Perception of malocclusion in 11-year-old children: a comparison between personal and parental awareness.

In orthodontic counselling an understanding of how individuals perceive their occlusal features is important to ensure effective communication and for provision of adequate advice on treatment need. The purpose of the present study was to assess personal and parental awareness of malocclusion in children, and to examine whether agreement existed between children and their parents on assessments of malocclusion traits. Of 104 randomly selected fourth-grade children 99 presented themselves at a public dental clinic. Ninety-three accompanying parents attended. Awareness was assessed by comparing the opinion of parents and children on the presence or absence of anterior malocclusion with direct measurements on dental study casts. The subjects' abilities to identify a polaroid of the child's dentition in a panel of 17 alternative photographs were also used as a measure of awareness. The findings revealed a moderate level of awareness among both the children and their parents. About half of the children and the parents identified the child's photograph on the first attempt. About three-quarters of the traits recorded as marked/severe malocclusion and about half of the mild/moderate traits were recognized. A significant association existed between the number of correct reports on traits given by the children and the parents. However, agreement across professional, child, and parental assessments varied for the different traits. The results indicated that the individual's comprehension of professional terms may be unclear and that professionally defined cut-off points often do not coincide with norms existing within the actual family unit.


Need and demand for orthodontic treatment in an adult Swedish population.

The prevalence of malocclusion, the need for and the demand for orthodontic treatment was studied in a randomly selected adult Swedish population > or = 20 years of age. Nine-hundred-and-twenty subjects were examined of whom 669 had their own teeth in occlusion. From those a group of 157 subjects was selected on the basis of objective need and/or subjective demand for orthodontic treatment. The various regimens of treatment required in this group were investigated. The prevalence of malocclusion ranged from 17 to 53 per cent in the various age groups. The spectrum of malocclusion was similar to that previously reported in Swedish children. The awareness of their malocclusion was higher among younger than older subjects and among those who had severe malocclusion. Objective treatment need, evaluated by two experienced orthodontists, was estimated at 11 per cent of the total population, whilst orthodontic treatment was requested by approximately 5 per cent of the population studied.


Ectopic eruption of maxillary first permanent molars and association with other tooth and developmental disturbances.

The associations between tooth and eruption disturbances in four groups of children selected primarily with only one diagnosed eruption or developmental disturbance in each group was analysed. Ninety-two children were primarily diagnosed with ectopic eruption of maxillary first permanent molars, 93 children with infra-occlusion and ankylosis of primary molars, 91 children with ectopic eruption of maxillary canines, and 97 children with aplasia of premolars. Of the children studied 69-79 per cent had only a single one of the four disturbances studied. In 18-28 per cent there was one additional disturbance and in 2-3 per cent two additional disturbances. From chi square contingency tests, it was found that infra-occlusion of primary molars and aplasia of premolars exhibited a higher prevalence in both directions compared to the expected population prevalence. Ectopic eruption of maxillary canines showed a significantly higher prevalence than expected in all the other three groups. Our interpretation is that these results support the hypothesis of a common, presumably hereditary, aetiology. Thus, the four conditions studied would be different manifestations of one syndrome, each manifestation having an incomplete penetrance. With closer follow-up of the maxillary canines during the eruption period in children with some of the other three disturbances, prophylactic, or early interceptive measures may be taken and complicated orthodontic treatment be reduced or avoided.


Nuclear bodies (NBs): a newly "rediscovered" organelle.

Nuclear bodies (NBs) were first described in detail some 30 years ago, by conventional electron microscopy, as prominent interchromatin structures found primarily in the nuclei of malignant or hyperstimulated animal cells. Subsequent studies have shown that NBs are ubiquitous organelles, but they are numerically and morphologically quite varied. With the recent discovery of human autoantibodies against several key nuclear antigens present in some NBs, these structures are once again the subject of much attention. At least one class of NBs, coiled bodies, has been shown to be nucleolus-derived and to contain not only nucleolus-associated antigens, but also many of the snRNP components involved in pre-mRNA splicing. These data suggest that coiled bodies, and perhaps other NBs as well, are multifunctional and may be involved in the processing or transport of both pre-mRNA and pre-rRNA. Further evidence is provided showing that NBs constitute distinct nuclear domains whose functional significance is just now emerging.


Reactivation of DNA replication in erythrocyte nuclei by Xenopus egg extract involves energy-dependent chromatin decondensation and changes in histone phosphorylation.

Reactivation of chicken erythrocyte nuclei for DNA replication in Xenopus egg extracts involves two phases of chromatin remodelling: a fast decondensation leading to a small volume increase and chromatin dispersion occurring within a few minutes (termed stage I decondensation), followed by a slower membrane-dependent decondensation and enlargement of up to 40-fold from the initial volume (stage II decondensation). Chromatin decondensation as measured by nuclear swelling and micrococcal nuclease digestion required ATP. We observed a characteristic change in the phosphorylation pattern of erythrocyte proteins upon incubation in egg extract. While histones H5, H2A, and H4 became selectively phosphorylated during decondensation, the phosphorylation of histone H3 and of several nonhistone proteins was prevented. Furthermore, histone H5 was selectively released from erythrocyte nuclei in an energy-dependent reaction. These molecular changes already occurred during stage I decondensation and they persisted during stage II decondensation. DNA replication was confined to nuclei of stage II decondensation which incorporated lamin LIII from the egg extract. These results show that initiation of DNA replication in chicken erythrocytes requires in addition to ATP-dependent chromatin remodelling (stage I), further changes in chromatin structure that correlates with lamin LIII incorporation, and stage II decondensation.


Splicing thermotolerance maintains Pre-mRNA transcripts in the splicing pathway during severe heat shock.

Thermotolerance, the ability of cells and organisms to withstand severe elevated temperatures after brief exposure to mild elevated temperatures, has been studied in numerous laboratories. Survival thermotolerance is defined as the increase in cell or organism survival at severe elevated temperatures after a pretreatment at mild elevated temperatures. This study examines splicing thermotolerance in Drosophila melanogaster, the ability to splice pre-mRNAs made at the severe temperature (38 degrees C) after a brief pretreatment at a milder temperature (35 degrees C). It is probably one of a number of mechanisms by which cells adapt to heat shock. These experiments demonstrate that pre-mRNAs synthesized at the severe temperatures in splicing thermotolerant cells, although protected in splicing-competent complexes, are not actually processed to mature mRNAs until the cells are returned to their normal temperature. We have also studied the kinetics of acquisition and loss of splicing thermotolerance. As little as 10 min of pretreatment at 35 degrees C was sufficient to provide full splicing thermotolerance to a 30-min severe heat shock of 38 degrees C. Pretreatments of less than 10 min provide partial splicing thermotolerance for a 30-min severe heat shock. Full splicing thermotolerance activity begins to decay about 4 h after the cessation of the 35 degrees C incubation and is completely lost by 8 h after the pretreatment. The kinetics experiments of pre-mRNAs synthesized during the 38 degrees C treatment in splicing thermotolerant cells indicate that one or more splicing thermotolerance factors are synthesized during the 35 degrees C pretreatment which interact with pre-mRNA-containing complexes to keep them in a splicing-competent state. These kinetic experiments also indicate that in cells which are partially splicing thermotolerant, the pre-mRNAs synthesized early during the 38 degrees C incubation are protected, whereas those synthesized late are not. In the absence of splicing thermotolerant factors, the pre-mRNA-containing complexes leave the normal splicing pathway and are allowed to exit to the cytoplasm.


Heat shock-induced redistribution of a 160-kDa nuclear matrix protein.

In this paper we describe a 160-kDa protein (p160) which is present in the nuclear matrix of rat, mouse, and human cells. Biochemical and ultrastructural analysis shows that p160 is associated with the internal matrix and is not present in the lamina-pore complex. Immunoelectron microscopy shows that the protein is part of the extranucleolar, fibrogranular network of the nuclear matrix. During an in vivo 42 degrees C heat treatment of HeLa cells, A431 human epidermoid cells, and T24 human bladder carcinoma cells, p160 transiently formed large clusters inside the nucleus. These p160 clusters are associated with the nuclear matrix network, as judged by immunolabeling on isolated nuclear matrices. The percentage of cells showing p160 clusters increased proportionally with longer heat treatments, reaching a maximum after a period of 3 h. At this time 70 +/- 5% of the cells displayed these clusters. Clustering decreased after longer heat treatments and the anti-p160 staining pattern became diffuse granular again. Other nuclear components, such as the A1 antigen of hnRNP (ribonucleoprotein), the Sm antigen of snRNPs, and lamins A and C, did not cluster during the 42 degrees C treatment, indicating that this reallocation is characteristic for the p160 matrix protein. These results demonstrate that p160 is an internal nuclear matrix element with a dynamic spatial distribution.


Temporal and spatial differences in glycosaminoglycan synthesis by fetal lung fibroblasts.

In studies of the ontogeny of fibroblast-epithelial interactions during late fetal lung rat lung development, we have identified two subpopulations of fibroblasts which differed in their ability to promote epithelial cell proliferation or differentiation. As glycosaminoglycans (GAGs) have been implicated in the regulation of these processes we have tested whether the two fibroblast populations synthesize different GAGs and whether the GAG pattern changes with development. Fibroblasts incorporate more [3H]glucosamine and Na2 35SO4 into GAGs than epithelial cells. Both cell types deposited a significant amount of newly synthesized GAGs in the cell-matrix layer. GAGs were lost faster from the cell-matrix layer of fibroblasts (t1/2 = 12 h) than from that of epithelial cells (t1/2 = 48 h). Total GAG synthesis by fibroblasts did not change with advancing gestation, but synthesis of sulfated GAGs by epithelial cells declined with advancing gestation. Independent of gestational age epithelial cells synthesized predominantly heparan sulfate. Depending on their proximity to the epithelium, fibroblasts differed in their production of GAGs. Fibroblasts in close proximity to the epithelium mainly produced and secreted hyaluronan. More distant fibroblasts, from the pseudoglandular stage of lung development synthesized primarily heparan sulfate and chondroitin sulfate. This same population of fibroblasts from the canalicular stage of lung development, produced more hyaluronan. As the shift to hyaluronan occurs with the thinning of the alveolar septal wall, this finding suggests that developmentally regulated GAG production by fibroblasts may facilitate epithelial-fibroblast interaction, thus influencing fetal lung growth and differentiation.


Deletions in the regulatory or kinase domains of protein kinase C-alpha cause association with the cell nucleus.

We have constructed expression plasmids carrying protein kinase C (PKC) cDNAs with deletions in the coding region. Two truncated molecules, consisting only of the kinase domain of PKC-alpha, were generated by removing parts of the cDNA coding for the regulatory region. Another mutant molecule was created by deleting 95 amino acids from the C-terminal part of the molecule. The full-length cDNA coding for PKC-alpha and its deletion constructs was expressed in COS cells. Using cell fractionation experiments and immunofluorescence staining, we demonstrate here that in contrast to the cytosolic localization of full-length PKC-alpha, the truncated forms, coding only for the kinase domain, were found exclusively in the cell nucleus. Further subfractionation of nuclei isolated from these transfected cells indicated partial association with the nuclear envelopes. Expression of the cDNA lacking the C-terminal part of the molecule in COS cells encoded a truncated molecule that was found both in the cytosol and in the nucleus. We also show that translocation of full-length PKC-alpha molecules to the cell nucleus occurred in response to phorbol ester treatment. Thus, it appears that accumulation of PKC-alpha in the nucleus results either by phorbol ester activation or by deletions of specific regions of the molecule. A molecular mechanism for the nuclear translocation of phorbol ester-activated PKC-alpha or its truncated molecules is suggested.


The attachment of nematocytes from the primitive invertebrate Hydra to fibronectin is specific and RGD-dependent.

The transient attachment of cells to components of the extracellular matrix is an important step in the complex molecular mechanisms involved in amoeboid cell locomotion. We have analyzed the attachment of nematocytes from the freshwater cnidarian Hydra to fibronectin which is a constituent of the mesoglea, the extracellular matrix, of the polyps. The percentage of attaching cells increased gradually in a concentration-dependent manner and reached a plateau value at a fibronectin concentration of 50 micrograms/ml. Attachment was inhibited by exposure of the fibronectin-coated surfaces to antibodies against the cell binding domain of fibronectin or by incubating the cells with peptides containing the recognition sequence Arg-Gly-Asp (RGD) known from vertebrate cells. This, together with data obtained by affinity chromatography, indicates that RGD-dependent binding to fibronectin, mediated by a receptor which possibly belongs to the integrin family, already occurs in Hydra, a member of an evolutionary low invertebrate phylum.


Expression of the calcium binding protein calretinin in WiDr cells and its correlation to their cell cycle.

Ca2+ ions intervene during different phases of the progression of the cell cycle, but only one calcium-binding protein, calmodulin, has been shown to be associated with dividing cells. We therefore screened cancer cells for the presence of other related calcium-binding proteins. Using molecular biological and immunohistochemical techniques we show that human tumor cells of epithelial origin, express calretinin. Calretinin immunoreactivity can be demonstrated at precise moments of the cell cycle and, in particular, in phase G1 and during mitosis. During mitosis calretinin is localized both in the cytoplasm and in the mitotic spindle. In the cytoplasm we find calretinin after prophase and until telophase. In the spindle apparatus, calretinin is already present in cells in prometaphase and persists in all the succeeding mitotic phases. It is associated with the kinetochore microtubules but, in contrast to calmodulin, also with the polar microtubules. The role that calretinin plays in well-defined moments of the cell cycle of these cells is as yet unknown, but our results strongly suggest that, in collaboration with other molecules, calretinin intervenes in the dynamic phenomena regulating the separation of the chromosomes.


Cytochalasans and PMA induce IL-2 receptors on CD8+ lymphocytes.

The cytochalasans, fungal metabolites that interact with actin, can affect lymphocyte proliferation; high concentrations inhibit lectin-induced proliferation and low concentrations augment it. The phorbol ester tumor promoter, PMA, alone is not mitogenic for primary lymphocytes but enhances the activity of mitogenic lectins. Because the cytochalasans have been reported to increase intracellular Ca2+ and because PMA activates protein kinase C, lymphocytes were treated with PMA and cytochalasin B (CyB) to determine if this combination would induce DNA synthesis. While this treatment by itself did not cause proliferation, lymphocytes cultured with PMA and CyB overnight, washed, and recultured with IL-2 proliferated to the same degree as lymphocytes stimulated with Con A. Three different cytochalasans, cytochalasin B, cytochalasin D, and chaetoglobosin C, all of which bind to cellular actin with different affinities and only one of which affects glucose transport, induced IL-2 receptors in combination with PMA. Flow cytometric analysis with an antibody to the IL-2 receptor alpha subunit confirmed the induction of receptors on CD8+ cells. However, no IL-2 was produced after the exposure of lymphocytes to the combination of cytochalasans and PMA. Therefore, there was sufficient signal to induce IL-2 receptor expression but not to induce IL-2.


Effects of TGF beta 1 on the proliferation and differentiation of an immortalized astrocyte cell line: relationship with extracellular matrix.

The astrocyte cell line (C.LT.T.1.1.), which is immortalized and has retained a normal density-dependent regulation of growth, is a suitable model for studying the relationships between proliferation, differentiation, and the production of extracellular matrix. The growth factor TGF beta 1 was used to modulate these processes. When added to proliferative cells, it inhibited growth and caused morphological changes. It also suppressed the growth arrest at confluence, so that the cells formed multilayers of parallel spindle-shaped cells. Whereas untreated control cells expressed progressively the glial fibrillary acidic protein (GFAP) after arrest of multiplication, the addition of TGF beta 1 to proliferative cells prevented GFAP expression and accumulation of its mRNA. Concomitantly, it increased the amounts of laminin, fibronectin, and collagens synthesized during the growth phase and greatly altered the composition and the structure of the matrix deposited at confluence. In contrast, when added after cell differentiation had begun, TGF beta 1 did not alter the appearance of the matrix whereas it still stimulated, but to a lesser extent, extracellular matrix components production. The results show that TGF beta 1 prevents the transition from the proliferating to the differentiating state and correlatively alters the composition and structure of the extracellular matrix.


Transferrin-receptor-independent but iron-dependent proliferation of variant Chinese hamster ovary cells.

The purpose of this study is to clarify the role of iron, transferrin, an iron-binding protein in vertebrate plasma, and transferrin receptors in cell proliferation. Transferrin, which is indispensable for most cells growing in tissue culture, is frequently referred to as a "growth factor". Proliferating cells express high numbers of transferrin receptors, and the binding of transferrin to their receptors that is needed for cells to initiate and maintain their DNA synthesis is sometimes regarded as analogous to other growth factor-receptor interactions. Although numerous previous experiments strongly indicate that the only function of transferrin in supporting cell proliferation is supplying cells with iron, they did not completely rule out some direct or signaling role transferrin receptors could play in cell proliferation. To address this issue, we exploited transferrin-receptor-deficient mutant Chinese hamster ovary (CHO) cells (McGraw, T. E., Greenfield, L., and Maxfield, F. R., 1987, J. Cell. Biol. 105, 207-214) in which various aspects of iron and transferrin metabolism in relation to their capacity to proliferate were investigated. Variant cells neither specifically bind transferrin nor do their extracts contain any detectable functional transferrin receptors, yet they proliferate and synthesize DNA with rates comparable to those observed with parent CHO cells. Desferrioxamine, an iron chelating agent, inhibits growth and DNA synthesis of both variant and control CHO cells. This inhibition can be fully alleviated, in both cell types, by ferric pyridoxal isonicotinoyl hydrazone, which can supply cells with a utilizable form of iron by a pathway not requiring transferrin and their receptors. Studies of 59Fe uptake and 125I-transferrin binding revealed that parent cells can take up iron by at least three mechanisms: from transferrin by receptor-dependent and -independent (nonspecific, nonsaturable, not requiring acidification) pathways and from inorganic iron salts (initially present in the medium as FeSO4). Although variant CHO cells are unable to acquire transferrin iron via the receptor pathway, two remaining mechanisms provide these cells with sufficient amounts of iron for DNA synthesis and cell proliferation. In conclusion, although transferrin receptors are dispensable in terms of their absolute requirement for proliferating cells, a supply of iron is still needed for their DNA synthesis. Transferrin-receptor-deficient CHO cells may be a useful model for investigating receptor-independent iron uptake from transferrin and nontransferrin iron sources.


Phosphorylation of a 225-kDa centrosomal component in mitotic CHO cells and sea urchin eggs.

Components of centrosomes are those among cellular proteins that are phosphorylated at the transition from interphase to mitosis. Using an anti-phosphoprotein antibody (CHO3) directed against isolated mitotic CHO spindles, we identified a 225-kDa centrosomal phosphocomponent in mitotic CHO cells and in cleaving sea urchin eggs. The 225-kDa protein is tightly attached to the centrosome, which allowed us to separate it from other spindle-associated factors by high salt extraction. Phosphorylation of the 225-kDa protein occurred during mitosis. This was shown by isotope labeling on gels as well as by visualization of thiophosphorylated centrosomes with an anti-thiophosphoprotein antibody (M. Cyert, T. Scherson, and M. W. Kirschner, 1988, Dev. Biol. 129, 209) after preincubation with ATP-gamma-S in vivo and in vitro. Mitotic spindles isolated from CHO cells retained their ability to phosphorylate the centrosomal component, whereas sea urchin spindles did not, possibly due to loss or inactivation of protein kinase(s) during spindle isolation. The enzyme associated with isolated CHO spindles was extractable by high salt treatment and was capable of phosphorylating many spindle components, including the 225-kDa centrosomal protein of CHO cells and sea urchin embryos. Such high salt extracts contain protein kinases, including cell cycle control protein kinase p34cdc2, suggesting that the enzyme responsible for centrosomal phosphorylation could be p34cdc2 or other downstream mitotic kinases activated by the action of p34cdc2.


Differential regulation of thyroid cell-cell and cell-substrate adhesion by thyrotropin.

Preservation of cell aggregation is necessary for thyroid follicular differentiation in vitro and requires stimulation by thyrotropin (TSH). We have tested the hypothesis that TSH preferentially increases thyroid cell-cell adhesion relative to cell-substrate adhesion. Cell-cell adhesion was measured in short-term suspension cultures by the decrease in the fraction of single cells remaining in culture (free cell ratio, FCR). When incubated in medium alone freshly isolated cells showed a progressive fall in FCR but this was accelerated by TSH and the cyclic AMP analog, 8-(4-chlorophenylthio)cyclic AMP. Aggregation was dependent upon extracellular Ca2+ and also promoted by a cell-free membrane extract. In contrast, attachment of cells to plastic dishes treated for tissue culture was not affected by TSH. We conclude that thyroid cells possess a TSH-sensitive cell adhesion system. The preferential increase in cell-cell adhesion may be one mechanism by which TSH stimulates the formation and preservation of follicles in vitro.


Reduced tyrosine phosphorylation in polyamine-starved cells.

Onset of cell proliferation is associated with enhanced turnover of the polyamines putrescine, spermidine, and spermine, particularly evident in the massive increase in the activity of the rate-limiting enzyme in their production, ornithine decarboxylase (ODC). The physiological functions of these polyamines, however, have remained unclear. Here we report that treatment of LSTRA cells for 2-18 h with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, decreased the amount of phosphotyrosine in several cellular substrates including the T cell protein tyrosine kinase p56lck. No reductions in the amount of p56lck, overall synthesis of protein and DNA, or cell viability were observed until much later. DFMO did not affect the catalytic activity of p56lck in vitro and the activity of p56lck immunoprecipitated from DFMO-treated cells was unaltered. Addition of putrescine, the reaction product of ODC, completely reversed the effect of DFMO on tyrosine phosphorylation. Finally, we provide evidence that polyamines reduce the activity of cellular protein tyrosine phosphatases toward endogenous substrates. Our results suggest that polyamines may influence the extent of tyrosine phosphorylation during cell proliferation and malignant transformation, perhaps by modulating the rate of dephosphorylation of specific target proteins.


Hydrogen peroxide lethality is associated with a decreased ability to maintain positive DNA supercoiling.

Hydrogen peroxide is more toxic to mammalian cells at 37 degrees C than 0 degree C at all concentrations studied. Histone-free nuclei (nucleoids) extracted from treated cells have a reduced ability to maintain positive DNA supercoiling, with the maximum effect at the higher temperature. Prior exposure of cells to sodium ascorbate at 0 degree C increased both toxicity and the inhibition of nuclear supercoil rewinding. After exposure at 0 degrees C, normal levels of supercoiling returned with both a fast and a slow component, kinetics characteristic of DNA single-strand break repair; the fast component was eliminated when cells were exposed at 37 degrees C due to in situ rejoining. At least a portion of the lethal lesions induced by hydrogen peroxide are DNA double-strand breaks (dsb) because the dsb repair-deficient mutant, xrs-5, is approximately two to three times more sensitive than wild-type cells over the initial portion of the survival curve. However, the increased toxicity found after exposure at 37 degrees C is observed equally in both cell lines, indicating that temperature-dependent cell killing is not directly linked to DNA dsb. It is suggested that cell killing at 37 degrees C is mediated through two linked processes. First, hydrogen peroxide may disrupt cation-stabilized nuclear supercoiling by direct ion oxidation. Second, as a part of the oxidation process, hydrogen peroxide will produce potentially cytotoxic free radicals close to the DNA-linked metal site, limited in extent only by the presence of chemicals capable of reducing metal ions prior to reoxidation.


Stimulation of DNA synthesis in senescent human cells following incubation with plasma membranes.

DNA synthesis and mitosis were increased in mitogen-stimulated senescent WI-38 cells following incubation with plasma membranes prepared from young or senescent WI-38 cells, A431 cells, 3T3 cells, or NR6 cells. The percentage of [3H]thymidine-labeled nuclei in senescent cultures was two- to fivefold greater than that seen in controls in which cells were incubated in the absence of membranes or in the presence of boiled membranes. The effect was trypsin sensitive, suggesting that a protein moiety is necessary for stimulation of DNA synthesis. As the culture age increased, basal levels of DNA synthesis, as well as maximal stimulation of DNA synthesis following incubation with plasma membranes, decreased. These observations are consistent with the hypothesis that different subpopulations exist in senescing cultures and suggest a complex pattern of inhibitory and stimulatory regulation of cell proliferation.


Inhibition of cell adhesion to plastic substratum by phosphorothioate oligonucleotide.

Antisense oligonucleotides have been widely used to achieve specific inhibition of targeted gene expression. However, the mechanism of action is not well understood and in many systems sequence-independent effects occur. We have recently shown that chronic administration of an antisense c-myc phosphorothioate oligonucleotide can specifically inhibit expression of the c-myc protein and growth in human breast cancer cells. We now identify an additional effect of the same oligonucleotide on cell adhesion. Transient delivery through electroporation of 2.5 microM antisense-myc oligonucleotide to MCF-7 cells results in 85% inhibition of adhesion to plastic substratum within 24 h. Both the onset of this effect and the subsequent recovery occur without a change in cell viability, growth, or alteration of adhesion to Matrigel, collagen IV, laminin, or fibronectin. However, no parallel changes in c-myc mRNA or protein expression are detectable, suggesting that in this instance inhibition of adhesion caused by antisense-myc oligonucleotide may involve a mechanism independent of the target sequence.


Intracellular localization of terminal transferase during the cell cycle.

Changes in the localization of terminal transferase during the cell cycle in random cultures of human pre-T leukemia line RPMI-8402 were examined by light and electron microscopy on immunoperoxidase-stained preparations. Paraformaldehyde-fixed and saponin-permeabilized human cells were used with a monoclonal anti-human terminal deoxynucleotidyl transferase (TdT) primary reagent to demonstrate changes in enzyme distribution occurring between interphase and mitosis. Nuclear localization is found uniformly during interphase. At metaphase, however, the majority of TdT staining appears randomly distributed in the cytoplasm and traces of TdT staining remain associated with mitotic chromatin. At later phases, when the daughter cells are forming, the enzyme again appears to be restricted to the new nuclear structure.


Stimulation by GH of IGF1 proforms synthesized by rabbit chondrocytes cultured with bFGF in serum-free medium.

The IR-IGF1 production by rabbit epiphyseal chondrocytes cultured in serum-free medium was analyzed. Cell proliferation was induced by the addition of 10 ng/ml basic fibroblast growth factor (bFGF) without or with 100 ng/ml recombinant human growth hormone (hGH). GH alone induced no cell multiplication. Chondrocytes treated with bFGF alone secreted an IR-IGF1 activity proportional to the mitotic activity of the cells. A specific positive IGF1 immunostaining was localized in the Golgi of control and hGH-treated cells. The IR-IGF1 activity recovered into culture medium was mainly composed of three fractions of apparent MW 6-8 kDa, 9-14 kDa, and 16-18 kDa. [35S]Methionine pulse-chase experiments indicated that the radiolabeled 16-18 kDa IR-IGF1 fraction was partly converted into the 9-14 kDa and 6-8 kDa fractions. At equilibrium, 70% of the chondrocyte IR-IGF1 activity was recovered as 9- to 18-kDa forms which contained high IR-proIGF1A activity. The 6-8 kDa fraction had biochemical characteristics similar to those of the mature IGF1 peptide. Similar results were observed when 4% fetal calf serum was added to the culture. The addition of 100 ng/ml of hGH significantly and specifically increased IGF1 precursor material, which thus represented 90% of total IR-IGF1 activity. On Day 16 of the culture, when cells stopped dividing, the amount of chondrocyte IR-IGF1 was significantly lower than during cell proliferation, and hGH had no effect on this production. These data indicate that cultured chondrocytes produce more IGF1 precursors than mature IGF1 and that GH specifically stimulates biosynthesis of IGF1 precursors but not IGF1 per se. A GH-dependent biological function of IGF1 proforms in chondrocytes remains to be demonstrated.


Membrane aging during cell growth ascertained by Laurdan generalized polarization.

The sensitivity of the fluorescent probe Laurdan to the phase state of lipids has been utilized to detect modifications in the composition and physical state of cell membranes during cell growth. In phospholipid vesicles, the Laurdan emission spectrum shows a 50-nm red shift by passing from the gel to the liquid-crystalline phase. The Generalized Polarization (GP) value has been used for the data treatment instead of the ratiometric method common in investigations utilizing other fluorescent probes that display spectral sensitivity to medium properties. The GP value can be measured easily and quickly and possesses all the properties of "classical" polarization, including the additivity rule. Once Laurdan limiting GP values have been established for the gel and the liquid-crystalline phase of lipids, the quantitative determination of coexisting phases in natural samples is possible. In the present work the observation of a relevant decrease in the fractional intensity of the liquid-crystalline phase in K562 cell membranes during 5 days of asynchronous growth is reported. A decrease in the "fluidity" of cell membranes in K562 cells kept in culture for several months is also reported. The procedure developed for labeling cell membranes with Laurdan is reported and the influence of cell metabolism on fluorescence parameters is discussed. Also discussed is the influence of cholesterol on Laurdan GP.


Bradykinin and muscarine induce Ca(2+)-dependent oscillations of membrane potential in rat glioma cells indicating a rhythmic Ca2+ release from internal stores: thapsigargin and 2,5-di(tert-butyl)-1, 4-benzohydroquinone deplete InsP3-sensitive Ca2+ stores in glioma and in neuroblastoma-glioma hybrid cells.

Continuous superfusion of rat glioma cells with medium containing bradykinin (from 0.2 nM) induced a transient hyperpolarization followed by regular hyperpolarizing oscillations of the membrane potential. Similar repetitive hyperpolarizing oscillations were caused by extracellularly applied bradykinin or muscarine or by intracellularly injected GTP-gamma-S. The frequency of the oscillations was 1 per minute at bradykinin concentrations ranging from 0.2 nM to 2 microM, but the amplitude and duration increased with rising peptide concentration. The muscarine-induced oscillations were blocked by atropine. In the presence of extracellular Ca2+, the substances thapsigargin, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), and ionomycin reversibly suppressed the bradykinin-induced oscillations. Thapsigargin and tBuBHA, which are known to block the Ca2+ ATPase of endoplasmic reticulum, caused a transient rise in cytosolic Ca2+ activity, monitored with Fura-2, in suspensions of rat glioma cells or of mouse neuroblastoma-rat glioma hybrid cells. After a transient Ca2+ rise caused by thapsigargin, tBuBHQ, or ionomycin, the Ca2+ response to bradykinin which is known to be due to release of Ca2+ from internal stores was suppressed. This indicates that thapsigargin and tBuBHQ deplete internal Ca2+ stores as already seen previously for ionomycin. Thus, the inhibition of the membrane potential oscillations by thapsigargin, tBuBHQ, and ionomycin indicates that the oscillations are associated with activation of InsP3-sensitive Ca2+ stores. In some cells composite oscillation patterns which consisted of two independent oscillations with different amplitudes that overlapped additively were seen. We discuss that this pattern and the concentration dependency of the oscillations could be due to "quantal" Ca2+ release from stores with different inositol 1,4,5-triphosphate sensitivities. Subsidence of the oscillations after omission of extracellular Ca2+ seems to be due to a lack of replenishment of the intracellular stores with Ca2+, which comes from the extracellular compartment.


Mitotic apparatus formation and cleavage induction by micromanipulation of the nucleus and centrosome: the centrosome forms a spindle together with only the chromosomes at a short distance.

We micromanipulated the nucleus and centrosomes in the zygote of the starfish, Asterina pectinifera, in order to investigate their roles in mitotic apparatus formation and cleavage induction. The zygote cleaved without spindle formation when its nucleus was removed. When one or two centrosomes were transplanted, they formed asters in the recipient cell, which cleaved into three or four blastomeres so that each blastomere might contain one centrosome or aster. When one centrosome was removed, a half-spindle formed in the manipulated cell, which did not cleave until the other centrosome was duplicated. When both centrosomes were removed, no microtubular structures such as the spindle and the aster appeared in the manipulated cell, which failed to cleave. These results indicate that two centrosomes or more in the cell induce cleavage with or without the nucleus and that one centrosome or less does not induce cleavage. It is also concluded that the centrosome(s) together with the nucleus forms a half-spindle or bipolar spindle. However, from the experiments of nucleus transplantation and displacement, spindle formation is found to depend on the distance between chromosomes and centrosomes. The half-spindle formed when the distance from the centrosome to the chromosomes was shorter than 22 microns; on the other hand, when the distance was longer than 22 microns, the nucleus remained apart from the aster, which means that the functional range of the astral microtubule's ability to engage chromosomes was 22 microns from the centrosome.


Enolase is present at the centrosome of HeLa cells.

Antibodies raised against the C-terminus and N-terminus region of gamma gamma enolase, as well as a polyclonal antibody raised against bovine brain gamma gamma enolase, were used to study the distribution of this glycolytic enzyme during the cell cycle in HeLa cells. Enolase was found to be present throughout the cytoplasm of both interphase and dividing cells. In addition, a portion of cellular enolase was detected at the centrosome throughout the cell cycle. The capacity of glycolytic enzymes to play a structural as well as a glycolytic role suggests that the presence of enolase at the centrosome may be correlated with the organization of both the interphase cytoskeleton and the mitotic spindle.


Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator.

Regulation of the activity of proteolytic enzymes is of major importance in the turnover of connective tissues. The search for physiologically relevant activation mechanisms of principal tissue-degrading enzymes, e.g., metalloproteinases, has therefore been of wide interest. We have now studied whether the initiating factor of the fibrinolytic system, urokinase plasminogen activator (u-PA), may also function in the early steps of activation of one of the metalloproteinases, the M(r) 72,000 gelatinase/type IV collagenase produced by cultured fibroblasts. Treatment of the secreted M(r) 72,000 proteinase by u-PA yielded a cleavage product of M(r) 62,000 as revealed by fluorography of radioactively labeled proteins as well as by gelatin zymography SDS-PAGE gels. The u-PA-catalyzed cleavage of the M(r) 72,000 proteinase was blocked by anti-u-PA antibodies, but was unaffected by the plasmin inhibitor aprotinin, thus indicating a specific action for the activator. On the contrary, the tissue activator of plasminogen, t-PA, did not cleave the type IV collagenase in similar assays. u-PA-catalyzed cleavage of recombinant type IV collagenase, produced in a baculovirus expression system, yielded a similar M(r) 62,000 activity in gelatinolysis assay. Zymograms of the isolated pericellular matrices of cultured fibroblasts also revealed M(r) 72,000 gelatinolytic polypeptide that was converted to an M(r) 62,000 form by u-PA. Both polypeptides were recognized in immunoblotting by antibodies against the gelatinase/type IV collagenase, suggesting immunological identity with the secreted enzyme. Thus the M(r) 72,000 gelatinase/type IV collagenase is not only secreted, but also deposited into the pericellular fibroblast matrix, and both forms are substrates for u-PA. The results suggest a new potential role for u-PA as a direct regulator of metalloproteinase-mediated extracellular proteolysis via the cleavage of the M(r) 72,000 gelatinase/type IV collagenase to an M(r) 62,000 form.


Mitosis-arresting effect of the calcium channel inhibitor SK&F 96365 on human leukemia cells.

The effect of SK&F 96365 (1-(beta-[3-(4-methoxyphenyl)propoxyl]-4- methoxyphenethyl)-1H-imidazole hydrochloride), a recently synthesized inhibitor of receptor-mediated calcium entry, was investigated on human hematopoietic cell lines. We found that treatment of the T-cell leukemia line Jurkat with SK&F 96365 inhibited the Ca2+ influx triggered by antibodies against the CD3/TCR complex, while the inositol trisphosphate-dependent Ca2+ release from intracellular stores remained intact. A 50% inhibition of the Ca2+ influx was obtained with 5 microM SK&F 96365, while higher concentrations of the drug blocked the CD3-dependent Ca2+ influx completely. In addition to its blocking of the Ca2+ influx, treatment with SK&F 96365 was found to accumulate mitotic cells. The drug (5 microM) imposed a total cell cycle arrest in G2/M. The mitosis block could be reversed by removal of the inhibitor from the cultures, while elevation of intracellular or extracellular Ca2+ did not restore cell cycle progression. This suggests that the cell cycle block induced by SK&F 96365 is not directly related to its action as an inhibitor of receptor-mediated calcium entry. Our findings indicate that SK&F 96365, in addition to its ability to inhibit receptor-triggered Ca2+ influx, offers a new method for imposing a reversible mitosis arrest in hematopoietic cell lines.


Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones.

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.


Spermidine nuclear acetylation in rat hepatocytes and in logarithmically growing rat hepatoma cells: comparison with histone acetylation.

Spermidine acetylation has been studied in nuclear homogenates and in entire nuclei from rat hepatocytes and rat hepatoma tissue culture (HTC) cells, isolated at different stages of logarithmic growth, and compared to histone acetylation. Under all experimental conditions, N8-acetylspermidine was the predominant product of the reaction (90%). Unlike histone, spermidine acetylation in HTC cell and hepatocyte entire nuclei was almost absent or strikingly reduced relative to acetylation using nuclear homogenates as the enzyme sources. This was due to the lack of a free minor pool of spermidine, most likely lost during the purification of entire nuclei. Thus, preincubation of intact nuclei in the presence of spermidine restored activities to values observed using nuclear sonicates. Spermidine acetylation in HTC cell nuclei fluctuated moderately during cell growth, being stimulated immediately after initiation of proliferation and decreasing progressively as cultures reached high cell density. This pattern corroborated that of N8-acetylspermidine intracellular accumulation induced by culturing cells in the presence of 1 mM 7-amino-2-heptanone, a competitive inhibitor of N8-acetylspermidine deacetylase. Histone acetylation during HTC cell growth was not markedly different qualitatively from that of spermidine. Moreover, spermidine and histone acetylations in hepatocyte nuclei were of the same order of magnitude as those seen in rat hepatoma cell nuclei. Finally, inhibition of deacetylation of N8-acetylspermidine had no apparent deleterious effects on cell and growth. It remains to be determined whether the acetylation step is of higher physiological importance, in particular, and as discussed in nuclear spermidine turnover.


The relationship between hsp 70 localization and heat resistance.

Using indirect immunofluorescence we have investigated the kinetics of nuclear accumulation and removal of hsp 70 in HA-1 Chinese hamster fibroblasts exposed to elevated temperatures. The kinetics of accumulation of hsp 70 in the nuclei were found to be time/temperature dependent at all temperatures tested (42-45 degrees C). At a given temperature, the fraction of cells manifesting nuclear localization of hsp 70 increased with exposure time. For a given duration of heating, the fraction of cells manifesting nuclear localization of hsp 70 increased with the temperature. The kinetics of the nuclear accumulation of hsp 70 were similar for normal HA-1 cells, their heat-resistant variants, and transiently thermotolerant cells (triggered by prior exposure to a brief heat shock or to sodium arsenite). Upon return to 37 degrees C after heat shock, the kinetics of removal of the hsp 70 associated with the nucleus was dependent on the severity of the initial heat challenge. However, for a given heat dose, the decay of nuclear localization of hsp 70 was more rapid in thermotolerant and heat-resistant cells than in their normal counterparts. These results suggest that the increased levels of hsp 70 associated with the transient or permanently heat-resistant state may play a direct role in restoring and/or repairing heat-induced nuclear and nucleolar alterations associated with heat-induced cell killing. Furthermore, they also suggest that the heat-resistant state may involve ameliorated repair of heat-induced cellular alterations.


Basal levels of max are sufficient for the cotransformation of C3H10T1/2 cells by ras and myc.

The ras and myc oncoproteins cooperate to transform the established murine fibroblast cell line C3H10T1/2. To determine the impact of overexpression of the myc oncoprotein on the phenotype of C3H10T1/2 cells, two C3H10T1/2-myc clonal cell lines, SVc-myc 11A and myc neo 13A, were isolated and characterized. Although both C3H10T1/2-myc cell lines are morphologically indistinguishable from wild-type C3H10T1/2 cells and possess growth properties similar to those of C3H10T1/2 cells, each displays a predisposition to transformation following transfection with the activated form of the human H-ras gene. In C3H10T1/2 cells overexpressing the v-myc or H-ras oncogenes, the levels of mRNA encoding max, the recently identified oligomerization partner of myc, remain unchanged, suggesting that the endogenous level of max in C3H10T1/2 cells is sufficient for a high frequency of transformation by ras and myc. Based on these studies, the C3H10T1/2-myc clonal cell lines we describe are suitable model systems for examining the molecular role of the myc protein in transformation and for characterizing additional factors that synergize with myc in multistep transformation.


Induction of peroxisomal enzymes and a 64-kDa peptide in cultured mouse macrophages treated with clofibrate.

The presence of catalase-containing particles was demonstrated in mouse macrophages of the cell line J774.3. These cells were incubated with clofibrate, a hypolipidemic drug known to cause peroxisome proliferation in rodent hepatocytes. The specific activities of catalase and fatty acid oxidase were significantly increased after 48 h incubation with 2 mM clofibrate. This response proved to be time and dose dependent. Other organelle marker enzymes showed little change in activity. Unexpectedly, clofibrate treatment also produced an increase in a 64-kDa peptide in these cells.


Phosphorylation of Xenopus elongation factor-1 gamma by cdc2 protein kinase: identification of the phosphorylation site.

The cdc2 protein kinase phosphorylates elongation factor-1 gamma (EF-1 gamma) during meiotic maturation of Xenopus oocytes. A synthetic peptide P2: PKKETPKKEKPA matching the cDNA-deduced sequence of EF-1 gamma was an in vitro substrate for cdc2 protein kinase and inhibited phosphorylation of EF-1 gamma. Tryptic hydrolysis of EF-1 gamma and the P2 peptide, both phosphorylated by cdc2 protein kinase, resulted in multiple partial digestion products generated by the presence of barely hydrolysable bonds. The two peptides obtained from the hydrolysis of EF-1 gamma comigrated exactly in two-dimensional separation with two of the P2 peptide hydrolysates. EF-1 gamma therefore contains one unique phosphoacceptor for cdc2 protein kinase, identified as threonine-230.


The phase of sperm flagellar beating is not conserved over a brief imposed interruption.

We have studied the phase component of flagellar beating by holding the head of a sea urchin sperm in the tip of a sinusoidally vibrating micropipet and then abruptly displacing the pipet laterally at a speed of 2.5 microns/ms for various durations. This rapid displacement of the pipet delayed the initiation of the next bend for as long as the displacement continued, up to a duration of 1 beat cycle, corresponding to a delay of 0.5 beat cycle. At the end of this displacement, the movement of the pipet was stopped completely without resumption of the initial vibration. Analysis of the flagellar waveform showed that immediately when the pipet was stopped, the flagellum started to beat by spontaneously initiating the bend that had been delayed. The flagellum then continued steady-state beating, with normal waveform and a new phase that was independent of the original phase of beating. These data suggest that the information on the phase of beating is located only at the basal end of the flagellum, and not in oscillators distributed along the axoneme. After this information has been lost, the flagellum can resume beating at any arbitrary phase relative to its original phase.


Streptococcal pharyngitis in general practice. 1. Some unusual features of the epidemiology.

This report is based on a study of acute infections of the upper respiratory tract in 1965 and detailed records of such infections in 1963 and 1964. A change from illnesses mainly yielding viruses to illnesses mainly yielding group A streptococci was noted around the age of 5 years. A positive culture for group A streptococci in patients over 4 years of age was highly correlated with a complaint of sore throat and with serological evidence of streptococcal infection. A bimodal age distribution curve for pharyngitis associated with a positive culture for group A streptococci was consistently noted. The incidence was highest in children aged 5-9 but a second smaller peak occurred among adults in the 30-39 age group. The evidence suggests that being female increases the risk of acquiring group A streptococci and of experiencing sore throat.


Streptococcal pharyngitis in general practice. 2. A note on dual infection and transient urinary abnormalities.

In an incomplete study in 1965 microscopic haematuria in the second or third week after acute pharyngitis was found four times more often in patients with either microbiological or clinical evidence of dual infection with both group A streptococci and a virus than in patients with evidence only of infection with group A streptococci. Prospective studies of the role of viruses in the aetiology of transient haematuria and of acute post streptococcal glomerulonephritis are feasible in general practice and would be most productive if concentrated in children 5-9 years of age.


Splenomegaly in acute infections due to group A streptococci and viruses.

Over a period of 9 years in general practice temporary enlargement of the spleen was found in 29 episodes of pharyngitis or tonsillitis, in 2 episodes of acute upper respiratory tract infection other than pharyngitis and in 6 episodes of acute cervical lymphadenitis. In five patients more than one episode of illness associated with splenomegaly was recorded. In 26 of the 37 episodes a possible aetiology was identified. Evidence only of infection with group A streptococci was found in 14 episodes, adenoviruses or coxsackie B viruses were isolated alone in 4 episodes and in 4 episodes the only finding was the presence in the blood of more than occasional atypical mononuclear cells; in 4 episodes there was evidence of both streptococcal and viral infection. Episodes with evidence of streptococcal infection only tended to be of shorter duration and to be more evenly distributed over the year than were episodes without such evidence. Temporary splenomegaly was noted also in two children with varicella (one of whom also had streptococcal infection) and in an adult with probable rubella.


A one-year study of streptococcal infections and their complications among Ethiopian children.

Post-streptococcal complications are known to be common among Ethiopian children. Little is known, however, about the epidemiology of beta-haemolytic streptococci in Ethiopia. A total of 816 children were studied during a one-year period: 24 cases of acute rheumatic fever (ARF), 44 chronic rheumatic heart disease (CRHD), 44 acute post streptococcal glomerulonephritis (APSGN), 143 tonsillitis, 55 impetigo, and 506 were apparently healthy children. Both ARF and APSGN occurred throughout the year with two peaks during the rainy and cold seasons. The female:male ratio among ARF patients was 1.4:1 and 1:1.9 among APSGN. The monthly carrier rate of beta-haemolytic streptococci group A varied from 7.5-39%, average being 17%. T type 2 was the most frequent serotype. Marked seasonal fluctuations were noted in the distribution of serogroups among apparently healthy children. Beta-haemolytic streptococci group A dominated during the hot and humid months of February-May. Strains were susceptible to commonly used antibiotics, except for tetracycline.


Transmission of Neisseria meningitidis among asymptomatic military recruits and antibody analysis.

Following the occurrence of a case of systemic meningococcal disease in a military camp in Norway, throat cultures and blood samples were collected from 33 healthy individuals belonging to the same troop as the patient (troop A) and from 29 individuals from a different troop (troop B) in the same camp. Serological studies showed that 91% of the recruits had bactericidal antibodies against the disease-causing strain. The isolates of Neisseria meningitidis recovered from the throat cultures were serogrouped, serotyped, and assigned to a clone on the basis of an analysis of the electrophoretic mobilities of 14 metabolic enzymes. None of the 23 carriers in troop A harboured the clone responsible for the case of disease, but 6 carried isolates of the same electrophoretic type, ET-7, which was not identified in any of the 19 carriers of troop B. Individuals in troop A were resampled 2 and 17 weeks after the meningococcal disease episode. Five of the carriers had acquired different clones and one of them changed clone twice in that period. Four of the six newly acquired clones had previously been identified in other carriers of troop A, demonstrating transmission of clones among individuals living and working in close proximity.


The serological grouping system for Serpulina (Treponema) hyodysenteriae.

Lipopolysaccharide from serostrains of Serpulina (Treponema) hyodysenteriae for serogroups A to I was characterized using sodium dodecylsulphate polyacrylamide gel electrophoresis and silver staining. All strains had lipopolysaccharide components ranging from 10 to 16 kDa that represented lipid A-core polysaccharide regions, and short O-antigen side chain were also recognized in certain immunoblots. Serological reactions between lipopolysaccharide and antisera against each of these serostrains were examined by Western immunoblotting. There was relatively little antigenic cross-reactivity between LPS from the nine strains, thus confirming their suitability as serostrains. Using cross-absorbed sera, isolates within serogroups A and E were shown to possess unique epitopes on the core lipopolysaccharide, distinct from serogroup reactivities. These isolates were therefore identified as serovars within the serogroups. This study confirmed the usefulness of the serotyping scheme for S. hyodysenteriae, in which the bacteria can be placed into serogroups using unabsorbed sera, and into serovars within these using cross-absorbed sera.


Concordance of auxotype/serovar classes of Neisseria gonorrhoeae between sexual contacts.

One hundred and three known sexual-contact pairs of patients with culture-proven gonorrhoea who attended St Mary's Hospital, London between May 1989 and February 1991 were identified. All isolates from these patients were serotyped and auxotyped and compared for type concordance within sexual-contact pairs. Serotype was concordant in 80 (78%) of 103 sexual-contact pairs, auxotype in 88 (85%) and auxotype/serovar (A/S) class in 66 (64%) on the first screening. All pairs of isolates showed concordance in both serotype and auxotype when typing was repeated using a single set of serotyping reagents and of auxotyping media. Seventeen serovars, 9 auxotypes and 36 A/S classes were found in this population. Our results suggest that both serotyping and auxotyping may be used as markers to allow tracing of sexual-contact pairs, but that a single set of reagents should be used to ensure maximum reliability.


Virulence factors of enteric Escherichia coli in young Aboriginal children in north-west Australia.

Enterotoxigenic Escherichia coli (ETEC) were the most frequently identified enteric pathogens associated with diarrhoea in 0-5 year old Aboriginal children in tropical north-west Australia with an incidence similar to those from other tropical regions. Heat-stable toxin-producing (ST+) strains were associated with diarrhoea throughout the year but heat-labile toxin-producing (LT+) strains were more important in the monsoonal summer season. ST+ strains were commonest in children with diarrhoea between 6 and 18 months of age while LT+ strains were associated with diarrhoea in children aged 18-24 months. Vero-toxigenic E. coli (VTEC) which produced VT1, but not VT2, and enteroadherent (EAF+) E. coli were significant causes of diarrhoea, mainly in children below 18 months but without a seasonal pattern.


Serological cross-reaction between Legionella pneumophila and campylobacter in the indirect fluorescent antibody test.

Sera from 50 patients with culture-proven campylobacter gastroenteritis were examined for the presence of antibodies to Legionella pneumophila. Ten patients (20%) had a positive titre (> or = 16) as measured by indirect immunofluorescence. Antibodies were detected in only 1 of 36 acute sera but in 10 of 14 (71%) sera obtained more than 10 days after the onset of symptoms. All positive sera contained specific IgM antibodies but specific IgG or IgA could not be detected in any sample. No legionella antibodies could be detected in sera from 42 similar patients with salmonella gastroenteritis. These results were shown to be due to serological cross-reaction between L. pneumophila and campylobacter.


Serological responses of chickens experimentally infected with Salmonella enteritidis PT4 by different routes.

Commercially reared chickens were challenged with Salmonella enteritidis phage-type (PT) 4 by aerosol, or via the conjunctiva. Inhalation of 2.9 x 10(2) or 4.2 x 10(3) S. enteritidis resulted in the production of IgG antibodies to the lipopolysaccharide (LPS) of S. enteritidis PT4. When the aerosol inoculum was increased to 2.4 x 10(5) bacteria per bird the antibody produced were predominantly of the IgM-class. Chickens challenged with 10(3) S. enteritidis PT4 via the conjunctiva mounted only a poor immune response. Increasing the challenge dose to 10(8) S. enteritidis resulted in the production of high-titre serum antibodies of both the IgG and IgM classes. Results from this study suggest that aerosols containing small numbers of S. enteritidis PT4 might be responsible for intraflock infection of poultry.


Accumulation of crystallin in developing chicken lens.

Separation and quantitation of crystallin subunits in embryonic and post-hatched chicken lens were carried out by two-dimensional gel electrophoresis and an image analysing system in order to elucidate detail in the accumulation process of each crystallin subunit in lens differentiation. Complete separation of the subunits was possible when 7 M urea was included in the second dimension gel of the electrophoresis. In particular, beta-crystallin could be separated into more than 24 spots on the gel. These experiments showed that delta-crystallin accumulated rapidly during early development up to more than 80% of total crystallins, while beta-crystallin accumulated quickly only after hatching. In contrast with the contents of beta- and delta-crystallins, alpha-crystallin content in total crystallins was kept at approximately 18% throughout lens development. Therefore, it was concluded that crystallins accumulated in several different ways. This suggests that different regulation mechanisms work on the accumulation of each crystallin subunit and that the subunit composition of lens proteins is specific to each state of lens development.


Oxygen kinetics in preretinal perfluorotributylamine.

Previous studies have relied on various electrodes or probes to monitor preretinal oxygen tension in an effort to gain insight into retinal oxygenation. In order to corroborate and extend the results of such studies, we developed a relatively non-invasive method of determining preretinal oxygen tension using 19F nuclear magnetic resonance (NMR) spectroscopy. Small liquid perfluorocarbon (LPFC) droplets were injected into the preretinal vitreous space of the rabbit eye. The T1 value obtained from the fluorine nuclide could then be used to determine preretinal oxygen tension (PO2) with a high degree of sensitivity, since the fluorine spin-lattice relaxation rate (T1)-1 in LPFCs is directly proportional to PO2 under conditions of no flow and known temperature. In the present study, we investigated the oxygen uptake and clearance rates from small preretinal droplets of the LPFC perfluorotributylamine (FTBA) in response to step changes in arterial PO2. At all FTBA volumes examined (2, 10 and 100 microliters), the oxygen uptake and clearance curves were well approximated by a simple exponential equation with mean time constants 9.8/15.3, 21.4/19.4 and 77.7/45.3 min (uptake/clearance), respectively. Following return to normoxemic (baseline) conditions, FTBA droplets provided a preretinal PO2 of 39.4 +/- 9.2 mmHg (mean +/- S.D., n = 12). The 19F NMR method provides a measure of steady-state preretinal PO2 that independently verifies and complements information obtained using oxygen-sensitive microelectrodes or probes. However, the long time constants for oxygen uptake and clearance, particularly in FTBA volumes on the order of 10 microliters and greater, may represent a practical limitation of this method for determining rapid oxygen flux in the preretinal vitreous space.(ABSTRACT TRUNCATED AT 250 WORDS)


Structural studies on beta H-crystallin from bovine eye lens.

Bovine lens beta H-crystallin, isolated at pH 6.7, undergoes reversible dissociation into dimers and an intermediate size of oligomer (peak A) at pH 5.4. Peak A is enriched in the beta B1 subunit but lacks beta B2, whereas beta B2 is a major component of the dimers. A method for isolation of beta B1 from peak A is described. The pH dependence of the dissociation-reassociation suggests that histidines on the surface of the dimers become buried in the assembly of beta H-crystallin. The positions of the four histidines on the surface of the compact domains of each subunit of the beta B2 homodimer are shown. The beta B1-enriched oligomer has a much lower solubility compared with the beta B2 containing beta H-crystallin. It is possible that beta B2 plays a role in solubilizing beta-crystallin aggregates.


Concerning the symmetry of the 'hexagonal' cells of the corneal endothelium.

Modeling studies are presented to show how the figure coefficient and hexagon deviation index values decrease as a six-sided endothelial cell is systematically distorted from a completely symmetrical geometric shape (a regular hexagon). By use of scanning electron micrographs of rabbit corneal endothelium and an analysis of published specular micrographs of rabbit corneal endothelium, it is shown that the six-sided cells are not necessarily very symmetrical. Such cells should thus be reported as six-sided cells rather than as 'hexagons' which carries the inherent risk of implying regularity (and thus supposed stability in a tesselated mosaic) of such cells.


Changes in Müller cell plasma membrane specializations during subretinal scar formation in the rabbit.

The aim of this study was to identify changes in Müller cell plasma membrane specializations during experimentally induced subretinal gliosis in rabbits. When rabbits are dosed with sodium iodate, large expanses of retinal pigment epithelium and photoreceptors are destroyed. They are replaced by a subretinal scar consisting mainly of the ascending processes of Müller cells. These processes transform from the slender, highly polarized structures seen in normal animals into irregular processes that form a glia limitans along the basement membrane of the pigment epithelium, left bare following its degeneration. As the scar processes extend through the subretinal space and contract this basement membrane, they undergo dramatic changes in shape that are especially apparent in three-dimensional computer reconstructions of serial thick sections examined by high-voltage electron microscopy. Other changes involve the intercellular junctions and apical microvilli normally associated with the external limiting membrane. These structures become scattered over the surfaces of the ascending processes and are eventually lost. Loss of microvilli is associated with disappearance of immunostaining for a specific glycoconjugate normally associated with the microvillar plasma membrane. The observations document profound changes in Müller cell structural and functional polarity during subretinal scar formation.


CD68 antigen expression by human retinal pigment epithelial cells.

Although a primary role of the retinal pigment epithelium (RPE) is the phagocytosis of aged outer segment membranes, the RPE may also phagocytize particulates via several specific receptors that are characteristically present on mononuclear phagocytes of bone marrow origin. In recent immunophenotypic studies, CD68 monoclonal antibodies (mAb) have been shown to react selectively with a specific 110 kDa cytoplasmic glycoprotein present in mononuclear phagocytes from various sources. Designated as anti-macrophage antibodies that react with this macrophage-associated antigen, CD68 antibodies are now widely used for immunohistochemical identification of mononuclear phagocytes. Using a panel of CD68 mAb (KP1, EMB11, Ki-M6, Y1/82A, and Y2/131) we performed immunohistochemistry on three cytospin preparations of freshly isolated human RPE cells, three primary human RPE cultures, and 12 human RPE cell lines maintained in culture for up to 40 passages. Cytospin preparations of freshly isolated RPE cells demonstrated heavy reactivity in 5% of cells. Five- to 7-day-old primary RPE cultures exhibited uniform, heavy staining of all cells. Strong immunohistochemical reactivity persisted in all 12 cell lines at various passages up to and including passage 40. Stimulation of cultured RPE cells with interferon-gamma (100 U ml-1) for 24 and 48 hr did not produce observable differences in CD68 staining. RPE cells failed to stain when control mAb or mouse serum were substituted for the primary antibody. The constitutive expression of CD68 by neuroectodermally-derived RPE cells extends their immunophenotypic similarities with mesenchymally-derived mononuclear phagocytes and provides an additional antigenic marker to identify RPE cells in vitro.


Transplantation of embryonic retina to the subretinal space in rabbits.

Embryonic rabbit retina can be transplanted to the subretinal space of adult rabbit with a new method, which gives a high rate of successful short-term transplants. Embryonic (stage E 15) neural retina cells were injected through an incision just behind the sclerocorneal border with a thin (inner diameter 0.15-0.4 mm, outer diameter 0.3-0.5 mm) plastic tube attached to a specially designed instrument, by which the length of the protruding plastic tip could be controlled. The retina was penetrated from the vitreous side and the donor tissue was injected into the subretinal space. The cells survived in the host for at least 5 months, although the long-term survival rate tended to decrease. The transplanted cells matured and differentiated, forming an approximation of the layered, retinal structure with some anomalies (e.g. rosettes). The subretinal location offers an interesting and convenient way of studying the development of retinal cell transplants in rabbits. Large transplants can be produced, and the risk for failures due to erroneous vitreous placement is small.


Ischaemia- and reperfusion-induced Na+, K+, Ca2+ and Mg2+ shifts in rat retina: effects of two free radical scavengers, SOD and EGB 761.

Using Sprague-Dawley rats with transient (90-min) regional ischaemia induced by retinal artery occlusion in the eye, we have shown that superoxide dismutase (SOD) and EGB 761 (IPSEN, France), two free radical scavengers, can dramatically reduce the reperfusion-induced sodium and calcium gains, and potassium loss in retinal tissue. Investigating whether this was a 'direct' protective effect, operating during reperfusion, or an 'indirect' effect arising from the action of SOD or EBG 761 on the tissue during ischaemia. SOD (15,000 U kg-1) and EGB 761 (100 mg kg-1) were added to the rats at the moment of reperfusion (after an ischaemic insult). Eyes were subjected to 90 min ischaemia followed by 4 and 24 hr of reperfusion, respectively. In the drug-free control group, 90 min of ischaemia resulted in an accumulation of retinal sodium (2-fold) and calcium (3-fold), and a loss of cell potassium (by 40%) and magnesium (by 40%). During the first 4 hr of reperfusion the ionic imbalance was unchanged, while after 24 hrs of reperfusion a normalization was observed and the ion content of the retina almost returned to their preischaemic values. SOD and EGB 761 treatment significantly reduced the reperfusion-induced ionic imbalance (magnesium was an exception) and improved the recovery of retinal ion contents. Our results indicate that the elimination of oxygen radicals by free radical scavengers may reduce the reperfusion-induced ionic imbalance and improve the ionic homeostasis in the injured retinal cells.


Two cone types of rat retina detected by anti-visual pigment antibodies.

The presence of two distinct cone types was demonstrated in the retina of the rat using two cone-specific monoclonal anti-visual pigment antibodies. Cones labelled by antibody COS-1 constituted the large majority (about 93%) of cones, and are most probably responsible for the green photopic sensitivity of the rat. About 7% of the cones were recognized by antibody OS-2, and are thought to be blue-sensitive elements. While OS-2 positive cones were evenly distributed throughout the retina, there were slight differences in the distribution of COS-1 positive cones. The cones made up about 0.85% of all photoreceptor cells. Although the OS-2 positive cones occur in a very low number (0.05% of all photoreceptors) and probably do not appreciably contribute to the photopic system of the rat, their presence in the rat strengthens the presumption that most mammalian species exhibit a dual cone system with a shortwave and a middle-to-longwave sensitivity.


Kynurenine identified as the short-wave absorbing lens pigment in the deep-sea fish Stylephorus chordatus.

A number of deep-sea fish have bright yellow lenses whose coloration is attributable to a variety of largely unidentified short-wave absorbing pigments. Here the pigment of the deep-sea fish Stylephorus chordatus has been isolated and identified by NMR and mass spectroscopy as kynurenine; a pigment also found in the human lens. The degree of this pigmentation is greater in older animals. The fact that the lenses of both a deep-sea fish and man contain the same pigment is of interest, given the vastly different light environments they inhabit.


The effects of isovolumic hemodilution on ocular blood flow.

Techniques by which retinal blood flow may be increased safely are potentially important in the treatment of retinal vascular disease. It was hypothesized that hemodilution, which increases cerebral blood flow, would also increase retinal blood flow. To investigate the physiological effects of hemodilution in the eye, ocular blood flow was measured in 14 cats using the radioactively labeled microsphere method. After the animals were anesthetized with halothane and oxygen, intraocular and systemic arterial pressure were recorded; blood flows were measured before and after isovolumic hemodilution to a hematocrit of 20-22% using 6% hydroxyethyl starch (a synthetic plasma expander with a molecular weight of 450 in 0.9% saline). In hemodiluted cats, retinal blood flow increased 71% from its baseline value (36.7 +/- 6.4 ml 100 g-1 min-1 to 62.9 +/- 6.4 ml 100 g-1 min-1, mean +/- S.E.M., P < 0.0001). Calculated retinal O2 delivery remained approximately constant, as the increased blood flow countered a significant decrease in arterial O2 content. Choroidal blood flow decreased (1297 +/- 140 ml 100 g-1 min-1 to 1051 +/- 144 ml 100 g-1 min-1) but the change was not statistically significant. Blood flows in the iris and sclera were not significantly altered. Hemodilution increased retinal blood flow without causing a redistribution in ocular blood flow.


Morphologic and molecular changes during the post-natal development of the rabbit vitreous.

Rabbits aged 1, 4, 10, 15, and 20 days, and 4 months were anesthetized and perfused with 4% formaldehyde. One eye of each rabbit was processed for paraffin embedding, while the other eye was embedded intact in methacrylate. Rabbits aged 1 and 15 days and 4 months were perfused with 2.5% glutaraldehyde, and the eyes were processed for Epon embedding. The paraffin sections were immunostained to allow detection of a high molecular weight cartilage matrix glycoprotein (CMGP), which is synthesized by the ciliary body and found in the vitreous in adult animals, using a specific mouse monoclonal antibody. CMGP was identified in the vitreous and in the inner layer of the ciliary epithelium only after the fifteenth day of life in amounts comparable to those detected in adult rabbits. Before this time immunostaining with the monoclonal antibody was seen only in the apical region of the inner ciliary epithelial cells. However, electron microscopic observations revealed that the cytoplasmic organelles responsible for the secretion of glycoproteins, i.e. the rough endoplasmic reticulum, Golgi apparatus, and vesicles, were present in the inner layer of ciliary epithelial cells as early as the first day of life. Anteroposterior sections of whole eyes embedded in methacrylate revealed a relatively dense meshwork of vitreous fibrils on the first day of life. The blood vessels were concentrated at the posterior region of the lens, and isolated cells were visible. The blood vessels were not seen after the age of 15 days, and the fiber meshwork and cells were inconspicuous by then.(ABSTRACT TRUNCATED AT 250 WORDS)


Relations among IOP reduction, ocular disposition and pharmacology of the carbonic anhydrase inhibitor ethoxzolamide.

We have measured sequentially the concentrations of ethoxzolamide (6-ethoxybenzothiazole-2-sulfonamide) in ocular tissues following its intravenous or topical administration to normal albino rabbits. This was done in parallel with determinations of intraocular pressure (IOP) measured by tonometer or direct manometry. Ethoxzolamide was used because of its very high activity against carbonic anhydrase and experience showing that there is little or no other receptor in tissues. During the course of these experiments it was discovered that the lipid-soluble ethoxzolamide is converted in vivo to a water-soluble metabolite, while retaining high activity against the enzyme. Presumably this is the 6-O-glucuronide adduct. At the minimal dose for maximal effect (4 mg kg-1 i.v. at 45 min) the IOP lowering was 4.2 mmHg, the concentration in anterior uvea was 2.5 mumol kg-1, and the fractional inhibition of the enzyme (i) was 0.9995. The effect of free drug in the anterior uvea and other tissues. Following topical administration i was measured as a function of drug and enzyme in ciliary process. IOP lowering at 1 hr was -1.9 mmHg and i = 0.9993. By 4 hr i = 0.9980 and the pharmacological effect disappeared. At 8 hr the concentration of ethoxzolamide in the ciliary process is 0.4 mumol kg-1, essentially that of enzyme, with no free drug present: drug is now a marker for enzyme. Ethoxzolamide also labels the red cell carbonic anhydrases in the rabbit as well as other species including man. There appears to be no ethoxzolamide receptor other than carbonic anhydrase.(ABSTRACT TRUNCATED AT 250 WORDS)


Ocular pharmacokinetics of lens epithelial cell-specific immunotoxin 4197X-RA.

The ocular clearance, stability and systemic distribution of a lens epithelial cell-specific immunotoxin were evaluated in adult Dutch Belted rabbits. Immunotoxin 4197X-RA was prepared from a murine monoclonal antibody conjugated by a disulfide linkage to ricin A. After intracameral (i.c.) injection, clearance of the immunotoxin from the eye followed a first-order process. The half-life (t1/2) of immunotoxin in the aqueous humor was 51 min. Accumulation of the immunotoxin in ocular tissues appeared to be associated with aqueous outflow tracts, as it was primarily confined to the iris and the limbal regions of the cornea. No acute metabolism of immunotoxin was observed in the anterior chamber. The immunotoxin was detected in the blood 20 min post-injection and achieved a steady-state level after 40 to 180 min. A slow decline in labeled protein was then observed from 180 min to 24 hr. Systemic clearance of the immunotoxin appeared to occur primarily by way of the kidneys. The present data indicate that immunotoxin 4197X-RA, like other large proteins, is passively cleared from the anterior chamber by aqueous flow, and that little or no acute breakdown occurs in the anterior chamber. It is anticipated that these data, along with results from cytotoxicity studies, will result in the determination of optimal doses and frequency of administration for the use of immunotoxin in human clinical trials.


Aqueous humor hydrogen peroxide analysis with dichlorophenol-indophenol.

Hydrogen peroxide is now reported to be a normal aqueous humor component present, in the low microM concentration range, in the animal species which have been studied. This finding was established with the exclusive use of the dichlorophenol-indophenol method of analysis. In this procedure, aqueous humor is added to a blue, oxidized dichlorophenol-indophenol solution. The 605 nm absorbance of this solution immediately decreases in response to the reducing action of ascorbate present in the sample. The extent of reoxidation of the solution upon the addition of peroxidase, as measured by the increase in its 605 nm absorbance, can be quantitatively related to the concentration of H2O2 in the sample. A close examination of this method revealed that reduced dichlorophenol-indophenol spontaneously reoxidizes at a rate of 0.03 nmol min-1 microM-1, with generation of H2O2. H2O2 generation was unequivocally established by analysis of the temporal dependency of the absorbance increase produced by peroxidase in the absence of added H2O2 and by the sensitivity of this phenomenon to catalase. This spontaneous production of H2O2, on the other hand, cannot be attributed to ascorbate auto-oxidation because added ascorbate quantitatively reacts with dichlorophenol-indophenol, provided that an excess of the latter is maintained. This method then has an enormous potential to overestimate H2O2 in any sample. On the other hand, the response of the assay system to a given level of H2O2 depends on the level of reduction previously produced by ascorbate. This results in an artifactual positive correlation between ascorbate and H2O2 levels in samples containing variable amounts of ascorbate. In spite of these serious limitations the method can still be useful to measure H2O2 if appropriate precautions are taken. When using it for the analysis of rabbit aqueous humor H2O2 without correcting for the H2O2 generated during the assay and ignoring differences in the level of ascorbate in the samples, we obtained an average value of 25.3 microM H2O2, which coincides with that reported in the literature for the rabbit, but is obviously incorrect. When analysing aqueous humor there was the additional variable of the aqueous humor itself inhibiting the rate of dichlorophenol-indophenol auto-oxidation and so the final, corrected figure for H2O2 concentration in the aqueous humor became uncertain, since the auto-oxidation of the substrate could not be properly subtracted.(ABSTRACT TRUNCATED AT 400 WORDS)


Docosahexaenoic acid increases in frog retinal pigment epithelium following rod photoreceptor shedding.

The vertebrate retina conserves docosahexaenoic acid (22:6n-3) during n-3 fatty acid deficiency. The mechanism of conservation is not known, although recycling of this fatty acid between the retinal pigment epithelium (RPE) and retina is one possibility. We examined the role of the RPE in conservation of 22:6n-3 by quantitating the fatty acids and phospholipid molecular species (PLMS) in frog RPE before and after light-stimulated shedding of rod outer segments (ROS). RPE cells were dissociated with brush agitation and purified by a discontinuous ficoll density gradient. One hour after the light-induced shedding of ROS, the phagocytosed ROS tip and opsin content of RPE had increased. Simultaneously, the levels of 22:6n-3 and 22:6(n-3)-containing PLMS were increased in the RPE. Within 8 hr following the shedding event, 22:6n-3 in the RPE had returned to the dark level. These findings indicate that the phagocytosed ROS tips contain 22:6n-3 and that the RPE metabolizes these ROS tips and eliminates 22: 6n-3 from the cell. Thus, the RPE is intimately involved in the metabolism of 22: 6n-3 in the retina. The recycling of 22: 6n-3 from the RPE to the retina is a possible means of conserving this important fatty acid in the retina.


Synaptic potentials produced in jaw-closer and jaw-opener motoneurons by palatal stimulation.

Excitation and inhibition of temporal and digastric motoneurons (Temp. and Dig. Mns) during transient jaw closing, the so-called jaw-closing reflex, were studied in cats. Application of diffuse pressure stimulation to the posterior palatal surface produced the jaw-closing reflex and it was found that mechanosensory inputs from the posterior palatal mucosa produce depolarizing potentials on the Temp. Mns responsible for jaw closure during the jaw-closing reflex. We have demonstrated that in one-third of 27 explored Temp. Mns the initial bursts of spikes were elicited before the onset of jaw closure, suggesting that these cells contribute to initiate jaw closure during the jaw-closing reflex. The remaining cells probably contributed to maintain the occlusal phase. Furthermore, it was found that mechanosensory inputs from the posterior palatal mucosa produce a hyperpolarization-depolarization sequence in the Dig. Mns responsible for the jaw-closing reflex. In addition, when pressure stimulation was applied to the anterior palatal mucosa, sustained jaw opening was elicited and an increase of firing frequency of Dig. Mns occurred 40 ms before the onset of jaw opening and continued for 80 ms.


Two modes of cerebellar input to the parietal cortex in the cat.

The characteristics of cerebellar input to the parietal cortex through the ventroanterior-ventrolateral (VA-VL) complex of the thalamus were investigated in the adult cat by using combined electrophysiological and anatomical methods. Two distinct parietal regions were activated by stimulation of the cerebellar nuclei (CN). In the first region located in the depth of the bank of the ansate sulcus, stimulation of the CN induced early surface positive-deep negative potentials and late surface negative-deep positive potentials. In this cortical area, potentials of similar shape and time course were evoked at a shorter latency by stimulation of the ventrolateral part of the VA-VL complex where large negative field potentials were evoked by stimulation of the CN. After injection of the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) in this part of the VA-VL complex, axon terminals of thalamocortical (TC) fibers were found in layers I, III and IV in the depth of the bank of the ansate sulcus and layers I and III in the motor cortex. In the second region located in the suprasylvian gyrus, late surface negative-deep positive potentials were evoked by stimulation of the CN and similar potentials were evoked at a shorter latency from the dorsomedial part of the VA-VL complex where large cerebellar-evoked potentials could be recorded. PHA-L injection in this thalamic region stained TC fibers and their terminals in layer I of the suprasylvian gyrus, and in layers I and III of the motor cortex. The laminar distribution of TC axon terminals in two different regions of the parietal cortex could account for the depth profiles of the cerebellar- and the thalamic-evoked potentials in each region. These results show that cerebellar information is conveyed to two separate areas in the parietal cortex by two different TC pathways.


Induction of high frequency activity in the somatosensory thalamus of rats in vivo results in long-term potentiation of responses in SI cortex.

Extracellular single-unit techniques were employed to record unitary activity simultaneously from the thalamic ventral posterior medial (VPM) nucleus and the ipsilateral primary somatosensory cortex of adult rats. Cross-correlation analysis triggered by the spontaneous firing of thalamocortical relay neurons in VPM and the discharge of layer IV neurons in the corresponding ipsilateral cortical barrel indicated that the paired-units included in this study were strongly correlated in their activity. The baseline responses of highly correlated cortical/thalamic pairs to a 10 ms deflection of a vibrissa on the contralateral side were measured using poststimulus time histograms. After establishing the baseline response, high frequency activity in VPM was induced in one of two ways: i) direct electrical stimulation of thalamic neurons or ii) whisker stimulation in the presence of bicuculline methiodide (BIC) released near the thalamic neurons. Both methods resulted in a conditioning stimulus (CS) paradigm consisting of "burst" of high-frequency activity (50-100 Hz) with an inter-burst interval of 150 ms (approximately 7 Hz). Almost immediately following the presentation of the CS, the response of layer IV cortical neurons to vibrissa stimulation increased by 37-62% over baseline values, which was maintained after the effects of BIC had worn off in VPM. This enhancement in the response of the cortical neurons was not accompanied by a concomitant increase in the thalamic responses. Thus, these results strongly suggest that the potentiation first occurred at the thalamocortical synapse.


Neurons of the motor trigeminal nucleus project to the hypoglossal nucleus in the rat.

The hypoglossal nucleus (Mo12) contains motoneurons that innervate the tongue, while the motor trigeminal nucleus (Mo5) contains motoneurons that elevate or depress the mandible. Previous studies have revealed lateral and medial tegmental field neuronal afferents to the Mo12 adjacent to, but not within, the motor trigeminal nucleus (Mo5). The current studies demonstrate the presence of retrogradely labeled neuronal afferents to the Mo12 within the Mo5 produced by as little as 10 nl of Fast Blue (FB) injected into the Mo12. Retrograde labeling of Mo5 afferents to the Mo12 with injections of Diamidino Yellow (DY) combined with injections of FB into the lumbar spinal cord showed these neuronal afferents to the Mo12 are not part of the diffuse projections to motoneurons from the nucleus subcoeruleus. Retrograde labeling of Mo5 afferents to the Mo12 with DY combined with injections of FB into the masseter revealed these neuronal afferents to the Mo12 are not trigeminal motoneurons. These results indicate that Mo5 interneurons are part of the lateral tegmental field projections to the Mo12, and are likely to comprise part of the neural substrate coordinating the motor activity of the jaw and tongue.


The area postrema is not involved in osmotic activation of neurosecretory cells in the supraoptic nucleus.

Experiments were performed to examine whether or not the area postrema (AP) is involved in the osmotic control of neurohypophysial hormone release. In control rats and in rats bearing extensive lesions of AP, extracellular action potentials were recorded from neurosecretory cells in the supraoptic nucleus (SON) and firing rates determined before and after the intraperitoneal (i.p.) injection of 1.5 M-NaCl solution. Lesion of the AP significantly (p less than or equal to 0.05, Mann-Whitney's U-test) lowered firing rate of putative vasopressin (phasic) cells but not that of oxytocin (non-phasic) cells. In lesioned animals, i.p. injection of hypertonic saline, however, caused similar changes in plasma osmotic pressure, plasma oxytocin concentration and mean firing rates of both putative oxytocin and vasopressin cells to those in control rats. The results suggest that the osmotic control of SON neurosecretory cell activity in the rat can take place in the absence of the AP.


Cortex, striatum and cerebellum: control of serial order in a grooming sequence.

Rats emit grooming actions in sequences that follow characteristic patterns of serial order. One of these patterns, a syntactic chain, has a particularly stereotyped order that recurs spontaneously during grooming thousands of times more often than could occur by chance. Previous studies have shown that performance of this sequence is impaired by excitotoxin lesions of the corpus striatum. In this study we examined whether the striatum is unique in its importance to this behavioral sequence or whether control of the sequence instead depends equally upon the cortex and cerebellum. In two experiments, a fine-grained behavioral analysis compared the effects of striatal ablation to the effects of motor cortex ablation, ablation of the entire neocortex, or ablation of the cerebellum. Cortical and cerebellar aspiration produced mere temporary deficits in grooming sequences, which appeared to reflect a general factor that was nonsequential in nature. Only striatal damage produced a permanent sequential deficit in the coordination of this syntactic grooming chain. We conclude that the striatum has a unique role in the control of behavioral serial order. This striatal role may be related to a number of sequential disorders observed in human diseases involving the striatum.


Similarities and differences between cholinergic systems in the superior colliculus of guinea pig and rat.

We studied the distribution of acetylcholinesterase activity and choline acetyltransferase immunoreactivity in the superior colliculus of the guinea pig and the albino rat, using enzyme histochemical and immunohistochemical methods. Choline acetyltransferase-like immunoreactivity was localized in the neuropil throughout the colliculi, but the density of the immunoreactive neuropil varied among layers as well as between species. In the intermediate collicular layers the pattern of choline acetyltransferase immunoreactivity was closely matched by the distribution of acetylcholinesterase activity in guinea pig and rat, confirming our previous findings in the cat. Furthermore, in the guinea pig, but not in the rat, choline acetyltransferase-like immunoreactivity was localized in a prominent population of perikarya of the superficial gray layer.


Supplementary eye field: influence of eye position on neural signals of fixation.

Single units were recorded in the supplementary eye field of monkeys performing visual-oculomotor tasks. Two patterns of unit activity were observed while the animals fixated photic stimuli. One consisted of a step in tonic firing frequency (either an increase or a decrease, depending on the cell), lasting as long as fixation. The occurrence of this pattern did not require the continuous presence of a stimulus but the animal had to be provided with an incentive to fixate a given location. The other pattern was a monotonically varying firing rate dependent on the eye orientation along a particular axis, indicative of eye eccentricity in orbit.


Longitudinal neuronal organization of defensive reactions in the midbrain periaqueductal gray region of the rat.

In a previous study we investigated the intraspecific defensive reactions evoked by excitation of neurons in the intermediate third of the midbrain periaqueductal gray matter (PAG) of the rat. Experiments revealed that activation of neurons in this region of the PAG mediated: (i) backward defensive behavior, characterized by upright postures and backward movements, and (ii) reactive immobility ("freezing"), in which the rat remained immobile, but reacted with backward defensive behavior to investigative, non-aggressive contact initiated by the partner. In the present study, we aimed to extend our understanding of PAG mediation of defensive behavior by observing: (i) in a non-aggressive social interaction test, the behavioral effects of microinjections of low doses of kainic acid (40 pmol in 200 nl) made in the caudal third of the PAG; and (ii) the behavioral and cardiovascular effects of microinjections of D,L-homocysteic acid (5-10 nmol in 50-100 nl) made in the PAG of the unanesthetized decerebrate rat. Kainic acid injections into the area lateral to the midbrain aqueduct in the caudal third of the PAG evoked: (i) forward avoidance behavior, characterized by forward locomotion and occasional hop/jumps; (ii) reactive immobility ("freezing"), in which the rat remained immobile, but reacted with forward avoidance behavior to investigative, non-aggressive contact initiated by the partner; and (iii) 22-28 kHz ultrasonic vocalizations. These injections also evoked a dramatic increase in defensive responsiveness to tactile stimuli on the half of the body contralateral, but not ipsilateral, to the site of injection. Electroencephalographic measurements indicated that none of these effects were secondary to seizure activity. In the decerebrate rat, D,L-homocysteic acid injections in the caudal third of the PAG evoked forward running movements along with increased blood pressure and heart rate, the strongest effects being evoked from the region lateral to the midbrain aqueduct. More rostrally, sites in the intermediate PAG evoked backward "defensive" movements, which were also associated with increased blood pressure and heart rate.(ABSTRACT TRUNCATED AT 400 WORDS)