Population dynamics and control of Triatoma infestans. Analysis of field populations of Triatoma infestans (Hemiptera: Reduviidae), after a 3-year study, shows that population growth rate is affected by both density-dependent and density-independent mortality. Although an equilibrium exists, apparently as a consequence of a density dependent-mechanism, population density fluctuates throughout the year because of the effect of monthly mean minimum temperature as a density-independent source of mortality. Simulation studies based on Moran curves shows that high population densities have an approximately constant extinction probability (around 0.20), independently of the season the population starts growing. However, at very low population densities, the extinction probability depends strongly on the season when the population begins to grow. Very low density populations beginning in winter or autumn have the highest extinction probability. The outcome of the simulation studies coincides with results observed in field populations affected by insecticide application at different seasons.


Responses of female New World screwworm flies, Cochliomyia hominivorax, to swormlure-4 in the laboratory. The behaviour of 4-day-old virgin female New World screwworm flies Cochliomyia hominivorax Coquerel was investigated in a wind tunnel using video. Addition of the screwworm attractant swormlure-4 to the airstream resulted in an increase in the number of take-offs and in the duration of flight. In the presence of swormlure-4 the flight speed decreased and the rate of turning increased. Take-off was significantly orientated upwind during the period when swormlure-4 was added to the airstream, and significantly orientated downwind in the period after the addition of swormlure-4. The possible means by which this species finds a point source of odour in the field are discussed and compared with the mechanisms used by other flies.


Snail predation by larvae of Sepedon scapularis Adams (Diptera: Sciomyzidae), a potential biocontrol agent of snail intermediate hosts of schistosomiasis in South Africa. Malacophagous larvae of the fly Sepedon scapularis Adams were shown experimentally to be effective predators of three species of aquatic pulmonate snails tested as prey: Bulinus africanus (Krauss) an important intermediate host of Schistosoma haematobium (Bilharz), Bulinus tropicus (Krauss) and the invasive species Physa acuta Draparnaud. Survival of S. scapularis larvae from instar to instar was negatively affected by the size of prey snails, since larvae tended to be asphyxiated by the mucous secretions of the snails, or by the larval hydrofuge hairs becoming entangled in snail faeces. In experiments to test the choice of S.scapularis larvae for different species and sizes of snails, B.africanus was significantly disfavoured compared with the other two snail species. Small snails (< 3 mm) were more frequently killed by all three larval instars of S.scapularis. First instars killed few, if any, large snails (> 7 mm), whereas second and third instars preyed effectively on all sizes of snails. Third instars killed significantly more snails than younger instars. For larvae offered only one species of snail as prey, the mean total number of snails killed per larva during its entire development was 49 B.africanus, 45 B.tropicus or 34 P.acuta. It is concluded that S.scapularis is potentially useful as a biological control agent for use against indigenous Bulinus and exotic Physa snails.


Patterns of sandfly distribution in tropical forest: a causal hypothesis. 1. In tropical rain forest, phlebotomine sandflies (Diptera: Psychodidae), such as Lutzomyia vespertilionis and L.ylephiletor, have an aggregated distribution on their tree buttress diurnal resting sites, as studied during 1987-88 at Finca la Selva in the Caribbean lowlands of Costa Rica. 2. Experimental transfer of flies to trees not used as resting sites indicated that many apparently suitable sites remain unoccupied. 3. Observations of sandflies on the buttresses revealed that males and females are juxtaposed more frequently than expected by chance alone. Courtship behaviour by three of the four species of sandfly studied was observed on the buttresses. 4. It is suggested that the use of buttresses as swarming sites for mating behaviour is more likely to account for the observed distribution patterns of sandflies than their use of buttresses simply as diurnal resting sites.


Host preferences of the phlebotomine sandfly Lutzomyia longipalpis in Amazonian Brazil. Experiments were undertaken to determine the relative attractiveness of humans, dogs and chickens to Lutzomyia longipalpis, the principal vector of Leishmania chagasi causing American visceral leishmaniasis. Field experiments in two villages on Marajó Island, Pará State, Brazil, showed that one boy attracted significantly more flies than one dog or chicken, and slightly fewer flies than a group of six chickens. Experiments with laboratory-bred female flies showed that a significantly greater number of flies engorged on a single human than on either a single dog or chicken, and man-biting catches demonstrated the willingness of flies to bite in the field. It appears that Lu.longipalpis has catholic feeding habits, the attractiveness of different hosts being largely a function of their relative sizes. These results are discussed with reference to the epidemiology of visceral leishmaniasis in Brazil.


Ootaxonomy and eggshell ultrastructure of Phlebotomus sandflies. The eggshell structure of four sandfly species: Phlebotomus perniciosus Newstead, P.perfiliewi Parrot, P.papatasi Scopoli and P.duboscqi Neveu-Lemaire, was examined by scanning and transmission electron microscopy (SEM and TEM). At the TEM level, the eggshell appears to have a homogeneous vitelline envelope and a thick chorion. At SEM level, the eggshell of all species is characterized by the outer chorion forming a series of fifteen to twenty longitudinal sinuous ridges, cross-linked in places to form a pattern of polygons, each line of the chorion consisting of columns arranged in a palisade. The aeropyle region of the egg is described for the first time in phlebotomine sandflies. Specific characters of the eggshell topography are described for distinguishing between these and other species of Phlebotomus.


Crossing type variability associated with cytoplasmic incompatibility in Australian populations of the mosquito Culex quinquefasciatus Say. An analysis of cytoplasmic crossing type variation in Australian populations of Culex quinquefasciatus, a member of the Culex pipiens complex of mosquitoes, revealed high levels of variability causing partial incompatibility between natural populations. Segregating crossing types were commonly found together within sampled sites. No correlation was evident between similarity of crossing type and environmental parameters of the sites, nor distance between sites. The nature of the observed variation did not support the hypothesis of paternally expressed nuclear 'restorer' genes. Such high levels of crossing type variation would be likely to impede attempts to control populations of the Culex pipiens complex using cytoplasmic incompatibility.


A comparison of African buffalo, N'Dama and Boran cattle as reservoirs of Trypanosoma congolense for different Glossina species. Teneral Glossina morsitans centralis Machado were fed on the flanks of the African buffalo (Syncerus caffer Sparrman), N'Dama (Bos taurus L.) or Boran (Bos indicus L.) cattle infected with Trypanosoma congolense Broden. The infected tsetse were maintained on rabbits and on day 30 after the infected feed, the surviving tsetse were dissected to determine the infection rates. The mean infection rates (% +/- SE) in the midgut of tsetse fed on buffalo, N'Damas and Borans were 23.5 +/- 3.3, 31.6 +/- 2.7 and 33.7 +/- 4.6, respectively. The differences were not significant. However, the mean mature infection rate in tsetse fed on the buffalo (13.2 +/- 2.1%) was significantly lower compared to the rates in tsetse fed on the N'Dama (20.4 +/- 1.4) or the Boran cattle (21.4 +/- 1.1). When groups of teneral G.m.centralis, G.pallidipes Austen, G.p.gambiensis Vanderplank, G.f.fuscipes Newstead, G.brevipalpis Newstead and G.longipennis Corti were fed simultaneously on either an infected buffalo, an N'Dama or a Boran steer, the mature infection rates ranged from 0 to 16.1%. Irrespective of the host species used, the T.congolense infection rate was highest in G.m.centralis, lowest in the palpalis and fusca group tsetse, with G.pallidipes being intermediate. Nevertheless, the trypanoresistant African buffalo and N'Dama may serve as reservoirs of T.congolense as can trypanosusceptible Boran cattle.


Carbohydrate-binding specificities of anti-erythrocyte lectins (haemagglutinins) in Anopheles gambiae gut extracts and haemolymph. Lectins that agglutinate red blood cells (RBC) were demonstrated in Anopheles gambiae mosquito haemolymph and gut extracts. No apparent differences in haemagglutinin titres were detected between male and female mosquitoes and overall agglutinin levels were not increased following a bloodmeal. Titres were highest in the haemolymph and midgut extracts versus human AB, horse, chicken and goat RBCs and in hindgut against human AB, chicken and sheep; foregut extract gave relatively low titres. Adsorption of haemolymph and gut extracts with selected RBCs coupled with carbohydrate inhibition and the use of enzyme-treated RBCs revealed the presence of multiple (hetero-) agglutinins. An.gambiae lectins were specific for (1-1)-, (1-4)- or (1-6)-linked glucose based disaccharides, glucose and its (1-2) or (1-3) linkages with fructose and, to a lesser extent, aminated or N-acetylated glucose, or galactose and its deoxy derivatives. This study presents the first report of the occurrence of heterogenous anti-RBC agglutinins in haemolymph and gut extracts of the mosquito An.gambiae, together with the sugar-binding specificities of these lectins.


Modelling the effect of feeding-related mortality on the feeding strategy of tsetse (Diptera: Glossinidae). Free-living haematophagous insects risk death through host grooming responses or through increased susceptibility to predation whenever they take a bloodmeal. In this paper we investigate the effects of these risks on the feeding strategy of tsetse. A model is presented that allows for death of tsetse by starvation if they do not succeed in feeding within a fixed time (set at 6 days in the first instance) and for mortality specifically associated with feeding. In addition there is background mortality that applies to all flies at all times. The model is used to compute the individual life-time fertility (number of female puparia per female) as a function of the probability of obtaining a meal (indicated by field data to be very high, usually > 0.85 per day) and the day on which flies start to search for a meal. We suggest that the feeding strategy that would be selected for is that which allows the maximum reproductive output. The model shows that this strategy involves making no attempts to feed for 3-4 days after the previous meal and then attempting to feed with the greatest possible probability until a meal is obtained. The predicted feeding interval, obtained independently of any trapping data, agrees closely with all previous estimates from field studies using a variety of methods. Preliminary results from a laboratory experiment reveal an increased risk of predation of recently fed as compared with hungry tsetse. The lower the actual feeding mortality the more frequently will flies be able to feed should conditions so demand. It is adaptive, however, for tsetse to delay attempting to feed for as long as they can, which is made possible by the near certainty of locating and feeding on a host within 1 day, using their sophisticated sensory systems.


Effects of hydroprene on development and reproduction in the Oriental cockroach, Blatta orientalis. Nymphs of the Oriental cockroach Blatta orientalis were exposed during the penultimate and final instars to vinyl tiles treated with the juvenile hormone analogue hydroprene. Adults moulting from these nymphs exhibited deformities such as twisted wings in both sexes and modified genitalia in females. Females with deformities were unable to produce viable oothecae. B.orientalis confined on tiles treated with hydroprene at rates of 25-100 mg/m2 did not reproduce. The treatment rate of 10 mg/m2 reduced fecundity and caused 47-57% of resultant adults to have deformities, but some adults reproduced successfully and population growth was not suppressed.


Acquired immunity to larvae of Amblyomma marmoreum and A.hebraeum by tortoises, guinea-pigs and guinea-fowl. A study of acquired resistance in guinea-pigs, guinea-fowl and tortoises to larvae of the ticks Amblyomma hebraeum Koch and A.marmoreum Koch (Acari: Ixodidae) showed that repeated infestations of the laboratory host resulted in a 29.3-49.4% reduction respectively in weight of engorged larvae. No resistance was induced in guinea-fowl, the natural host for larvae of both species. A small decline in the mean weight of engorged larvae was observed for both species of ticks after three successive infestations on tortoises. The reduction in weight was more pronounced in A.hebraeum (14.5%) which does not naturally feed on tortoises than for A.marmoreum (8.4%), a common tick on this host. The occurrence of acquired resistance in natural tick host relationships as opposed to unnatural tick host relationships is discussed.


Phoretic and parasitic mites infesting the New World screwworm fly, Cochliomyia hominivorax, following sterile insect releases in Libya. During the screwworm eradication programme in Libya in 1991, trapped specimens of Cochliomyia hominivorax (Coquerel) were found infested with phoretic mites, Macrocheles muscaedomesticae (Scopoli), or parasitic mites, Trichotromidium muscarum (Riley). The possible sources of infestation and potential effects of mites on screwworm flies are discussed.


Effects of flavonoids released naturally from bean (Phaseolus vulgaris) on nodD-regulated gene transcription in Rhizobium leguminosarum bv. phaseoli. Nine flavonoid aglycones released from black bean (Phaseolus vulgaris 'PI165426CS') seeds and roots induced nodC::lacZ transcription in Rhizobium leguminosarum bv. phaseoli strains containing extra cloned copies of the regulatory genes nodD1, nodD2, or nodD3 from that biovar. Individual flavonoids generally induced highest levels of nodC::lacZ transcription (Imax) with extra copies of nodD2, and the concentration required for half-maximum induction (I50) was lowest with extra copies of nodD1 genes. One apparently unique feature of R. l. bv. phaseoli is that naturally released flavonoids with very diverse structures induce nod genes. For all three nodD genes, two compounds exuded from roots, genistein and naringenin, produced much higher levels of nodC::lacZ transcription than other flavonoids, but this fact was not explained by increased transcription of the nodD genes themselves. The remaining seven flavonoid aglycones showed reproducibly different capacities to induce nodC::lacZ transcription, but all were considerably less powerful inducers than genistein and naringenin in strains with extra copies of each of the nodD genes. Tests with glycosides of the nod-gene inducers showed that glycosides, which are normally released by bean, had lower I50 values than the corresponding aglycones with all nodD genes. Additive interactions observed between the strong nod-gene inducer genistein and the weak inducer eriodictyol remain to be explained at the molecular level.


252Cf plasma-desorption mass spectrometry of lipid A from Enterobacter agglomerans. Endotoxins from gram-negative bacteria are believed to be causative agents of byssinosis, an occupational pulmonary disease associated with exposure to cotton dust in textile mills. Lipid A preparations from Enterobacter agglomerans, a gram-negative bacterium commonly found in cotton and cotton dust, have been analyzed using plasma-desorption mass spectrometry. Results indicate the existence of at least two lipid A types which differ only by the presence of an additional oxygen atom whose position has been localized to the acyloxyacyl ester-linked side-chain of the distal portion of the molecule. The lower molecular weight compound of the two structures has the same molecular weight and presumably the same empirical formula as a well-characterized lipid A from Salmonella minnesota. The mass spectra of lipid A compounds obtained from S. minnesota and E. agglomerans show strong similarities. Palmitoyl, hydroxymyristoyl, myristoyl, and lauroyl side-chains which are known to be present in the former are inferred from spectral evidence to be present in the latter.


An Xanthomonas citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhosts. The pathogenicity gene, pthA, of Xanthomonas citri is required to elicit symptoms of Asiatic citrus canker disease; introduction of pthA into Xanthomonas strains that are mildly pathogenic or opportunistic on citrus confers the ability to induce cankers on citrus (S. Swarup, R. De Feyter, R. H. Brlansky, and D. W. Gabriel, Phytopathology 81:802-809, 1991). The structure and the function of pthA in other xanthomonads and in X. citri were further investigated. When pthA was introduced into strains of X. phaseoli and X. campestris pv. malvacearum (neither pathogenic to citrus), the transconjugants remained nonpathogenic to citrus and elicited a hypersensitive response (HR) on their respective hosts, bean and cotton. In X. c. pv. malvacearum, pthA conferred cultivar-specific avirulence. Structurally, pthA is highly similar to avrBs3 and avrBsP from X. c. pv. vesicatoria and to avrB4, avrb6, avrb7, avrBIn, avrB101, and avrB102 from X. c. pv. malvacearum. Surprisingly, marker-exchanged pthA::Tn5-gusA mutant B21.2 of X. citri specifically lost the ability to induce the nonhost HR on bean, but retained the ability to induce the nonhost HR on cotton. The loss of the ability of B21.2 to elicit an HR on bean was restored by introduction of cloned pthA, indicating that the genetics of the nonhost HR may be the same as that found in homologous interactions involving specific avr genes. In contrast with expectations of homologous HR reactions, however, elimination of pthA function (resulting in loss of HR) did not result in water-soaking or even moderate levels of growth in planta of X. citri on bean; the nonhost HR, therefore, may not be responsible for the "resistance" of bean to X. citri and may not limit the host range of X. citri on bean. The pleiotropic avirulence function of pthA and the heterologous HR of bean to X. citri are both evidently gratuitous.


Broad host range and promoter selection vectors for bacteria that interact with plants. A plasmid vector, pGV910, and a derived cosmid, pRG930, have been constructed. Both contain the ColE1 and pVS1 origins of replication and are stably maintained in Escherichia coli, Agrobacterium tumefaciens, and Azorhizobium caulinodans ORS571. They are compatible with commonly used IncP cloning vectors, although pVS1 was classified as an IncP plasmid, unable to replicate in E. coli (Y. Itoh, J.M. Watson, D. Haas, and T. Leisinger, Plasmid 11:206-220, 1984). Promoter selection vectors were derived from both of these plasmids by using a promoterless beta-glucuronidase and/or beta-galactosidase gene. These vectors facilitate the study of gene expression in bacteria under particular environmental conditions. This is illustrated by the expression of the gusA gene under the control of a nod promoter in A. caulinodans nodulating stem-located infection sites on Sesbania rostrata.


Mass spectrometry of DNA mixtures by laser ablation from frozen aqueous solution. We report time-of-flight mass spectra of test mixtures of six single-stranded DNA segments. The segments range in size from 8 to 60 nucleotides (molecular weight range 2413 to 18,602 Da). The best mass spectra were obtained by pulsed laser ablation of thin frozen films of an aqueous solution of the mixture from an oxidized copper substrate. These mass spectra are dominated by the molecular-ion peak for each DNA segment, and show little evidence of fragmentation, peak broadening or cluster formation. In contrast, mass spectra obtained using UV laser ablation from an anthranilic acid matrix yield broad peaks with evidence of fragmentation, and DNA segments longer than 26 nucleotides are difficult to detect.


Isolation and gene disruption of the Tox5 gene encoding trichodiene synthase in Gibberella pulicaris. The trichodiene synthase gene (Tox5) was isolated from Gibberella pulicaris, and its nucleotide sequence was determined. Tox5 was disrupted through transformation with a plasmid carrying a doubly truncated copy of the coding region and a selectable marker for resistance to hygromycin B (Hygr). Analysis of 82 transformants for their ability to produce the trichothecene, 4,15-diacetoxyscirpenol (DAS), resulted in the identification of five DAS- strains. Southern hybridization analysis of DAS- Hygr transformants indicated that the plasmid integrated at the Tox5 locus. The disrupted Tox5 gene was shown to be mitotically stable. Analysis of nine tetrads revealed either the cosegregation of the disrupter plasmid and the DAS- phenotype or the loss of the disrupter plasmid. These results demonstrate the feasibility of using gene disruption in G. pulicaris and suggest a general method for obtaining Tox5- mutants in other trichothecene-producing fungi.


Determination of 15N enrichment of taurine in cat urine by high resolution fast-atom bombardment mass spectrometry. A method is described for measuring the stable isotopic enrichment of taurine in cat urine samples by high resolution fast-atom bombardment mass spectrometry, after 15N labelled taurine was given to cats for the purpose of investigating taurine metabolism. The 15N enrichment of taurine was measured after hydrolysis and purification of taurine by anion/cation exchange chromatography. The isotopic ratio of taurine was determined by measuring the [M+H]+ ion peaks in the spectra of the unlabelled and labelled compounds under multiple ion scan conditions. The overall standard deviation of the measurement is better than 4%. This method requires no derivation and uses only 500 microL of urine samples.


Expression of cell adhesion molecules during initiation and cessation of neural crest cell migration. Because of their distribution and known ability to promote neuronal adhesion, it has been proposed that N-CAM and N-cadherin are involved in the formation of the nervous system. Here, we examine the expression of these molecules during the initiation and cessation of trunk neural crest cell migration during the formation of the peripheral nervous system. Whereas other neural tube cells express N-cadherin, the dorsal neural tube containing neural crest precursors has little or no N-cadherin immunoreactivity. In contrast, N-CAM is expressed in the dorsal neural tube and on early migrating neural crest cells, from which it gradually disappears during migration. Both N-CAM and N-cadherin are absent from neural crest cells at advanced stages of migration. As neural crest cells cease migration and condense to form dorsal root and sympathetic ganglia, N-cadherin but not N-CAM is observed on the forming ganglia, identified by neurofilament expression and the aggregation of HNK-1 reactive cells. The results demonstrate that the absence of N-cadherin correlates with the onset of neural crest migration and its reappearance correlates with cessation of migration and precedes gangliogenesis.


Molecular analyses of carbonic anhydrase-II expression and regulation in the developing chicken lens. The expression of carbonic anhydrase-II (CA-II) in the developing chicken lens was examined and compared with that in the retina of the chicken embryo. CA-II expression was measured by immunohistochemistry and radioimmunoassay during development, and CA-II mRNA was quantified by Northern blot and densitometric scanning and localized by in situ hybridization. A functional promoter of the chicken CA-II gene was identified by transfection of primary embryonic chicken lens epithelial cells and analyzed in deletion mutants. The results establish that CA-II makes up about 0.1% of the total soluble protein of the embryonic chicken lens, an amount insufficient to make it a candidate for an enzyme crystallin in this species. Lens fiber differentiation coincided with a loss of CA-II mRNA and protein; by contrast, CA-II persisted in the epithelial cells of the embryonic and mature lens. This and previous studies showed that CA-II amounts to as much as 3% of the protein of the embryonic chicken retina and follows a different developmental time course of expression; like the lens, CA-II decreases until day 10 in the embryonic retina, but, unlike the lens, it increases thereafter and plateaus at hatching. Progressive deletions of the 5' flanking regions (from position -1314 to +32) of the CA-II gene fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene resulted in a gradual loss of promoter activity, consistent with an additive effect of putative cis-regulatory elements found in many crystallin genes. These experiments provide the foundation for a molecular analysis of the developmental and differential regulation of the CA-II gene in lens and retina.


Association interneurons of embryonic rat spinal cord transiently express the cell surface glycoprotein SNAP/TAG-1. SNAP/TAG-1 is a 135 kDa glycoprotein of the immunoglobulin superfamily that is transiently expressed upon the surfaces of developing axons. In the embryonic rodent spinal cord, this molecule is expressed by motor neurons, dorsal root ganglion cells, and commissural neurons (Yamamoto et al.: J. Neurosci. 6:3576-3594, 1986; Dodd et al.: Neuron 1:105-116, 1988). The commissural cells are a subset of early-forming dorsal horn interneurons whose axons follow a circumferential course in the embryonic spinal cord. The axons of commissural neurons cross the developing ventral commissure to terminate on contralateral synaptic targets, whereas those of the other subset of circumferential cells, the association interneurons, remain on the same side of the spinal cord to form ipsilateral, terminal synaptic fields. The difference between the axonal trajectories of these two subsets of nerve cells raised the question of whether or not association interneurons would also express the SNAP/TAG-1 epitope and, if so, how would this expression be related to that of the commissural cells. Immunocytochemistry for SNAP/TAG-1 and choline acetyltransferase (ChAT) was used to answer these questions. The results indicated that association interneurons expressed SNAP/TAG-1 epitopes and that this expression began later and lasted longer than that of the commissural neurons. Other new findings of this study included the identification of a lateral subgroup of commissural fibers that expressed SNAP/TAG-1 later than their more medially located counterparts, and these lateral fibers were more pronounced in the thoracic spinal cord than at cervical levels.(ABSTRACT TRUNCATED AT 250 WORDS)


Distinct regions of human fibronectin are essential for fibril assembly in an in vivo developing system. In early vertebrate development, the proper assembly of fibronectin into fibrils is crucial for embryonic cells to adhere and to migrate on the extracellular matrix. The molecular mechanisms by which such a process occurs in vivo are poorly understood. In the amphibian embryo Pleurodeles waltl fibronectin fibrils appear first at the blastula stage. They form a fibrillar matrix on the basal surface of animal cells facing the blastocoel. Using competition and perturbation experiments with purified proteolytic fragments and domain-specific monoclonal antibodies, we demonstrate that at least three fibronectin sites are essential for assembly of fibronectin fibrils in the blastula of Pleurodeles waltl. Two sites, the RGDS sequence and the synergistic domain in the 10th type III repeat, are both involved in receptor recognition. A third site that spans the 9th type I and 1st type III homology sequences is also likely to participate in fibronectin-fibronectin interactions.


Structural and functional analysis of two different nodD genes in Bradyrhizobium japonicum USDA110. Bradyrhizobium japonicum has two closely linked homologs of the nodulation regulatory gene, nodD; these homologs are located upstream of and in divergent orientation to the nodYABCSUIJ gene cluster. We report here the nucleotide sequence and mutational analyses of both nodD copies. The predicted NodD1 and NodD2 proteins shared 62% identical amino acid residues at corresponding positions and exhibited different degrees of homology with NodD proteins of other Bradyrhizobium, Azorhizobium, and Rhizobium strains. Induction of the nodYABCSUIJ operon, as measured by expression of a translational nodC'-'lacZ fusion, required the nodD1 gene, but not nodD2. A B. japonicum mutant deleted for both nodD copies (strain delta 1267) still showed residual nodulation activity; however, nodulation of soybean was significantly delayed, and nodulation of mung bean and siratro resulted in strongly reduced nodule numbers. Fully efficient nodulation of mung bean and siratro by strain delta 1267 was restored by genetic complementation with the nodD1 gene, but not with nodD2. We conclude from these data that nodD1 is the critical gene that contributes to maximal nodulation efficiency, whereas the nodD2 gene does not play any obvious role in nodulation of the host plants tested.


A cyclical, developmentally-regulated death phenomenon in a colonial urochordate. Botryllus schlosseri is a colonial ascidian whose asexually derived, clonally modular systems of zooids exhibit developmental synchrony. The blastogenic cycle culminates in a phase of programmed cell and zooid death called takeover, in which all functional zooids die over a 30 hr period, and are replaced by a new generation of individuals. Because of the weekly recurrence and magnitude of visceral death in this model organism, we have begun to characterize the mechanisms that govern takeover. Here we describe a monoclonal antibody (B3F12.9) that recognizes a novel 57 Kd polypeptide (under reducing conditions) localized to the perivisceral extracellular matrix (PVEM) of buds and zooids, as well as blood cells of Botryllus by immunofluorescence and immunogold labeling of tissue sections. During their active feeding phase, zooids exhibited a uniform labeling pattern of PVEM along their anteroposterior (A-P) axis. At the onset of takeover (T = 3 hr), B3F12.9 immunostaining became diffuse or absent at the anterior end, which paralleled the axis of contraction of the dying zooid, whereas the posterior end retained its labeling integrity. During mid (T = 15 hr) to late (T = 28 hr) takeover, issue damage was extensive, large blood macrophages and other B3F12.9 immunoreactive blood cells invaded the peribranchial cavity, whereas PVEM labeling gradually disappeared along the entire A-P axis. These findings indicate that takeover is a dynamic process in which extracellular matrix breakdown proceeds in a polarized fashion, beginning at the anterior end of each zooid and gradually propagating toward the posterior end.


Changes in the activity and the polypeptide composition of the oxygen-evolving complex in photosystem II of tobacco leaves infected with cucumber mosaic virus strain Y. The amount of 22- and 23-kDa polypeptides in a 23-kDa protein family, which plays a regulatory role in photosynthetic oxygen evolution, decreased significantly during the progress of appearance of chlorotic spots in cucumber mosaic virus strain Y (CMV[Y])-inoculated tobacco (Nicotiana tabacum 'Ky57') leaves (Plant Mol. Biol. 16:689-698, 1991). The present study was conducted to determine if the amount of other polypeptides of the oxygen-evolving complex and the oxygen-evolving activity also decreased in CMV(Y)-inoculated tobacco leaves. The amount of a 33-kDa polypeptide, which is essential to oxygen evolution, did not decrease in CMV(Y)-inoculated leaves showing early symptoms, although the amount of 22- and 23-kDa polypeptides began to decrease. However, comparative analysis of electron transport in thylakoid membranes indicated that the oxygen-evolving activity in CMV(Y)-inoculated tobacco leaves was only partly reduced, compared with the activity in CMV(O)-inoculated tobacco leaves which did not show clear symptoms. Partial inhibition of the oxygen-evolving activity, by a differential decrease in the amount of polypeptides of the oxygen-evolving complex, seems to be associated with the primary molecular process of symptom expression in CMV(Y)-inoculated tobacco leaves.


Localization of type II collagen, long form alpha 1(IX) collagen, and short form alpha 1(IX) collagen transcripts in the developing chick notochord and axial skeleton. In this study we compare, by in situ hybridization, the spatial and temporal expression patterns of transcripts of avian type II collagen and the long and short forms of the (alpha 1) chain of type IX collagen during the development of the notochord and axial skeleton. We observed type II collagen and short form type IX collagen transcripts in the developing (stage 25-28) nonchondrogenic notochord. Conversely, long form type IX transcripts were not detectable in the notochord or perinotochordal sheath. Interestingly, all three transcripts colocalized in the developing chondrogenic vertebrae of the axial skeleton as well as in the chondrocranium and Meckel's cartilage. The expression of the short form of type IX collagen in these regions was more restricted than that of the long form. This report provides additional support for a complex regulatory pathway of cartilage marker gene expression in chondrogenic vs. nonchondrogenic tissues during avian embryogenesis.


Cardiac expression of polysialylated NCAM in the chicken embryo: correlation with the ventricular conduction system. The neural cell adhesion molecule (NCAM) and its polysialic acid moeity (PSA) affect cellular interactions during the development of the nervous system and skeletal muscle. NCAM has also been identified in the embryonic heart of various species including humans. However, knowledge regarding the role of NCAM and its function-modulating PSA in cardiogenesis is limited. The distribution of NCAM and its PSA in the ventricular myocardium of chicken embryos was determined by indirect immunofluorescence staining. The NCAM polypeptide was found throughout the cardiac myocardium. In contrast PSA was located in discrete regions in stage 20 to 44 embryos (during and after septation). Myocardium at the subendocardial regions of the atrioventricular canal and ventricular trabeculae were PSA positive by stage 20. At later stages, transverse sections of the postseptation heart just below the level of the atrioventricular interface revealed a PSA-positive bundle of myocardium in the septum. This bundle was continuous with two branches at a more apical level which in turn were continuous with the PSA-positive subendocardial myocardium lining the left and right ventricles. This pattern of PSA in the myocardium was similar to that of the ventricular conduction system configuration defined in the adult heart. Electron micrographs of the subendocardium of the ventricular septum revealed PSA positivity on myofibril-containing cells with the ultrastructural location of Purkinje fibers. At later stages (35-44) a subset of cells within PSA-positive regions was stained by an antibody against an isoform of the myosin heavy chain found in adult Purkinje fibers. These cells and surrounding tissue lacked PSA in the adult heart. Thus polysialylated NCAM may be modulating cell-cell interactions during the development of the ventricular conduction system.


Colonization of the developing pancreas by neural precursors from the bowel. Neurons in ganglia of the myenteric plexus of the duodenum and stomach have recently been demonstrated to innervate pancreatic ganglia and transsynaptically to excite acinar and islet cells. The hypothesis that crest-derived cells first colonize the foregut and secondarily enter the pancreas by way of the pancreatic buds was tested. Studies were done with fetal rats (days E11-E15). Pancreatic rudiments and foregut were explanted separately and in co-culture. The development of neurons in the explants, identified by demonstrating the immunoreactivities of neurofilaments and growth-associated protein-43 (GAP-43), provided an indirect assay for the presence of neural precursors in the tissue at the time of explantation. Cells of putative neural crest origin were visualized immunocytochemically using the monoclonal antibody, NC-1. Additional markers included the immunoreactivities of dopamine-beta-hydroxylase (DBH), which is expressed by vagal crest-derived cells that colonize the bowel, neuropeptides (substance P and neuropeptide Y [NPY]) found in mature pancreatic neurons, and serotonin (5-HT), which is located in the cell bodies of enteric but not pancreatic neurons. Neurons were detected in cultures of foregut, but not pancreas, when these tissues were explanted by themselves at days E11 and E12. At E11 neural precursors did not leave explants of bowel or migrate into co-cultured pancreatic rudiments. When the foregut was explanted at E12, however, neural precursors migrated away from the bowel, giving rise both to distant ganglia and to neurons within co-cultured pancreatic rudiments. Intrapancreatic ganglia developed in the co-cultures even when the pancreatic attachment to the bowel was severed. Neurons appeared in pancreatic rudiments explanted by themselves on day E13. Neurons developing in pancreatic explants expressed the immunoreactivities of DBH, substance P, and NPY, but not 5-HT. These observations support the idea that pancreatic ganglia develop from crest-derived cells that first colonize the fetal rat foregut and there acquire the ability to colonize the pancreas. A later migration into the pancreatic rudiments of a subset of the original émigrés or their progeny between days E12 and E13 gives rise to a network of pancreatic ganglia that can be regarded as an extension of the enteric nervous system.


Adenosine levels in the postimplantation mouse uterus: quantitation by HPLC-fluorometric detection and spatiotemporal regulation by 5'-nucleotidase and adenosine deaminase. Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryo-decidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5'-nucleotidase (5'-NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno-derivatized and quantitated by high-performance liquid chromatography-fluorescence detection (0.03 pmol/mg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmol/mg protein) and day 5 (0.59 nmol/mg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmol/mg protein) and day 7 (0.10 nmol/mg protein). The periimplantation adenosine surge coincided with uterine expression of 5'-NT, an enzyme which catalyzes the irreversible dephosphorylation of 5'-AMP to adenosine. 5'-NT expression was shown by Northern blot analysis to peak in the embryo-decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5'-NT activity was localized to the primary decidual zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5'-NT appeared on giant trophoblast (days 7-13) and the metrial gland (days 11-13). Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S-adenosylhomocysteine hydrolase) were not detected in the embryo-decidual unit suggesting that the net flux of utero-placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation. The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6-11), secondary giant cells (days 7-13), and spongiotrophoblasts (days 8-13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5'-NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities.(ABSTRACT TRUNCATED AT 400 WORDS)


Coincident loss of mitochondria and nuclei during lens fiber cell differentiation. During normal differentiation, lens fiber cells lose their nuclei, mitochondria, and other membrane-bound organelles. In the present study, a slice preparation of the embryonic chicken lens was used with laser scanning confocal microscopy to study the spatial and temporal patterns of organelle breakdown during embryonic development. At all stages examined, mitochondria in lens epithelial cells were present in perinuclear clusters. In contrast, early in development, lens fiber cells contained extremely elongated mitochondria (> 100 microns) that were distributed throughout the cytoplasm and oriented along the long axis of the cells. By the 8th day of embryonic development (E8), the mitochondria in the central fiber cells began to fragment. At the same time, the nuclei in these cells became smaller and more spherical. By E10, mitochondrial staining in the central fibers became punctate. Electron microscopy of this region revealed swollen mitochondria with disrupted cristae. By E12, cells in the central region of the lens lacked mitochondria and nuclei. The loss of nuclei and mitochondria from a given cell was coincident and abrupt (2-4 hr), occurring in a previously unsuspected domain situated about 300 microns from the anterior surface of the lens. A cytoskeletal component, actin, persisted in the central cells indicating that organelle degradation represents a selective process and not simply the global degradation of supramolecular structures. Throughout embryonic development, the organelle-free region grew at approximately the same rate as the lens and, by the time of hatching, had expanded to match the diameter of the pupil.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of NCAM isoforms during skeletal myogenesis in the mouse embryo. We have examined the developmental patterns of neural cell adhesion molecule (NCAM) gene expression in embryonic mouse skeletal muscle cells by in situ hybridization. Moreover, by utilising exon-specific cRNA probes, we have examined tissue specific splicing of the NCAM gene. We show that there is a distinct sequence of NCAM isoform expression during skeletal muscle development. Since NCAMs are also expressed in other cell types, particularly neurons, NCAM mRNAs have been colocalised with acetylcholine receptor alpha (AChR alpha) gene transcripts to identify muscle-specific expression. NCAM is first detected in somites as they first form, prior to their differentiation into muscle and nonmuscle compartments. Myotomes, the first skeletal muscle masses to form in the embryo, express mRNAs for the transmembrane 180 and 140 kDa isoforms of NCAM. Both of these transcripts are also detected in the neural tube, and their spatial pattern of expression changes with development. Transcripts containing the muscle-specific domain (MSD) of the NCAM gene are not detected prior to 11 days postcoitum (p.c.), at a time when rostral somites already contain well-developed myotomes. As the level of MSD mRNAs increases at 12 days p.c., the 140 and 180 kDa transcript levels decrease in skeletal muscle masses. The level of all NCAM isoform transcripts declines between 13 and 15 days p.c. in muscle. However, the 180 and 140 kDa NCAM isoforms are expressed at a high level in neural tissue and in other locations in the developing embryo such as in smooth muscle, around vibrissae follicles, and in the perichondrial zone of digits.


High-intensity nociceptive stimuli minimize behavioral effects induced by restraining stress during the tail-flick test. Analgesia following exposure to various stressors is a well-documented phenomenon. Restraint of an animal during the tail-flick test (TFT) represents a potent stressor that can induce both altered baseline latencies and enhanced response to opioids. The present study shows that the use of higher stimulus intensities during TFT minimizes the stress influences produced by restraint on the animal's response rendering the test more sensitive to the pharmacological action of analgesic drugs.


Cell-growth quantitation methods for the evaluation of antiestrogens in human breast cancer cells in culture. The antiproliferative activity of the antiestrogen, tamoxifen, on the growth of MCF-7 human breast cancer cells was evaluated using the hemocytometric trypan blue exclusion method, [3H]-thymidine incorporation, and a total protein determination. Tamoxifen was evaluated over a concentration range from 10(-9) to 10(-6) M. The hemocytometric trypan blue exclusion method and [3H]-thymidine incorporation were sensitive enough to demonstrate the inhibitory influence of tamoxifen on the proliferation of MCF-7 cells at a concentration as low as 10(-9) M. A very good correlation of these two methods was observed in the submicromolar concentration range of tamoxifen. The total protein determination method was only sensitive enough to detect the antiproliferative influence of tamoxifen at concentrations above 10(-6) M. In conclusion, the [3H]-thymidine incorporation method was found to be effective and much less time consuming than the hemocytometric trypan blue exclusion method for evaluating the antiproliferative effects of antiestrogens in cultured MCF-7 cells. However, when evaluating antiestrogens, which are cell-cycle specific, the results of the [3H]-thymidine incorporation method should be interpreted with caution.


Quantitative autoradiography of 3H-paroxetine binding sites in rat brain. The use of 3H-paroxetine as a ligand for quantitative autoradiography of serotonin (5-HT) transport sites was optimized, and the distribution of 3H-paroxetine binding sites in rat brain was studied. Under the conditions described, 3H-paroxetine binding in forebrain sections was of high affinity and saturable, with a Kd of 0.18 +/- 0.02 nM (mean +/- SEM) and Bmax of 268 +/- 12 fmol/mg of protein (n = 3). Nonspecific binding was 10.7% +/- 1.0 of the total binding (n = 8). The distribution of 3H-paroxetine binding sites closely matched the regional distribution of 5-HT nerve terminals and cell bodies. The highest concentrations of 3H-paroxetine binding sites were found in the dorsal raphe nucleus (563 +/- 55 fmol/mg tissue, n = 4), and high densities of binding were also found in the locus coeruleus, medial forebrain bundle, substantia nigra, several limbic structures (amygdala, hippocampus, hypothalamus, olfactory tubercle, septum, and thalamus), and components of the visual relay system (superior colliculus and the lateral geniculate body). Although lesioning of 5-HT neurons with p-chloroamphetamine (PCA) drastically eliminated 3H-paroxetine binding in most regions of the rat brain, significant binding remained in the raphe nuclei and medial forebrain bundle suggesting that 3H-paroxetine binding in these regions was to presynaptic sites on cell bodies or axons relatively resistant to PCA.


Three-dimensional electrocardiography in the rat. Three-dimensional electrocardiography with computerized analysis provides a means to rapidly collect and quantitatively analyze electrocardiographic data from rats. Electrical activity at each point in time can be expressed quantitatively as a vector with magnitude and direction. Rats are fitted with plate and needle electrodes in three mutually perpendicular axes: X (right to left), Y (rostral to caudal), and Z (ventral to dorsal). Electrocardiographic signals are recorded on paper and also collected at 1000/sec using an IBM-compatible computer equipped with a fast A/D convertor. Signals are analyzed using an interactive program written in Turbo PASCAL which transforms data into an array consisting of vector magnitude and direction at each time point. The data are further used to produce both a tabular report and graphic output. This method retains simplicity while extending the accuracy of electrocardiographic (ECG) measurement. It literally adds another dimension of information to the three-lead ECG most commonly measured in rats.


Venous thrombosis produced in the vena cava of rabbits by vascular damage and stasis. The main objective of these experiments was to develop and characterize a new experimental model of venous thrombosis, and determine whether a combination of vascular wall damage (crushing with hemostat clamps) and prolonged stasis produced more reproducible clots than prolonged stasis per se. Rabbits were laparotomized, and a segment of the vena cava was dissected free and looped with two silk ligatures, 2.5 cm apart. The proximal tie was ligated 5 min after release of the clamps; the distal tie applied shortly thereafter, trapping a volume of blood in the isolated segment. At 2 hr after ligation, the isolated venous sac was excised and examined for the presence of a clot. Large, well-formed clots, which could be readily transferred and weighed, were invariably observed in the "clamp" and "no clamp" groups, the latter being a sham control. Mean clot weights did not differ in the two groups (23.1 +/- 1.6 versus 30.8 +/- 5.4 mg dry weight, clamped versus no clamp, respectively, p greater than 0.05). However, the precision of the method was improved significantly (p less than 0.005) by clamping as determined by homogeneity of variance testing. Time-course studies showed that a considerable lag period (about 60 min) preceded development of a detectable clot, and that the thrombus evolved rapidly during the interval of 60-90 min postligation. The location of the small clots at 60 min in clamped segments, as well as the failure of prolonged (120 min) stasis without caval isolation to cause substantial thrombi, strongly suggests that clot formation attributed to "stasis per se" is in fact due to focal vascular lesions created at the tie-down points. The present study is also the first report of blockade of localized venous thrombosis by recombinant tissue factor pathway inhibitor (rTFPI). When given as an i.v. bolus 20 min prior to ligation, rTFPI at 400 and 800 micrograms/kg completely blocked formation of the thrombus or greatly reduced its size in five of the six animals tested.


A computerized method for measurement and analysis of blood pressure in small animals. A method is described for computerized measurement of blood pressure (BP) in small animals. As described, the system measures BP in up to four experimental animals at one time by sequential sampling but can be expanded to take data from up to 10. It requires an Apple II computer, and is run from a Grass pen-recording system. Pulse rate and systolic and diastolic BP are measured, and these variables are used to calculate mean arterial pressure (MAP) and the time integral of MAP minute by minute. The data are displayed in real time and stored hourly on floppy disc for further analysis.


Gastric mucosal damage due to aspirin and copper aspirinate assessed by gastric mucosal potential difference changes. When the gastric mucosa is damaged by antiinflammatory agents, such as aspirin, gastric mucosal potential difference (GPD) decreases and may or may not return to predamage values after the agent is removed. The magnitude and time course of the gastric potential difference changes have been suggested as a measure of mucosal damage. Male Sprague-Dawley rats were fasted, anesthetized, and surgically prepared for measurement of GPD by placement of electrodes in the gastric lumen and spleen. Test mixtures of aspirin, copper aspirinate, copper sulfate, or mixtures of aspirin and copper sulfate were administered by gavage, and subsequent changes in GPD were recorded. The area between the extrapolated control (baseline) GPD and the damage GPD was determined. The product of this area and the maximum change in GPD, the Reizindex (RI), was calculated. Values for all copper (II)-containing systems, including those with no aspirin, were significantly greater than aspirin alone. It was concluded that factors other than mucosal damage may contribute to a reduction in GPD and, therefore, an increase in Reizindex.


Laparoscopic hernioplasty update.

Laparoscopic hernioplasty update. The Toy-Smoot laparoscopic hernioplasty has been performed on 75 patients with a total of 83 hernioplasties over the past 20 months. Sixty-nine patients were male and six were female. The age range was 20 to 75 years with an average age of 51.5 years. Twelve of the patients had bilateral hernias repaired: 55 direct hernias, 16 indirect hernias, and 5 pantaloon hernias. Eleven of the repairs were for recurrent hernias. The procedure was performed under general anesthesia. The abdomen was insufflated with carbon dioxide, establishing the pneumoperitoneum. Three 11 mm trocars were inserted, the first via the umbilicus, into which the 0 degrees endoscope was inserted. Two additional trocars were inserted at the level of the umbilicus at the anterior axillary lines. The hernia sacs were left in situ. The medial umbilical ligament was dissected medially, so as to identify directly the pubic tubercle and the Cooper's ligament. An expanded PTFE soft tissue patch, 1 mm thick and 7.5 x 10 cm in size, was attached to the Nanticoke Endo-patch spreader and introduced via the contralateral trocar and positioned over the hernia defect. The Endopath EMS stapler was then used to secure the PTFE patch over the hernia defect. This required secure anatomical fixation to the transversalis fascia anteriorly and laterally, the pubic tubercle, and the posterior rectus sheath, medially, Cooper's ligament, posteromedially, and the endoabdominal fascia, posterolaterally. There were a total of seven different complications, one major, which was a bladder injury that required an open repair of the bladder and then an open, conventional hernioplasty.(ABSTRACT TRUNCATED AT 250 WORDS)


A novel endoscopic transgastric fundoplication procedure for gastroesophageal reflux: an initial animal evaluation. Complications of gastroesophageal reflux are common in adults and children. A variety of surgical procedures have been successfully used to treat this condition. All current techniques require open surgery and are associated with significant morbidity and mortality, particularly in the high risk, neurologically damaged child. The authors have developed a novel method of creating a gastric fundoplication using an endoscopic transgastric approach in pigs. The procedure involves the creation of an esophageal intussusception into the stomach under direct vision with an endoscope. The gastric fundus is then plicated around the esophagus using a custom stapling device. The transgastric approach allows creation of a fundoplication, is easily performed, is potentially effective at preventing gastroesophageal reflux, and has the obvious advantages of minimally invasive surgery over open techniques of fundoplication.


Why catheterize the bladder for laparoscopic cholecystectomy? Bladder catheterization is widely employed for laparoscopic cholecystectomy. Fifty consecutive laparoscopic cholecystectomies were performed without bladder catheterization. Patients were asked to void shortly before induction of anesthesia to ensure that the bladder was empty. Postoperative bladder catheterization for urinary retention was required in 3 of the 50 patients. Routine bladder catheterization for laparoscopic cholecystectomy is unnecessary, and its elimination will reduce costs, urethral trauma, and nosocomial urinary tract infections.


Extraperitoneal endoscopic pelvic lymph node dissection vs. laparoscopic lymph node dissection in the staging of prostatic and bladder carcinoma. Eighteen patients undergoing laparoscopic pelvic lymphadenectomy were compared with eighteen patients undergoing lymph node dissection performed via a totally extraperitoneal approach called extraperitoneal endoscopic pelvic lymph node dissection. Operative time, nodal yield, and hospital stays were essentially the same in both groups. However, the laparoscopic approach had a greater incidence of morbidity, leading the authors to adopt a totally extra-peritoneal endoscopic approach to pelvic lymph node dissection. Advantages of using an extraperitoneal approach are presented.


Three-dimensional transrectal ultrasound: preliminary patient evaluation. Transrectal ultrasound is uniquely useful in diagnosing and localizing the extent of lower colon and rectal disease. This paper reports the preliminary evaluation of three-dimensional intraluminal ultrasound imaging of normal and diseased rectal segments. The three-dimensional reconstructions were produced using a computerized PC based image analysis system which aligns ultrasound images to produce the three-dimensional images. A unique perspective for displaying both normal and pathologic anatomy is achieved using this new technology and the method has promising diagnostic and therapeutic potentials.


Hernioscopy: laparoscopy via an inguinal hernia sac. The use of laparoscopy in general surgery has expanded to aid in the diagnosis of various intraabdominal conditions. A new application of the laparoscope is described to explore the abdominal cavity for potential gangrenous bowel after reduction of an incarcerated inguinal hernia. Hernioscopy allows for a good view of the entire abdomen including inspection of the colon for serosal changes of carcinoma. Exploration via this technique is accomplished without additional skin incisions or fascial disruptions and with no increased morbidity, when performed during the inguinal herniorrhaphy.


Aortic laceration: a rare complication of laparoscopy. Aortic laceration during laparoscopic procedures is a rare but well-known complication with a high mortality rate. Thus far, few cases which were recognized and treated successfully have been reported in the literature; the exact incidence is not known. Such a complication occurred after an elective laparoscopic sterilization in a 35-year-old woman. The situation was recognized early and successfully treated. The common complications of laparoscopy are usually of a minor nature but a few are life threatening. This case illustrates the need for emphasis on the prevention of complications and the appropriate course of action in the event of their occurrence.


Laparoscopy in the diagnosis and treatment of acute small bowel obstruction. The surgical correction of acute small bowel obstruction is conventionally performed through a vertical laparotomy incision. The increasing use of the laparoscope for elective general surgery has led to an increase in its use for the diagnosis and treatment of acute abdominal conditions. The authors report five cases of acute small bowel obstruction treated with the aid of the laparoscope. All five patients were able to leave the hospital in the early postoperative period and remain symptom free. Laparoscopy is a useful technique in the management of selected cases of small bowel obstruction.


Laparoscopic cholangiography: a new method and device. Laparoscopic cholangiography is invaluable in the detection of bile duct stones, visualization of ductal anomalies and discovery and prevention of iatrogenic injuries to the bile ducts. The use of this procedure is, however, limited by the technical difficulties of cystic duct cannulation. A method of laparoscopic cholangiography that avoids cystic duct cannulation is described. It consists of a Kumar I Cholangiography Clamp that is applied across the gallbladder just above the Hartmann's pouch and divides the gallbladder into a medial and lateral compartment. A Kumar Cholangio Catheter is then introduced through the side-channel of the clamp, puncturing the medial compartment (i.e. the Hartmann's pouch) with a short #22 gauge needle for aspiration, followed by dye injection. Cholangiography was obtained with remarkable ease in 49 of the first 50 cases (98%) without complication.


Percutaneous endoscopic implantation of Automatic Implantable Cardioverter/Defibrillator (AICD): an animal study of a new nonthoracotomy technique. The Automatic Implantable Cardioverter/Defibrillator (AICD) prevents death due to malignant ventricular arrhythmias but requires thoracotomy for the implantation of the preferred two-patch lead system. The purpose of this study was to develop and test a new percutaneous endoscopic method of the AICD lead implantation without the need for open chest surgery. A high resolution video endoscopy system and currently available endoscopic instrumentation were used to develop pleural-pericardial dissection technique in 7 pigs and to endoscopically implant custom-made AICD patches in 20 pigs. An examining 10 mm rigid endoscope inserted in the 6th intercostal space in the anterior axillary line provided direct visual control for endoscopic dissection of the parietal pleura from the pericardium, delivery, and implantation of the AICD patches. This was successfully carried out through two trocars (10 and 11 mm) inserted into the pleural-pericardial space via the subxyphoid approach in 18 of 20 pigs. Effective patch positioning was confirmed by attaining a defibrillation threshold of 20J or less in 13 pigs. Of those, three required lead polarity reversal, and three others required lead repositioning to lower defibrillation thresholds to 20J or less. In three pigs, defibrillation thresholds of 30J or higher were required. Defibrillation was unsuccessful in two pigs due to patch malfunction. The authors conclude that percutaneous endoscopy is a feasible method of AICD lead implantation.


Laparoscopic pericardial window.

Laparoscopic pericardial window. The patient with a malignant pericardial effusion can be extremely difficult to treat and provide optimal palliation. The performance of transabdominal laparoscopic pericardial window may prove to be a significant advance in the management of these individuals. No previously reported description of this procedure has been found in the world literature as far back as 1966. A presentation of the case, description of the surgical procedure, and discussion of the therapeutic implications are provided.


A technique for extracorporeal suturing. Advances in laparoscopic surgical procedures will be facilitated by the ability to perform suturing efficiently and effectively. A simple technique of extracorporeal suturing is described, which permits rapid and reliable knot placement for a wide variety of laparoscopic procedures.


Ultrastructural histopathology of human olfactory dysfunction. This paper presents electron-microscopic observations on biopsies of the olfactory mucosae of several classes of patients with smell disorders: 1) patients with loss of smell function following head injury (post-traumatic anosmics or hyposmics); 2) patients with loss of smell function following severe head colds and/or sinus infections (post-viral olfactory dysfunction, or PVOD); and 3) patients that have lacked smell function since birth (congenital anosmics). Of these, the traumatic anosmics' olfactory epithelia were quite disorganized; the orderly arrangement of supporting cells, ciliated olfactory receptor neurons, microvillar cells, and basal cells was disrupted. Although many somata of ciliated olfactory receptors were present, few of their dendrites reached the epithelial surface. The few olfactory vesicles present usually lacked olfactory cilia. The post-viral anosmics, too, had a greatly reduced number of intact ciliated olfactory receptor neurons, and most of those present were aciliate. The post-viral hyposmics had a larger population of intact, ciliated olfactory receptor cells. In the seven cases of congenital anosmia studied, no biopsies of olfactory epithelium were obtained, indicating the olfactory epithelium is either absent--or greatly reduced in area--in these individuals.


Fine structural aspects of secretion and extrinsic innervation in the olfactory mucosa. The mucus at the surface of the olfactory mucosa constitutes the milieu in which perireceptor events associated with olfactory transduction occur. In this review, the ultrastructure of olfactory mucus and of the secretory cells that synthesize and secrete olfactory mucus in the vertebrate olfactory mucosa is described. Bowman's glands are present in the olfactory mucosa of all vertebrates except fish. They consist of acini, which may contain mucous or serous cells or both, and ducts that traverse the olfactory epithelium to deliver secretions to the epithelial surface. Sustentacular cells are present in the olfactory epithelium of all vertebrates. In fish, amphibia, reptiles, and birds, they are secretory; in mammals, they generally are considered to be "non-secretory," although they may participate in the regulation of the mucous composition through micropinocytotic secretion and uptake. Goblet cells occur in the olfactory epithelium of fish and secrete a mucous product. Secretion from Bowman's glands and vasomotor activity in the olfactory mucosa are regulated by neural elements extrinsic to the primary olfactory neurons. Nerve fibers described in early anatomical studies and characterized by immunohistochemical studies contain a variety of neuroactive peptides and have several targets within the olfactory mucosa. Ultrastructural studies of nerve terminals in the olfactory mucosa have demonstrated the presence of adrenergic, cholinergic and peptidergic input to glands, blood vessels, and melanocytes in the lamina propria and of peptidergic terminals in the olfactory epithelium. The neural origins of the extrinsic nerve fibers and terminals are the trigeminal, terminal, and autonomic systems.


Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: ultrastructure and uptake of tracers. Mammalian olfactory neurons possess a well-developed system of endocytic vesicles, endosomes, and lysosomes in their dendrites and perikarya. Vomeronasal neurons are similar and also contain much perikaryal agranular endoplasmic reticulum (AER). Olfactory supporting cells contain endocytic vesicles and endosomes associated closely with abundant fenestrated AER, and vesicles and numerous large dense vacuoles are present basally. Vomeronasal supporting cells have little AER, and few dense vacuoles occur in their bases. In olfactory neurons, ultrastructural tracers (0.08% horseradish peroxidase, thorium dioxide, ferritin) are endocytosed by olfactory receptor endings and transported to the cell body, where their movement is halted in lysosomes. Higher concentrations (1%) of horseradish peroxidase penetrate olfactory receptor plasma membranes and intercellular junctions. In olfactory supporting cells, endocytosed tracers pass through endosomes to accumulate in dense basal vacuoles. These observations indicate that olfactory sensory membranes are rapidly cycled and that endocytosed materials are trapped within the epithelium. It is proposed that in the olfactory epithelium, endocytosis presents redundant odorants to the enzymes of the supporting cell AER to prevent their accumulation, whereas in the vomeronasal epithelium the receptor cells carry out this activity.


Ultrastructural studies of microtubules and microtubule organizing centers of the vertebrate olfactory neuron. The olfactory neuron is specialized along its length into highly determined morphological regions. These regions include the dendritic cilia, dendritic vesicle, dendritic shaft proper, perikaryon, axon, zone of transition where the axon widens as it approaches its termination, and the axon terminal. Except for the zone of transition and the terminal, characteristic populations of microtubules occur in these compartments. In the olfactory vesicle, three discrete microtubule organizing centers (MTOCs) nucleate microtubules: the basal body, the lateral foot associated with the body, and dense masses of nearby material. Little is known about MTOCs elsewhere in the neuron, although the polarity of the axonal microtubules indicate that they originate at or near the perikaryon. An attempt is made to summarize what is known of the origin, structure, distribution, and function of microtubules in vertebrate olfactory neurons, which are useful model systems in which to study microtubules. Information about olfactory neuron microtubules may be applicable to neurons in general (e.g., the discovery that axons contain microtubules of uniform polarity was first made in the olfactory neuron) or to microtubules in other eukaryotic cells.


Growth of olfactory epithelial tissue in vitro: lectin staining of axons. In vitro methods have been used to study several aspects of development of olfactory epithelium. In this paper, the value of growing olfactory tissue in explant cultures is reviewed and some experiments are reported on the identification of lectin receptors on olfactory axons by the use of lectin-gold complexes. Both concanavalin A-gold (con A-gold) and wheat germ agglutinin-gold consistently decorated olfactory axons in explant cultures. Con A-gold also bound to the tips of growth cone filopodia, suggesting the glycoconjugate molecules containing alpha-methyl-pyranoside are important in adherence of growth cones to their substrate. The wide range in density of lectin-gold particles suggested that axons, and the sensory cells from which they arise, are not a uniform population, i.e., they have different molecular fingerprints. This was supported by the observation that soybean agglutinin-gold stained some axons very well, but others remained unstained. Peanut agglutinin did not bind to any axons.


Lectins bind differentially to cilia and microvilli of major and minor cell populations in olfactory and nasal respiratory epithelia. Binding of colloidal gold-conjugated lectins was studied in cilia and microvilli of rat olfactory and respiratory epithelia. This was done in sections of rapidly frozen, freeze-substituted specimens embedded in Lowicryl K11M or, for wheat germ agglutinin (WGA) alone, in deep-etched replicas. Olfactory dendritic endings and cilia labeled with WGA and faintly with soybean agglutinin (SBA); olfactory supporting cell microvilli bound only Dolichos biflorus agglutinin (DBA). Microvilli of an infrequent cell bound peanut agglutinin (PNA), SBA, and WGA. These microvilli labeled more strongly with the last two lectins than the olfactory cilia. Respiratory cilia bound WGA and, somewhat more weakly, PNA; microvilli of ciliated respiratory cells bound all four lectins. Visualization of specific labeling improved after preincubation of sections with neuraminidase, except for DBA where lectin binding was abolished. PNA labeling was seen only after neuraminidase preincubation. The densities of membrane surface particles that labeled with WGA corresponded with those of fracture plane particles in a quantitative freeze-fracture, deep-etch analysis. Therefore, a considerable fraction of the WGA-bound particles could reflect transmembrane proteins in olfactory dendritic endings and cilia and in respiratory cilia. The possible nature of these particles is discussed.


Depo provera treatment for sex offending behavior: an evaluation of outcome. Forty men, ages 16 to 78 years, with sex-offending behavior, were treated with combined medroxyprogesterone acetate (MPA), group therapy, and individual psychotherapy. Twenty-three are pedophiles; seven, rapists; and 10, exhibitionists. Five had sex-offending behavior that began after head trauma. The duration of MPA therapy, usual intramuscular dose 400 mg/wk, ranged from six months to 12 years, usually more than two years. These men were compared with a control group of 21 men who refused MPA therapy. They had similar types of sex-offending behavior and were treated with psychotherapy alone with follow-up for a period that ranged from two to 12 years. MPA-related side effects included excessive weight gain, malaise, migraine headaches, severe leg cramps, elevation of blood pressure, gastrointestinal complaints, gallbladder stones, and diabetes mellitus. Of the 40 individuals who took MPA, 10 are still on therapy. Eighteen percent reoffended while receiving MPA therapy; 35 percent reoffended after stopping MPA. In contrast, 58 percent of the control patients, who refused and never received MPA, reoffended. Patients defined as regressed were much more likely to reoffend off therapy than the patients defined as fixated. Other risk factors for reoffense include elevated baseline testosterone, previous head injury, never forming a marriage relationship, and alcohol and drug abuse. In spite of significant medical side effects, maintenance MPA offers benefit for the compulsive sex offender by reducing the reoffense rate.


Incompetence, treatment refusal, and hospitalization. Psychiatrists have proposed broadened commitment statues based on need for care and treatment, and under which judges have no role in deciding cases of treatment refusal. The mental health bar has consistently opposed these proposals on constitutional and common law grounds. The authors propose new commitment criteria based on incompetency to decide about hospitalization, and inability to live safely in freedom. The proposed standards would meet the Constitutional requirements, and would permit hospitalization and/or treatment for many persons who are in need but who now go without. The authors recognize that new commitment law without adequate clinical resources will not greatly improve patient care.


Treatment boundary violations: clinical, ethical, and legal considerations. The observance of treatment boundaries maintains the integrity of the therapist-patient relationship. It is the therapist's professional duty to establish appropriate treatment boundaries. Basic boundary guidelines are reviewed. The principles underlying these boundary guidelines are explored. A clinical vignette describing the sexual exploitation of a patient by her therapist dramatically illustrates progressive boundary violations. Boundary violations involving money are particularly common. Double agent roles also are likely to lead to the establishment of dissonant treatment boundaries with patients. The clinical, ethical, and legal issues surrounding the maintenance of treatment boundaries are discussed.


Approaches to forensic assessment of false claims of sexual misconduct by therapists. The author offers systematic approaches to evaluation of claims of therapist-patient sexual misconduct, together with their rationales. False accusations should be considered in all such cases in order to maintain the balanced forensic perspective and to probe for malingering, as in all evaluations. Practical techniques are offered and the underlying reasoning described.


Collision between law and ethics: consent for treatment with adolescents. The dilemmas between legal obligations and ethical responsibilities can often create problems in clinical work. The treatment of minors, and particularly adolescents, can present special issues to the clinician that are becoming increasingly frequent and difficult. The issue of informed consent for treatment of adolescents raises serious questions for the clinical practitioner who is faced with both legal and ethical dilemmas in making decisions about treatment. There are an increasing number of cases where adolescents may seek treatment yet are in circumstances that preclude parental consent. This paper uses case material to illustrate some of the legal, ethical, and treatment considerations in the situation of adolescent treatment where parental consent is problematic.


The occupational hazards of jury duty.

The occupational hazards of jury duty. Jurors on criminal trials carry a considerable burden of responsibility. They determine the defendant's fate. Additionally, during trials they can be exposed to stressful, frightening, and sordid aspects of life. The stressfulness varies depending upon the nature of the trial, its length, the nature of the testimony and evidence, the jurors' interpersonal relationships, the difficulty establishing guilt or innocence, the public's attitude, etc. These experiences can create psychological and/or physical discomfort that can be transient and mildly or moderately intense, or more serious and constitute illness. The authors have studied juries of four criminal trials--two murder cases, one child abuse case, and one obscenity case. Forty jurors were interviewed. Twenty-seven had one or more discomforting physical and/or physiological symptoms. These involved gastrointestinal distress (10 jurors); generalized nervousness (4 jurors); heart palpitation (6 jurors); headaches (4 jurors); sexual inhibitions (4 jurors); depression (4 jurors); anorexia (4 jurors); faintness (2 jurors); and numbness, lump in throat, chest pain, hives, and flu (1 juror each). Seven of the jurors became clearly ill. Illnesses included: peptic ulcer reactivation and hives, phobic reaction, anxiety state and increased alcohol use, hypertensive episode and visual scotomata, sexual inhibition, chills, fever, and depression, and post-traumatic stress disorder.


The Tarasoff raid: a new extension of the duty to protect. The authors discuss the general outlines of the Tarasoff duty of psychotherapists to protect potential victims of their violent patients. They describe the flexible range of clinical responses that therapists have utilized, as well as their professional concerns about preserving patient confidentiality (or at least strictly circumscribing the scope of disclosure when confidentiality must be breached). A recent case is reported that illustrates a striking new extension of Tarasoff, involving a police search and seizure of a psychotherapist's confidential treatment records and tapes, in response to a third-party complaint that the records contained evidence of his patients' violent acts and propensities. The implications of this case are that the therapist's discretion in the assessment of his duty to protect, the selection of a proper course of action, and the implementation of specific responses may be taken out of his hands, for all intents and purposes, and expropriated by law and order officials. Moreover, regardless of whatever clinical approach he adopts and whether or not he issues a warning, his attempts to preserve patient confidentiality are bound to prove unsuccessful in any future legal proceedings. Patient communications are likely to lose their confidential status on the grounds that they caused or triggered the Tarasoff warning (or that they should have triggered it). If the patient directed serious threats against the therapist himself, the court may find that, as a consequence, a "genuine therapeutic relationship" ceased to exist and thereafter all patient disclosures were no longer confidential on that basis.(ABSTRACT TRUNCATED AT 250 WORDS)


Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts. Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways.


Ability of adenovirus 5 E1A proteins to suppress differentiation of BC3H1 myoblasts correlates with their binding to a 300 kDa cellular protein. We have used deletion mutants to define the regions in Ad5 E1A proteins necessary to suppress differentiation of mouse BC3H1 myoblasts. We examined the differentiation of cells infected at a low multiplicity with viruses containing the E1A deletions and constructed so as to produce only the smaller of the two major E1A proteins. Only four of the mutant viruses containing deletions within the N-terminal 69 residues failed to suppress differentiation as judged by changes in morphology and in levels of muscle-specific alpha-actin mRNA and creatine kinase activity. The results were confirmed by analyses of lines of cells stably transfected with representative E1A mutants. The mouse cellular proteins to which mutant E1A proteins bound were identified by immunoprecipitating E1A proteins specifically from infected BC3H1 cells and by analyzing the precipitates on denaturing gels. Bands of proteins of 300, 130, 107, 105 (the retinoblastoma product), and 60 kDa (cyclin A) were distinguished. Failure to suppress differentiation correlated with loss of binding to the 300-kDa protein but not to any of the others. The regions of E1A defined in this way have been shown to be required for several other activities, including enhancer repression and transformation. One function of the 300-kDa protein appears to be to facilitate the action of transcriptional enhancers of differentiation-specific genes.


Inhibition of c-Jun DNA binding by mitogen-activated protein kinase. Here we demonstrate that partially purified Xenopus p42 mitogen-activated protein (MAP) kinase phosphorylates bacterially expressed human c-Jun at a single site, serine 243. Several lines of evidence argue that this phosphorylation is due to p42 MAP kinase itself rather than some contaminating species. Phosphorylation of serine 243 markedly decreases the binding of c-Jun to oligonucleotides containing the 12-O-tetradecanoylphorbol-13-acetate response element. These findings suggest that MAP kinase may play a role in the down-regulation of c-Jun or in the cycle of transcriptional initiation and elongation.


Induction of NF-kappa B-like activity by platelet-derived growth factor in mouse fibroblasts. Nuclear factor kappa B (NF-kappa B) modulates the expression of numerous genes via interaction with a specific DNA sequence termed the kappa B site. Its activity is modulated by a cytosolic inhibitor protein termed I kappa B, and its activation occurs in response to a variety of agents in a variety of cell types, most notably B and T lymphocytes. Data presented here show that an activity (designated complex I) that binds specifically to the kappa B site is induced in density-arrested Balb/c-3T3 mouse fibroblasts by platelet-derived growth factor (PDGF), a potent mitogen for these cells. Increased levels of complex I, as evaluated by electrophoretic mobility shift assays of nuclear extracts, were observed in cells treated for 1-4 h (but not 15 min) with the BB isoform of PDGF. 12-O-tetradecanoylphorbol 13-acetate (TPA) and the AA isoform of PDGF also stimulated this response and both isoforms, but not TPA, were effective in cells depleted of protein kinase C. Complex I most likely is authentic NF-kappa B, a p50-p65 heterodimer, or a closely related factor because it exhibited properties characteristic of those previously described for NF-kappa B including inducibility by deoxycholate and cycloheximide and sensitivity to I kappa B. A second kappa B binding activity (complex II), which apparently contained p50 homodimers, displayed limited induction by PDGF, whereas a third complex (complex III) migrated faster than but behaved similarly to complex I. These studies suggest that NF-kappa B or an NF-kappa B-like factor may participate in the expression of PDGF-inducible genes.


Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Microtubule-associated proteins (MAP), such as tau, modulate the extent and rate of microtubule assembly and play an essential role in morphogenetic processes, such as axonal growth. We have examined the mechanism by which tau affects microtubule polymerization by examining the kinetics of microtubule assembly and disassembly through direct observation of microtubules using dark-field microscopy. Tau increases the rate of polymerization, decreases the rate of transit into the shrinking phase (catastrophe), and inhibits the rate of depolymerization. Tau strongly suppresses the catastrophe rate, and its ability to do so is independent of its ability to increase the elongation rate. Thus, tau generates a partially stable but still dynamic state in microtubules. This state is perturbed by phosphorylation by MAP2 kinase, which affects all three activities by lowering the affinity of tau for the microtubule lattice.


Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP. The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.


Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces. Osteopontin, a glycoprotein with a glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain, has been described in bone and is also known to be expressed in other organs, particularly kidney. The goal of the present work was to define the distribution of osteopontin synthesis and deposition in a wide variety of normal adult human tissues using a multifaceted approach that included immunohistochemistry, in situ hybridization, and Northern analysis. Immunohistochemical studies have revealed the unexpected finding that osteopontin is deposited as a prominent layer at the luminal surfaces of specific populations of epithelial cells of the gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lung, breast, salivary glands, and sweat glands. Northern analyses identified gallbladder as a major site of osteopontin gene transcription comparable in magnitude with that of kidney, and immunoblotting identified osteopontin in bile. In situ hybridization localized osteopontin gene transcripts predominantly to the epithelium of a variety of organs as well as to ganglion cells of bowel wall. Osteopontin of epithelial cell origin, like bone-derived osteopontin, promoted GRGDS-dependent cell spreading in attachment assays. We postulate that osteopontin secreted by epithelium binds integrins on luminal surfaces. Collectively, these findings suggest an important role for osteopontin on many luminal epithelial surfaces communicating with the external environment.


Interaction of dynamin with microtubules: its structure and GTPase activity investigated by using highly purified dynamin. We purified a large amount of dynamin with high enzymatical activity from rat brain tissue by a new procedure. Dynamin 0.48 mg was obtained from 20 g of rat brain. The purity of dynamin was almost 98%. Dynamin plays a role of GTPase rather than ATPase. In the absence of microtubules, Michaelis constant (Km) and maximum velocity (Vmax) for dynamin GTPase were 370 microM and 0.25 min-1, respectively, and in their presence, both were significantly accelerated up to 25 microM and 5.5 min-1. On the other hand, the ATPase activity was very low in the absence of microtubules, and even in their presence, Km and Vmax for dynamin ATPase were 0.2 mM and 0.91 min-1. Despite slow GTPase turnover rate in the absence of microtubules, binding of GTP and its nonhydrolizing analogues was very fast, indicating that GTP binding step is not rate limiting. Dynamin did not cause a one-directional consistent microtubule sliding movement just like kinesin or dynein in the presence of 2 mM ATP or 2 mM GTP. We observed the molecular structure of dynamin with low-angle rotary shadowing technique and revealed that the dynamin molecule is globular in shape. Gel filtration assay revealed that these globules were the oligomers of 100-kDa dynamin polypeptide. Dynamin bound to microtubules with a 1:1 approximately 1.2 molar ratio in the absence of GTP. Quick-freeze deep-etch electron microscopy of the dynamin-microtubule complex showed that dynamin decorates the surface of microtubules helically, like a screw bolt, very orderly and tightly with 11.4 +/- 0.9 (SD)nm period. Contrary to the previous report, microtubules make bundles by the attachment of the dynamin helixes around each adjacent microtubule, and no cross-bridge formation was observed.


Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection. Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites. The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells. When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection. Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur. These results indicate that sites of polarized growth are specified before the end of G1. Sites of polarized growth can be influenced by external conditions. Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites). Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner. These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.


Myosin II phosphorylation and the dynamics of stress fibers in serum-deprived and stimulated fibroblasts. The actin-based cytomatrix generates stress fibers containing a host of proteins including actin and myosin II and whose dynamics are easily observable in living cells. We developed a dual-radioisotope-based assay of myosin II phosphorylation and applied it to serum-deprived fibroblasts treated with agents that modified the dynamic distribution of stress fibers and/or altered the phosphorylation state of myosin II. Serum-stimulation induced an immediate and sustained increase in the level of myosin II heavy chain (MHC) and 20-kDa light chain (LC20) phosphorylation over the same time course that it caused stress fiber contraction. Cytochalasin D, shown to cause stress fiber fragmentation and contraction, had little effect on myosin II phosphorylation. Okadaic acid, a protein phosphatase inhibitor, induced a delayed but massive cell shortening preceded by a large increase in MHC and LC20 phosphorylation. Staurosporine, a kinase inhibitor known to effect dissolution but not contraction of stress fibers, immediately caused an increase in MHC and LC20 phosphorylation followed within minutes by the dephosphorylation of LC20 to a level below that of untreated cells. We therefore propose that the contractility of the actin-based cytomatrix is regulated by both modulating the activity of molecular motors such as myosin II and by altering the gel structure in such a manner as to either resist or yield to the tension applied by the motors.


Phosphorylation of p90 and p52 in response to phorbol-esters in Swiss/3T3 cells overexpressing protein kinase C-alpha. Cell lines stably overexpressing protein kinase C (PKC)-alpha were previously described by us. These cell lines were generated by the introduction of the full length cDNA coding for PKC-alpha into Swiss/3T3 cells. Here we show that activation of PKC-alpha by phorbol-esters induced in these cells specific phosphorylation of two cellular proteins p90 and p52. Phosphorylation of p80 (MARCKS protein), previously identified as a substrate for PKC, was also enhanced. Phosphorylated p90 and p52 proteins were associated with particulate membrane-enriched fractions and were extractable with the use of nonionic detergents. Time course analysis of phorbol-ester induced phosphorylation of p90 and p52 revealed maximal stimulation of phosphorylation after 15-30 min. Phosphamino acid analysis showed that phosphorylation of p90 and p52 occurred mainly on serine residues. Phosphorylation of p52 was also on threonine residues. Whereas, phorbol ester activation induced phosphorylation of both p90 and p52, the mitogens platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) enhanced phosphorylation of p90, but not p52. Thus, our studies showed the involvement of PKC-alpha in the regulation of p90 and p52 phosphorylation and provided direct evidence for the role of PKC-alpha in cellular signaling by PDGF and FGF. Moreover, the fact that phosphorylation of p52 was specific to phorbol ester activation may suggest its involvement in tumor promotion. Characterization of p90 and p52 will enable us to reveal the phosphorylation cascade activated downstream to PKC-alpha and to determine their role in mitogenic signaling and tumor promotion.


Chromosome loss, hyperrecombination, and cell cycle arrest in a yeast mcm1 mutant. The original mcm1-1 mutant was identified by its inability to propagate minichromosomes in an ARS-specific manner, suggesting that it is defective in the initiation of DNA synthesis at ARSs. This mutant is also defective in expression of alpha-mating-type-specific genes. Further genetic and biochemical studies confirmed that Mcm1 is a transcription factor that mediates the transcriptional regulation of a number of genes, including genes outside of the mating type complement, by interacting with different cofactors. Although MCM1 is an essential gene, none of the previously characterized mcm1 mutants exhibits significant growth defects. To assess which of the many roles of Mcm1 is essential for growth, we constructed and characterized a temperature-sensitive conditional mutant of mcm1, mcm1-110L. This mutant exhibits a temperature-dependent cell-cycle arrest, with a large, elongated bud and a single, undivided nucleus that has a DNA content of close to 2n. In addition, it shows elevated levels of chromosome loss and recombination. In spite of the severity of the mcm1-110L mutation, this mutant still retains an ARS-specific pattern of minichromosome instability. All of these phenotypes are precisely those exhibited by mutants in three MCM genes, MCM2, MCM3, and MCM5/CDC46, that have been shown to play interacting roles in the early steps of DNA replication.


Signal transduction by the platelet integrin alpha IIb beta 3: induction of calcium oscillations required for protein-tyrosine phosphorylation and ligand-induced spreading of stably transfected cells. We demonstrate an example of signal transduction by an integrin and have begun to define the pathway through which this signaling is achieved. We constructed a stably transfected derivative of 293 cells (ATCC 1573) that expresses the platelet integrin GPIIbIIIa (alpha IIb beta 3). This cell line, clone B, adheres to and spreads on fibrinogen, a ligand for alpha IIb beta 3, while the parent cell line does not. Stimulation of these cells either by adhesion to fibrinogen or with antiserum directed against alpha IIb beta 3 results in induction of calcium oscillations, followed by tyrosine phosphorylation of at least one protein of molecular weight approximately 125 kDa. We establish that this phosphorylation, as well as the morphological rearrangements, requires the mobilization of calcium.