 +/- 15.4) greater than or equal to 25 (45.5 +/- 18.1) greater than 21 (112.2 +/- 33.2) greater than or equal to 24 (126.7 +/- 24.2). Structure-activity relationships are also discussed.


Conformationally restrained, chiral (phenylisopropyl)amino-substituted pyrazolo[3,4-d]pyrimidines and purines with selectivity for adenosine A1 and A2 receptors. Two modes of tethering a chiral (phenylisopropyl)amino substituent in pyrazolo[3,4-d]pyrimidines and purines have been explored. One mode gave (S)-2,7-dihydro-7-phenyl-2-(phenylmethyl)-5- propoxy-3H-imidazo[1,2-c]pyrazolo-[4,3-e]pyrimidine (12a) and its corresponding R-enantiomer 12b, which were selective for A2 and A1 adenosine receptors, respectively. The corresponding diimidazo[1,2-c:4',5'-e]pyrimidines 12e and 12f were analogously selective. This is the first example where a single chiral recognition unit provides enantiomers with opposite selectivities for adenosine receptors. The second mode gave (2S-trans)-2,7-dihydro-2-methyl-3,7-diphenyl-5- propoxy-3H-imidazo[1,2-c]-pyrazolo[4,3-e]pyrimidine (12c) and its corresponding R-enantiomer 12d. Compounds 12c and 12d were significantly less potent than 12a and 12b at A1 receptors, and were nonselective.


Urea and 2-imidazolidone derivatives of the muscarinic agents oxotremorine and N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide. Some urea and 2-imidazolidone analogues of the muscarinic agents oxotremorine (1) and N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide [10; BM-5] have been synthesized and assayed for muscarinic and antimuscarinic activity on the isolated guinea pig ileum. The new compounds (15-24) were found to be muscarinic agonists, partial agonists, or antagonists. The compounds were also tested for in vitro receptor binding to homogenates of the rat cerebral cortex using the muscarinic antagonist [3H]-3-quinuclidinyl benzilate ([3H]QNB) as the ligand. They were found to be less potent than 1 in this assay. On the guinea pig ileum, the N-3-methyl substituted imidazolidone analogue 20 was the most potent agonist of the new compounds studied; 20 was 5-fold more potent in inducing contractions of the ileum and had 4-fold higher affinity for ileal muscarinic receptors than the 3-methyl substituted 2-pyrrolidone 6. However, the N-3-unsubstituted urea and imidazolidone derivatives 15 and 19 were several-fold less potent than the parent acetamide N-methyl-N-(4-pyrrolidino-2-butynyl)acetamide [9; UH-5] and 1, respectively. The urea analogue (16) of the partial muscarinic agonist 10 was devoid of intrinsic activity and displayed 3-fold lower affinity than 10 for ileal muscarinic receptors.


Terminal disorder: a common structural feature of the axial proteins of bacterial flagellum? We report, based on proteolytic experiments and high resolution 1H nuclear magnetic resonance studies that the terminal regions of the monomeric hook protein are highly mobile and exposed to the solvent. The disordered parts of the hook protein span approximately the first 70 and the last 30 amino acid residues. Although the amino acid sequences of flagellin and hook protein do not resemble each other at all, both proteins have now been shown to contain large disordered terminal regions. Sequential similarities of flagellin and hook protein, especially near the NH2 and COOH termini, to other axial components of bacterial flagellum suggest that terminal disorder may be a common structural feature of the axial proteins of the bacterial flagellum.


Interdependence of translation, transcription and mRNA degradation in the lacZ gene. We have constructed a collection of Escherichia coli strains which differ by point mutations in the ribosome binding site (RBS) that drives the translation of the lacZ gene. These mutations affect the Shine-Dalgarno sequence or the initiation codon, or create secondary structures that sequester these elements, and result in a 200-fold variation in beta-galactosidase expression. Surprisingly, these variations of expression are paralleled by nearly equivalent changes in the lacZ mRNA level. The ratio of the beta-galactosidase expression to the mRNA level reflects the average spacing between translating ribosomes: hence, paradoxically, mutations that affect translation initiation do not correspondingly change this spacing. Further analysis of the mRNA level variations shows that they originate from two independent mechanisms. When beta-galactosidase expression exceeds a threshold corresponding roughly to one translation event per transcript, the variations in the efficiency of translation initiation affect largely the chemical and functional lifetimes of the mRNA. We further show that the rate-limiting step in the chemical decay process is an RNase E-dependent cleavage, which is outcompeted by translation initiation. Below this expression threshold, the mRNA lifetime levels out and strain-to-strain variations in mRNA level arise solely from polarity effects. We suggest that, in this activity range, most mRNA molecules that escape polarity are crossed by a single ribosome, and hence are identical from the viewpoint of degradation. Altogether, the tight couplings between translation initiation on one hand, polarity and/or mRNA degradation on the other, result in translation initiation events being closely spaced in time even from inefficient RBS, at the expense of the mRNA level. Finally, we evocate the possible beneficial consequences of a coupling between translation, transcription and mRNA degradation, for the management of cellular resources.


Culture conditions differentially affect the translation of individual Escherichia coli mRNAs. Our aim is to investigate whether changes in growth conditions can differentially affect the initiation of translation from individual Escherichia coli mRNAs that are not subjected to specific translational control. As a model system, we have constructed a series of point-mutated lacZ genes which differ in their Shine-Dalgarno (SD) sequence, their initiator codon, or the secondary structure around these elements. Alterations in growth conditions produced large (up to 8-fold) changes in the relative expression from these genes, which, we argue, stem from changes in their relative efficiencies of translation initiation. In particular, compared to genes bearing mutations outside the SD or initiator codon, genes mutated in these elements experience a significant decrease in their expression when cells are grown in minimal rather than rich medium; at 42 degrees C rather than 37 degrees C; or under amino acid starvation. We discuss the mechanisms underlying these effects, and evocate their possible generality.


Translation inhibition by an mRNA coding region secondary structure is determined by its proximity to the AUG initiation codon. In the present study we investigate the impact of highly stable coding region secondary structures on mRNA translation efficiency. By introducing antisense segments into the 3'non-translated region of human alpha-globin mRNA we are able to synthesize a series of transcripts in which site-specific secondary structures are introduced without altering the primary structure of the 5' non-translated region, the coding region, or the encoded protein product. Coding region duplexes in close proximity to the AUG initiation codon are found to inhibit translation severely to a degree equal to that of a duplex that extends into the 5' non-translated region. In contrast, mRNAs containing duplexes positioned further 3' in the coding region translate at levels that are significantly higher although are still below those of native alpha-globin mRNA. The primary determinant of translation inhibition by coding region duplexes appears to be the proximity of the duplex to the AUG initiation codon and reflects a parallel inhibition of monosome formation. These data demonstrate that extensive coding region secondary structures suppress translation to a minimal or to a substantial degree depending on their distance from the initiation codon.


Functional structures of the recA protein found by chimera analysis. We developed a novel genetic method for finding functional regions of a protein by the analysis of chimeras formed between homologous proteins. Sets of chimeric genes were made by intramolecular homologous recombination in a linearized plasmid DNA carrying both recA genes of Escherichia coli and Pseudomonas aeruginosa. A recBCsbcA strain of E. coli was used for isolation of plasmids carrying recombinants between these genes. Examination of properties of E. coli strains deleting the recA gene and carrying a plasmid with a chimeric gene shows that chimera formation at certain positions inactivates a RecA function. Frequently, all chimeras with a junction in a certain region of the protein inactivate a function. Rather than a direct effect of the presence of the junction at a particular position, mismatching of the regions both sides of the junction that are derived from the different species is responsible for the inactivation. For a chimeric protein to be functional, certain pairs of sequences in different regions of the protein must derive from the same parent. Four pairs of such sequences were found: two are involved in activities for genetic recombination and for resistance to ultraviolet light irradiation and the others in formation of active oligomers. Regions defined by these sequences are located in the looped regions of the protein. A pair of regions may co-operate to form a functional folded structure.


Alu RNA transcripts in human embryonal carcinoma cells. Model of post-transcriptional selection of master sequences. Alu master sequences colonized the human genome using RNA as amplification intermediate. To understand this phenomenon better we isolated and analyzed Alu RNA from NTera2D1 pluripotential cells. Northern hybridization, primer extension, cDNA cloning and sequencing data are congruent and demonstrate a low level of Alu specific transcription. These bona fide RNA Polymerase III Alu transcripts, although enriched in the cytoplasm, are not dominated by a single master species but rather originate from a variety of loci. However, when compared with the genomic average, or to repeats from RNA Polymerase II co-transcripts, they belong to the youngest group of Alu subfamilies (p less than 0.001) and have a higher content of intact CpG-dinucleotides. This suggests that Alu transcription is influenced both by mutations and the genomic context, and points to a possible role of DNA methylation in silencing the bulk of genomic repeats. Because of the heterogeneity of Alu transcripts a post-transcriptional selection mechanism recruiting Alu master sequences for retroposition is required. We propose that Alu RNA masters could have evolved as selfish satellites to a more complex retroposition system equipped with a reverse transcriptase activity and that their structure was conserved through "phenotypic" selection of the RNA level.


Activation of recA protein. The open helix model for LexA cleavage. RecA protein is induced by the binding of DNA and ATP to become active in the hydrolysis of ATP and the cleavage of repressors. These reactions appear to depend on the structural state of the protein polymerized along the DNA, i.e. a helical coat of six RecA per turn of 95 to 100 A pitch. In support of this model of the active conformation, it was shown that high concentrations of salt also induce this helical polymerized state as well as the enzymatic activities. Here, we describe that, in vitro and with the non-hydrolyzable analogue ATP gamma S, RNA and heparin can also induce both the structural transition and the enzymatic activation of RecA to LexA cleavage in accordance with the model. RNA and heparin do not support the reaction in the presence of ATP, and they do not induce the hydrolysis of ATP either, suggesting that, in contrast to ATP gamma S, the nucleotide is not bound stably enough, and that the combined affinities of polynucleotide and ATP actually modulate the discrimination of RecA for the various possible inducers in vivo.


Association of AUUUA-binding protein with A+U-rich mRNA during nucleo-cytoplasmic transport. Resealed nuclear envelope (NE) vesicles from rat liver containing entrapped exogenous RNA were used to study the effect of adenosine+uridine binding factor (AUBF), present in cytosolic cell extracts, on ATP-dependent transport of A+U-rich RNA (AU+RNA) and A+U-free RNA (AU-RNA) across the NE. This factor specifically binds to A+U-rich sequences present in the 3' untranslated regions of lymphokine and cytokine mRNAs, containing overlapping AUUUA boxes (granulocyte-macrophage colony stimulating factor, interleukin-3). Addition of AUBF to the extravesicular compartment markedly increased the efflux of the in vitro transcribed, capped and polyadenylated AU+ RNAs. Export of entrapped AU- control RNA, such as beta-globin RNA, was not affected by AUBF, in contrast to chimeric AU+ beta-globin RNA containing the A+U-rich sequence of human interferon-alpha mRNA (6 reiterated AUUUA motifs). Competition experiments revealed that AUBF enhances the affinity of poly(A)-containing AU+ RNAs to the NE poly(A)-binding component (poly(A)-recognizing mRNA carrier p106), and thereby accelerates nuclear export of these RNAs. We could demonstrate that AUBF added to the extravesicular space forms stable complexes with polyadenylated AU+ RNA with relative molecular masses of about 45,000, 62,000 and 70,000 inside the vesicles or during ATP-dependent export. In addition we determined that AUBF may affect mRNA stability by protecting A+U-rich RNA against degradation by trans-acting, nuclear matrix-associated and A+U-specific endoribonuclease V.


The rhombotin gene family encode related LIM-domain proteins whose differing expression suggests multiple roles in mouse development. The rhombotin (RBTN1 or Ttg-1) gene was first identified at a chromosome translocation in a T-cell acute leukaemia and later used to isolate two related genes (RBTN2 or Ttg-2 and RBTN3). Complete characterization of these genes in man and mouse shows that all three encode cysteine-rich proteins with typical LIM domains. RBTN1 and RBTN3-derived proteins have 98% identity in the LIM domains but are located on separate chromosomes in man and in mouse while RBTN1 and RBTN2, both located on human chromosome 11p but are on separate chromosomes in mouse, are only 48% identical in this part of the protein. The exon organization of RBTN1 and RBTN3 genes are similar, both having an intron, absent from the RBTN2 gene, in the LIM2-encoding region. The remarkable similarity between rbtn-1 and rbtn-3 proteins is parallelled in their expression patterns in mouse development, since both genes show high expression in restricted areas of the brain, but little lymphoid expression. rbtn-2 expression, however, is more ubiquitous. This gene shows a low level of thymus expression but high expression in fetal liver, adult spleen and B-cell lines, consistent with a role in B-cell development. These results suggest multiple cellular targets for the action of these proteins during development.


Quantitation of molecular densities by cryo-electron microscopy. Determination of the radial density distribution of tobacco mosaic virus. We have determined the absolute mass and radial scattering density distribution of tobacco mosaic virus in the frozen-hydrated state by energy-filtered low-dose bright-field transmission electron microscopy. The absolute magnitude of electron scattering from tobacco mosaic virus in 150 nm of ice was within 3.0% of that predicted, with inelastic scattering accounting for approximately 80% of the scattering contrast. In order to test the accuracy of the radial reconstruction, a computer model of tobacco mosaic virus was built from the atomic co-ordinates assuming uniform solvent density. The validity of the model was confirmed by comparison of X-ray scattering and predictions of the model (R factor = 0.05). First-order corrections for the microscope contrast transfer function were necessary and sufficient for conversion of the cryo-electron microscopy images into accurate representations of the mass density. At 1.9 nm resolution the compensated reconstruction and model had density peaks of similar magnitude at 2.4, 4.2, 6.0 and 7.8 nm radius and a central hole of 2 nm radius. Equatorial Fourier transforms of the corrected electron images were in excellent agreement with predictions of the model (R factor = 0.12). Thus, the uniform solvent approximation was adequate at 1.9 nm resolution to describe quantitatively X-ray scattering in liquid water and electron imaging in vitreous ice. This is the first demonstration that cryo-electron microscopy images can be used to quantitate the absolute mass, mass per unit length and internal density distributions of proteins and nucleic acids.


Conformational sub-states in B-DNA.

Conformational sub-states in B-DNA. Theoretical studies of the sequence-dependent conformation of B-DNA have been carried out using Jumna, a helicoidal co-ordinate minimization algorithm. The results obtained for a series of six oligomers with repetitive sequences show that, with the exception of the homopolymers (dA)n.(dT)n and (dG)n.(dC)n, all sequences can adopt a variety of conformations characterized by considerable changes in helicoidal parameters and also in sugar puckers which adopt C(2')-endo (falling into 2 classes) or, in the case of pyrimidine nucleotides, O(1')-endo forms. These studies lead to an improved understanding of the role of base sequence on DNA conformation and point to a number of interesting correlations between the various structural parameters describing the double helix.


Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin. In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.


Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. II. Plastocyanin. In an attempt to understand the earliest events in the protein folding pathway, the complete sequence of French bean plastocyanin has been synthesized as a series of short peptide fragments, and the conformational preferences of each peptide examined in aqueous solution using proton n.m.r. methods. Plastocyanin consists largely of beta-sheet, with reverse turns and loops between the strands of the sheet, and one short helix. The n.m.r. experiments indicate that most of the peptides derived from the plastocyanin sequence have remarkably little propensity to adopt folded conformations in aqueous solution, in marked contrast to the peptides derived from the helical protein, myohemerythrin (accompanying paper). For most plastocyanin peptides, the backbone dihedral angles are predominantly in the beta-region of conformational space. Some of the peptides show weak NOE connectivities between adjacent amide protons, indicative of small local populations of backbone conformations in the a region of (phi,psi) space. A conformational preference for a reverse turn is seen in the sequence Ala65-Pro-Gly-Glu68, where a turn structure is found in the folded protein. Significantly, the peptide sequences that populate the alpha-region of (phi,psi) space are mostly derived from turn and loop regions in the protein. The addition of trifluoroethanol does not drive the peptides into helical conformations. In one region of the sequence, the n.m.r. spectra provide evidence of the formation of a hydrophobic cluster involving aromatic and aliphatic side-chains. These results have significance for understanding the initiation of protein folding. From these studies of the fragments of plastocyanin (this paper) and myohemerythrin (accompanying paper), it appears that there is a pre-partitioning of the conformational space sampled by the polypeptide backbone that is related to the secondary structure in the final folded state.


Modeling of the structure of bacteriorhodopsin. A molecular dynamics study. Secondary structure predictions for membrane proteins are relatively reliable and permit the construction of model structures that may serve as initial conformations for molecular dynamics simulations. This might provide a scheme to predict the three-dimensional structures of membrane proteins. The feasibility of such an approach is tested for bacteriorhodopsin. We were not able to fully predict the kidney-shaped structure of bacteriorhodopsin. However, features compatible with this structure developed in a simulation starting from a circular arrangement of the seven predicted helices. When instead we started from the kidney shape, assigning the seven predicted helices in different ways to those on the structure, we could distinguish between the different assignments on the basis of energy and tilt of the helices. In this way we could select the correct assignment from a few others. For the correct assignment, the helices spontaneously adopted a tilt that agrees remarkably well with the experimental model structure derived by others. The root-mean-square deviation between our best molecular dynamics structure and the experimental model structure is 3.8 A, caused mainly by deviations in the internal degrees of freedom of the helices.


Crystal structure of Escherichia coli malate dehydrogenase. A complex of the apoenzyme and citrate at 1.87 A resolution. The crystal structure of malate dehydrogenase from Escherichia coli has been determined with a resulting R-factor of 0.187 for X-ray data from 8.0 to 1.87 A. Molecular replacement, using the partially refined structure of porcine mitochondrial malate dehydrogenase as a probe, provided initial phases. The structure of this prokaryotic enzyme is closely homologous with the mitochondrial enzyme but somewhat less similar to cytosolic malate dehydrogenase from eukaryotes. However, all three enzymes are dimeric and form the subunit-subunit interface through similar surface regions. A citrate ion, found in the active site, helps define the residues involved in substrate binding and catalysis. Two arginine residues, R81 and R153, interacting with the citrate are believed to confer substrate specificity. The hydroxyl of the citrate is hydrogen-bonded to a histidine, H177, and similar interactions could be assigned to a bound malate or oxaloacetate. Histidine 177 is also hydrogen-bonded to an aspartate, D150, to form a classic His.Asp pair. Studies of the active site cavity indicate that the bound citrate would occupy part of the site needed for the coenzyme. In a model building study, the cofactor, NAD, was placed into the coenzyme site which exists when the citrate was converted to malate and crystallographic water molecules removed. This hypothetical model of a ternary complex was energy minimized for comparison with the structure of the binary complex of porcine cytosolic malate dehydrogenase. Many residues involved in cofactor binding in the minimized E. coli malate dehydrogenase structure are homologous to coenzyme binding residues in cytosolic malate dehydrogenase. In the energy minimized structure of the ternary complex, the C-4 atom of NAD is in van der Waals' contact with the C-3 atom of the malate. A catalytic cycle involves hydride transfer between these two atoms.


Structure-function relationships in the low-affinity mutant haemoglobin Aalborg (Gly74 (E18)beta----Arg). Haemoglobin Aalborg (Gly74 (E18)beta----Arg) has a reduced oxygen affinity, in both the absence and the presence of organic phosphates; it has a raised affinity for organic phosphates, and it is moderately unstable. By contrast, haemoglobin Shepherds Bush (Gly74 (E18)beta----Asp) has an increased oxygen affinity in both the absence and the presence of organic phosphates, a diminished affinity for organic phosphates and is also unstable. We have determined the crystal structure of deoxyhaemoglobin Aalborg at 2.8 A resolution and compared it to the structures of deoxy- and oxyhaemoglobin A and of deoxyhaemoglobin Shepherds Bush. The guanidinium group of Arg74(E18)beta protrudes from the haem pocket and donates hydrogen bonds to the E and F helices. The carboxylate group of Asp74(E18)beta forms a hydrogen bond only with residue EF6 and is partially buried, which may be why haemoglobin Shepherds Bush appears to be more unstable than haemoglobin Aalborg. To discover why the latter has a low oxygen affinity, we superimposed the B, G and H helices of haemoglobin A, whose conformation is known to be unaffected by ligand binding, on those of haemoglobin Aalborg. This also brought helices E and the haems into superposition, but revealed a shift of the F helix of deoxyhaemoglobin Aalborg towards the EF-corner. This shift is opposite to that which occurs on ligand binding and on transition to the quaternary oxy-structure, and is linked to an increased tilt of the proximal histidine residue away from the haem axis. Since the relative positions of helices E and F and of the haem group are thought to be the main determinants of the changes in oxygen affinity, the shift of helix F may account for the reduced oxygen affinity of haemoglobin Aalborg. The shift may be due to a combination of steric and electrostatic effects introduced by the arginine residue's side-chain. The effects of the arginine and aspartate substitutions at position E18 beta on the 2,3-diphosphoglycerate affinity are equal and opposite. They can be quantitatively accounted for by the electrostatic attraction or repulsion by the oppositely charged side-chains.


Selection of phage antibodies by binding affinity. Mimicking affinity maturation. We describe a process, based on display of antibodies on the surface of filamentous bacteriophage, for selecting antibodies either by their affinity for antigen or by their kinetics of dissociation (off-rate) from antigen. For affinity selection, phage are mixed with small amounts of soluble biotinylated antigen (less than 1 microgram) such that the antigen is in excess over phage but with the concentration of antigen lower than the dissociation constant (Kd) of the antibody. Those phage bound to antigen are then selected using streptavidin-coated paramagnetic beads. The process can distinguish between antibodies with closely related affinities. For off-rate selection, antibodies are preloaded with biotinylated antigen and diluted into excess unlabelled antigen for variable times prior to capture on streptavidin-coated paramagnetic beads. To mimic the affinity maturation process of the immune system, we introduced random mutations into the antibody genes in vitro using an error-prone polymerase, and used affinity selection to isolate mutants with improved affinity. Starting with a small library (40,000 clones) of mutants (average 1.7 base changes per VH gene) of the mouse antibody B1.8, and using several rounds of affinity selection, we isolated a mutant with a fourfold improved affinity to the hapten 4-hydroxy-5-iodo-3-nitrophenacetyl-(NIP)-caproic acid (mutant Kd = 9.4(+/- 0.3) nM compared with B1.8 Kd = 41.9(+/- 1.6) nm). The relative increase in affinity of the mutant is comparable to the increase seen in the anti-4-hydroxy-3-nitrophenylacetyl/NIP-caproic acid murine secondary immune response.


Crystallization of recombinant chitinase from the cloned chiA gene of Serratia marcescens. The chiA gene encoding for the chitinase enzyme from Serratia marcescens was efficiently overexpressed under the pL promoter and the enzyme was secreted into the growth medium. The chitinase was purified to homogeneity using affinity chromatography on a Phenyl-Sepharose column and the protein was successfully crystallized. The crystals are presently in the form of small needles in space group C222(1) and have unit cell dimensions a = 204(+/- 0.5) A, b = 134(+/- 0.5) A, c = 60(+/- 0.5) A. The crystals diffract X-rays to about 3 A resolution and are suitable for three-dimensional structural analysis.


Sequence of lamprey vitellogenin. Implications for the lipovitellin crystal structure. The amino acid sequence of lamprey vitellogenin has been predicted from the nucleotide sequence of cloned cDNA. The sites of proteolytic cleavage that produce the lipovitellin complex from the vitellogenin have been located by comparing the N-terminal sequences of two lamprey lipovitellin polypeptides with the predicted sequence. These results also confirm that the vitellogenin sequence derived here corresponds to the lipovitellin complex for which the crystal structure has been solved previously. Predictions of secondary structure indicate that the region most likely to correspond to the large alpha-helical domain of the crystallographic model consists of vitellogenin residues 300 to 600. Similar to the lipovitellins of Xenopus laevis, lamprey lipovitellin appears to lack approximately 200 C-terminal residues that are present in vitellogenin. However, the lamprey lipovitellin differs from those of Xenopus and chicken in two respects. First, most of the serine-rich domain that is present as the phosvitin polypeptide in the lipovitellins of the higher vertebrates appears to be lost in the maturation of lamprey vitellogenin to lipovitellin. Second, the domains that constitute the large lipovitellin-1 polypeptide in Xenopus and chicken are present in two polypeptides in lamprey, owing to an additional proteolytic processing event.


Histology of the esophagus of the adult frog Rana perezi (Anura: Ranidae). Study of the esophageal microscopic morphology of adult Rana perezi by light and electron microscopy discloses some large folds throughout the esophagus that are in themselves ringed. Glandular ostia open in the furrows of the luminal surface. The esophageal wall is made up of a connective adventitia rich in melanocytes, a muscular tunica, a connective and glandular subepithelial layer, and a pseudostratified ciliated epithelium. This epithelium basically consists of ciliated, goblet, basal, microvillous-apex, and migratory cells. Two types of goblet cells are distinguished with regard to the granular ultrastructure. The microvillous-apex cell has not been found in other amphibians. It shows a very differentiated morphology with a high number of mitochondria. The basal cells give the epithelium a pseudostratified morphology, and they have a proliferative function. Glands are branched and drain through an excretory duct that has a monolayered mucosecreting epithelium. The glandular units are formed by two principal types of cells: mucosecretory and serous.


Electron microscopic structure and innervation of the carotid baroreceptor region in the rock hyrax (Procavia capensis). Semi-thin plastic sections reveal that the carotid baroreceptor region in the rock hyrax comprising the origin of the internal carotid artery has a preponderantly elastic structure and a thick tunica adventitia. In contrast, the common carotid artery has a musculoelastic structure, whereas the cranial segment of the internal carotid artery (immediately distal to the baroreceptor areas) shows the features of a muscular artery. Electron microscopy discloses the presence of sensory nerve endings within the parts of the tunica adventitia adjoining the preponderantly elastic zone of the internal carotid artery. These nerve endings are characterized by varicose regions containing a large quantity of mitochondria. Bundles of collagen fibers in the tunica adventitia form convolutions or whorls around the nerve terminals and often terminate on the surface of the elastic fibers or into the basement membranes of the neuronal profiles. The large content of elastic tissue in the tunica media of the baroreceptor region may render the vessel wall highly distensible to intraluminal pressure changes. This, in turn, would facilitate the transmission of the stimulus intensity to the sensory nerve terminals located in the tunica adventitia. It is suggested that the stretching of elastic fibers may form the main mechanical event leading to the distortion of the associated nerve terminals. However, a change in the geometrical configuration of the bundles of collagen under the influence of the elastic fibers may provide a better insight into the mechanisms of distortion of the baroreceptors related to and/or in contact with collagen fibers.


Edgeworth's legacy of cranial muscle development with an analysis of muscles in the ventral gill arch region of batoid fishes (Chondrichthyes: Batoidea). A series of studies by Edgeworth demonstrated that cranial muscles of gnathostome fishes are embryologically of somitic origin, originating from the mandibular, hyoid, branchial, epibranchial, and hypobranchial muscle plates. Recent experimental studies using quail-chick chimeras support Edgeworth's view on the developmental origin of cranial muscles. One of his findings, the existence of the premyogenic condensation constrictor dorsalis in teleost fishes, has also been confirmed by molecular developmental studies. Therefore, developmental mechanisms for patterning of cranial muscles, as described and implicated by Edgeworth, may serve as structural entities or regulatory phenomena responsible for developmental and evolutionary changes. With Edgeworth's and other studies as background, muscles in the ventral gill arch region of batoid fishes are analyzed and compared with those of other gnathostome fishes. The spiracularis is regarded as homologous at least within batoid fishes, but its status within elasmobranchs remains unclear; developmental modifications of the spiracularis proper are evident in some batoid fishes and in several shark groups. The peculiar ventral extension of the spiracularis in electric rays and some stingrays may represent convergence, probably facilitating ventilation and/or feeding in both groups. The evolutionary origin of the "internus" and "externus" remains uncertain, despite the fact that a variety of forms of the constrictor superficiales ventrales in batoid fishes indicates an actual medio-ventral extension of the "externus." The intermandibularis is probably present only in electric rays. The "X" muscle occurs only in electric rays and is considered to be Edgeworth's intermandibularis profundus. Its association with the adductor mandibular complex in narkinidid and narcinidid electric rays may relate to its functional role in lower jaw movement. Contrary to common belief, in most batoid fishes as well as some sharks, muscles that originate from the branchial muscle plate and extend medially in the ventral gill arches do exist: the medial extension of the interbranchiales in most batoid fishes and some sharks and the "Y" muscle in the pelagic stingrays Myliobatos and Rhinoptera. The latter is another example of the medial extension of the "internus." Whether the interbranchiales and "Y" muscle are homologous within elasmobranchs and whether homologous with the obliques ventrales and/or transversi ventrales of osteichthyan fishes await further research. Four hypobranchial muscles are recognized in batoid fishes: the coracomandibularis, coracohyoideus, coracoarcualis, and coracohyomandibularis. The coracohyoideus is discrete from the coracoarcualis; its complete structural separation from the latter occurs in several groups of batoid fishes.(ABSTRACT TRUNCATED AT 400 WORDS)


Middle-ear development: II. Morphometric changes in the conducting apparatus of the chick. The ontogeny of various middle-ear structures was examined in 11 groups of chicks between 10 days embryonic and adult. Measurements of the tympanic membrane surface area and height, columella length, and that of the columella footplate, annular ligament, and oval window area were obtained using video micrographs and computer digitization techniques. The oval window matures first at 53 days post-hatching, whereas the columella achieves adult size at 74 days. The tympanic membrane surface area is the last middle-ear variable studied to reach adult size (79 days post-hatch). The columella increases its length from 0.63 mm (10 days embryonic) to 2.73 mm in the adult. The tympanic membrane area expands by 280% whereas the columellar footplate area increases by 11x. As a result, the pressure amplification of the middle ear due to the tympanic membrane/columellar footplate area ratio improves by over 400%. These data further contribute to our understanding of the functional development of the middle ear.