Morphological analysis of the neurons in the area of the hypothalamic magnocellular dorsal nucleus of the guinea pig

Summary In the guinea-pig hypothalamus, a group of enkephalinergic cells forms a well-circumscribed nuclear area called the magnocellular dorsal nucleus (MDN). This nucleus gives rise to a prominent projection to the lateral spetum: the hypothalamo-septal enkephalinergic pathway. In the present study, MDN neurons visualized by Golgi impregnation were subjected to morphological analysis in order to define the potential segregation of cellular types within the MDN. This study was complemented by additional observations of MDN neurons intracellularly injected by Lucifer yellow (LY) or horseradish peroxidase (HRP) during the in vitro incubation of hypothalamic slices. The following results were obtained from the analysis of 200 neurons: 163 Golgi-impregnated cells plus 37 injected cells (LY=14; HRP=23). Thirteen HRP-injected cells were precisely located in the MDN and 10 were located in the perifornical area surrounding the MDN. Four different cellular types were identified. Type-I neurons (41%) displayed a globular perikaryon, a variable number of primary dendrites that were poorly ramified, no preferential orientation, and an axon emerging from the perikaryon. Type-II neurons (30.5%) had a triangular perikaryon, three well-ramified primary dendrites, an orientation perpendicular to the third ventricle, and an axon emerging from the perikaryon. Type-III neurons (22%) exhibited a spindle-shaped perikaryon, two opposed well-ramified primary dendrites, an orientation perpendicular to the third ventricle, and an axon emerging from a primary dendrite. Type-IV neurons (6.5%), showed a globular perikaryon, a variable number of primary dendrites, poorly ramified dendrites, an orientation parallel to the third ventricle, and an axon whose orientation could not be identified. Neurons labeled after intracellular injection belonged to the first three cellular types.     

Effect of acetylcholine on the structuro-functional characteristics of bush-like interoceptors of the urinary bladder of the frog

In the preparation of the frog (Rana temporaria) isolated urinary bladder, it has been stated by morphological and physiological experiments that under an exogenic acetilcholine influence certain structural-functional changes take place in the bushy interoceptor. They are manifested as a sharp deterioration in coloration the receptor with methylene blue, the receptory plates decrease in their size, character of granuloformation changes in them. Simultaneously, an increased electrical activity is observed and, on the contrary, under the influence of atropine the impulse activity drops. It should be suggested that acetilcholine acts as an activator of metabolic and as a modulator of physiological processes in the receptor.     

Reversal of the effect of the allosteric ligands of dCMP-aminohydrolase and stabilization of the enzyme in the T form

The 5-mercury derivative of dCMP is a substrate of deoxycytidylate aminohydrolase in the presence of mercaptoethanol. With this substrate a reversal of the effect of the allosteric ligands of the enzyme is observed. dCTP, which is an allosteric activator for aminohydrolysis of dCMP, becomes an inhibitor for the mercury substrate, whilst dTTP, an allosteric inhibitor for dCMP, becomes an activator for the mercury substrate.This observation has been interpreted by assuming that dCMP-Hg-S-CH₂-CH₂-OH is a substrate of the T form of the enzyme. By reacting dCMP-aminohydrolase in the T form (in the presence of dTTP) with glutaraldehyde, an enzyme has been isolated that is no longer active with dCMP, while it is fully active with the mercurated analog. Gel electrophoresis demonstrated that glutaraldehyde does not produce intermolecular crosslinks, but fixes 95% of the enzyme in a stable hexameric form by intramolecular crosslinks. The data are explained by assuming that glutaraldehyde stabilizes the enzyme in the T conformation.     

Acetylation increases the alpha-helical content of the histone tails of the nucleosome

The nature of the structural changes induced by histone acetylation at the different levels of chromatin organization has been very elusive. At the histone level, it has been proposed on several occasions that acetylation may induce an alpha-helical conformation of their acetylated N-terminal domains (tails). In an attempt to provide experimental support for this hypothesis, we have purified and characterized the tail of histone H4 in its native and mono-, di-, tri-, and tetra- acetylated form. The circular dichroism analysis of these peptides shows conclusively that acetylation does increase their alpha-helical content. Furthermore, the same spectroscopic analysis shows that this is also true for both the acetylated nucleosome core particle and the whole histone octamer in solution. In contrast to the native tails in which the alpha-helical organization appears to be dependent upon interaction of these histone regions with DNA, the acetylated tails show an increase in alpha-helical content that does not depend on such an interaction.     

Characterization of the structure and the amyloidogenic properties of the Josephin domain of the polyglutamine-containing protein ataxin-3

Expansion of the polyglutamine (polyQ) region in the protein ataxin-3 is associated with spinocerebellar ataxia type 3, an inherited neurodegenerative disorder that belongs to the family of polyQ diseases. Increasing evidence indicates that protein aggregation and fibre formation play an important role in these pathologies. In a previous study, we determined the domain architecture of ataxin-3, suggesting that it comprises a globular domain, named Josephin, and a more flexible C-terminal region, that includes the polyQ tract. Here, we have characterised for the first time the biophysical properties of the isolated Josephin motif, showing that it is an autonomously folded unit and that it has no significant interactions with the C-terminal region. Study of its thermodynamic stability indicates that Josephin has an intrinsic tendency to aggregate and forms temperature-induced fibrils similar to those described for expanded ataxin-3. We show that, under destabilising conditions, the behaviours of the isolated Josephin domain and ataxin-3 are extremely similar. Our data therefore strongly suggest that the stability and aggregation properties of non-expanded ataxin-3 are determined by those of the Josephin domain, which is sufficient to reproduce the behaviour of the full-length protein. Our data support a mechanism in which the thermodynamic stability of ataxin-3 is governed by the properties of the Josephin domain, but the presence of an expanded polyQ tract increases dramatically the protein's tendency to aggregate.     

On the role of reinfection in the transmission of infectious diseases

We introduce a spatial stochastic model for the spread of tuberculosis. After a primary infection, an individual may become sick (and infectious) through an endogenous reinfection or through an exogenous reinfection. We show that even in the absence of endogenous reinfection an epidemic is possible if the exogenous reinfection parameter is high enough. This is in sharp contrast with what happens for a mean field model corresponding to our spatial stochastic model.     

Constraints on the deformation of the vibrissa within the follicle

Nearly all mammals have a vibrissal system specialized for tactile sensation, composed of whiskers growing from sensor-rich follicles in the skin. When a whisker deflects against an object, it deforms within the follicle and exerts forces on the mechanoreceptors inside. In addition, during active whisking behavior, muscle contractions around the follicle and increases in blood pressure in the ring sinus will affect the whisker deformation profile. To date, however, it is not yet possible to experimentally measure how the whisker deforms in an intact follicle or its effects on different groups of mechanoreceptors. The present study develops a novel model to predict vibrissal deformation within the follicle sinus complex. The model is based on experimental results from a previous ex vivo study on whisker deformation within the follicle, and on a new histological analysis of follicle tissue. It is then used to simulate whisker deformation within the follicle during passive touch and active whisking. Results suggest that the most likely whisker deformation profile is “S-shaped,” crossing the midline of the follicle right below the ring sinus. Simulations of active whisking indicate that an increase in overall muscle stiffness, an increase in the ratio between deep and superficial intrinsic muscle stiffness, and an increase in sinus blood pressure will all enhance tactile sensitivity. Finally, we discuss how the deformation profiles might map to the responses of primary afferents of each mechanoreceptor type. The mechanical model presented in this study is an important first step in simulating mechanical interactions within whisker follicles.     

Effects of chronic exercise on the ultrastructure of the adrenocortical cells in the rat

Summary Four groups of male rats were exercised for periods of 2, 4, 6, and 8 weeks with controls in each group. As a result of chronic exercise there was an increase in the width of the zona fasciculata of the adrenal cortex. Also, there was an increase in the number and size of the mitochondria, and an increase in the quantity of smooth endoplasmic reticulum, and during the first 4 weeks of exercise an increase in the number of lipid droplets in the zona fasciculata. The close relationship between the smooth endoplasmic reticulum and the mitochondria, and the relationship between the smooth endoplasmic reticulum and the lipid droplets suggests a possible means for a transport mechanism for movement of precursors between these organelles.This research was supported in part by a Public Health Research Career Development Award KO4 GM42,355 from the National Institute of General Medical Sciences     

Heterogeneity of purinergic P2 receptors at the level of the caudal artery of the rat and the saphenous vein of the dog

Phentolamine (10(-5) M) and an inhibitor of the lipoxygenase pathway, nordihydroguaiaretic acid (N. D. G. A.; 8 10(-6) M) antagonized the ATP induced contraction but not antagonized the UTP induced contraction on both rat tail artery and dog saphenous vein. We conclude that the receptors to ATP are distinct from receptors to UTP and that the P2 purinoceptors are an heterogeneous group.     

Inhibiting effect of the testis on the ovary of the chick embryo]

The testicular hormone responsible for the retrogression of the mullerian ducts in the female chick embryo submitted to an embryonic testicular graft, causes an atrophy of the two, cortical and medullary, parts of this gonad, which is more marked at the level of the cortex.     

Redesigning the Industrial Ecology of the Automobile

This article explores the potential of industrial ecology to inform the redesign of an existing industry: that which is concerned with the production, sale, and support of automobiles. In so doing, it brings together the concepts embedded in industrial ecology with issues of economic scale, product design or technology, process technology, and the way in which new combinations of these features can result in an alternative structure for the automotive industry that has the potential to enhance sustainability performance. In so doing, the article advances the general argument that the economic, technical, and spatial organization of production and consumption are co-determined in a manner that collectively shapes the industrial ecology of an industry. In contradistinction to the prevailing industry, the article then advances the concept of micro factory retailing as an alternative framework for the industry that would result in significantly different performance in terms of industrial ecology.     

Fine structure of the spermatozoon of the viviparous teleost, Cymatogaster aggregata

The approximately 50 μm long sperm of Cymatoguster aggregata is composed of an elongate head (4 μm), an elongate mitochondria1 midpiece (3.5 μm) and a tail flagellum (roughly 40 μm). The sperm lacks an acrosome. Contained within depressions on one surface of the compressed head are a proximal centriole and a distal centriole separated by an electron dense, intercentriolar body. The anterior portion of the tail flagellum originates at the basal body (distal centriole) and is contained within an extracellular, flagellar tunnel within the mitochondria1 midpiece. The morphological similarity of C. uggregutu sperm to sperm of other internally fertilizing fishes supports the hypothesis that spermatozoan morphology is related to the mode of fertilization and that an elongate head and midpiece are specializations for internal fertilization.     

Orientation of the amino-terminal domain of ClpB affects the disaggregation of the protein

ClpB/Hsp104 efficiently reactivates protein aggregates in cooperation with the DnaK/Hsp70 system. As a member of the AAA+ protein family (i.e. an expanded superfamily of ATPases associated with diverse cellular activities), ClpB forms a ring-shaped hexamer in an ATP-dependent manner. A protomer of ClpB consists of an N-terminal domain (NTD), an AAA+ module, a middle domain and another AAA+ module. In the crystal structures, the NTDs point to two different directions relative to other domains and are not visible in the single-particle cryo-electron microscopy reconstruction, suggesting that the NTD is highly mobile. In the present study, we generated mutants in which the NTD was anchored to other domain by disulfide cross-linking and compared several aspects of ClpB function between the reduced and oxidized mutants, using the wild-type and NTD-truncated ClpB (ClpBΔN) as references. In their oxidized form, the mutants and wild-type bind casein with a similar affinity, although the affinity of ClpBΔN for casein was significantly low. However, the extent of casein-induced stimulation of ATPase, the rate of substrate threading and the efficiency of protein disaggregation of these mutants were all lower than those of the wild-type but similar to those of ClpBΔN. These results indicate that the NTD supports the substrate binding of ClpB and that its conformational shift assists the threading and disaggregation of substrate proteins.     

Sequence analysis of the COOH terminus of the alpha-chain of the fourth component of human complement. Identification of the site of its extracellular cleavage

The predominant form of the fourth component of complement in human and murine plasma (C4p) has an Mr approximately 5,000 less than C4 secreted by hepatocytes or macrophages (C4s). Here we demonstrate that the difference in Mr results from excision of a peptide from the COOH terminus of the alpha-chain of C4s. The site of truncation of the alpha-chain of C4 in plasma was established by sequence analysis of the COOH-terminal cyanogen bromide fragment isolated by high performance liquid chromatography. Sequential Edman degradation, digestion with carboxypeptidase Y, analysis of amino acid composition, and partial sequence analysis of an overlapping tryptic peptide established the sequence of this peptide to be Glu-Ala-Asn-Glu-Asp-Tyr-Glu-Asp-Tyr-Glu-Tyr-Asp-Glu-Leu-Pro-Ala. Comparison of our results with reported sequences establishes the COOH-terminal alanine to be residue 748 in the alpha-chain. This identifies the site at which C4s is cleaved by an extracellular protease and suggests that the protease has an elastase-like activity.     

The pro-sequence domain of streptopain directs the folding of the mature enzyme

The cysteine endopeptidase streptopain, an extracellular enzyme from pathogenic Streptococcus pyogenes, is synthesized as a precursor containing an NH2-terminal pro-sequence. The pro-sequence of streptopain was expressed in Escherichia coli and subjected to structural and functional investigation. Heat-induced denaturation of the pro-sequence studied using circular dichroism spectroscopy revealed that it forms a compact structure and represents an independently folded domain. The isolated pro-sequence exhibits high affinity towards mature streptopain and associates with its cognate enzyme by forming an equimolar complex. Refolding of denatured streptopain in the presence of pro-sequence in vitro facilitated recovery of active enzyme. Expression of the mature streptopain in E. coli either alone, or in trans with its pro-sequence as an independent polypeptide, led to the formation of insoluble protein aggregates or functionally active enzyme, respectively. These results demonstrate that the pro-sequence domain acts as an intramolecular chaperone that directs the correct folding of the mature streptopain.     

Stabilizing the Central Part of Tropomyosin Increases the Bending Stiffness of the Thin Filament

A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.     

Structure of the O-chain of the lipopolysaccharide of Haemophilus pleuropneumoniae serotype 1

By phenol-water extraction an aqueous-phase soluble cellular lipopolysaccharide was isolated from Haemophilus pleuro-pneumoniae serotype 1. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hydrolysis, methylation, and both one- and two-dimensional 1H and 13C nuclear magnetic resonance studies to be an S-type lipopolysaccharide, which could be cleaved to yield a lipid A and an O-chain polysaccharide identified as a high molecular weight branched polymer of a tetrasaccharide repeating unit having the structure: (Formula: see text).     

Crystal structure of the beta-apical domain of the thermosome reveals structural plasticity in the protrusion region

The crystal structure of the beta-apical domain of the thermosome, an archaeal group II chaperonin from Thermoplasma acidophilum, has been determined at 2.8 A resolution. The structure shows an invariant globular core from which a 25 A long protrusion emanates, composed of an elongated alpha-helix (H10) and a long extended stretch consisting of residues GluB245-ThrB253. A comparison with previous apical domain structures reveals a large segmental displacement of the protruding part of helix H10 via the hinge GluB276-ValB278. The region comprising residues GluB245-ThrB253 adopts an extended beta-like conformation rather than the alpha-helix seen in the alpha-apical domain. Consequently, it appears that the protrusions of the apical domains from group II chaperonins might assume a variety of context-dependent conformations during an open, substrate-accepting state of the chaperonin. Sequence variations in the protrusion regions that are found in the eukaryotic TRiC/CCT subunits may provide different structural propensities and hence serve different roles in substrate recognition.     

Mitosis in the formation and function of the primary hypoblast of the chick

Summary The present work demonstrates that a normal stage-XIII hypoblast, if submitted to an X-ray dose of 6,000 rads (sufficient to stop cell division), retains the capacity to induce axis formation on the chick epiblast. Examination of the process of hypoblast formation indicates, in agreement with previous findings, that a hypoblastic layer can form in the absence of cell division from an irradiated stage-X blastoderm. Furthermore, it is observed that this hypoblastic layer, formed in the absence of cell division, can induce the development of an embryonic axis when combined with a normal stage-XIII epiblast.     

Ultrastructural localization of ependymins in the endomeninx of the brain of the rainbow trout: possible association with collagen fibrils of the extracellular matrix

In the rainbow trout, ependymins represent the predominant protein constituents of the cerebrospinal fluid (CSF) and perimeningeal fluid (PMF). Synthesis of these glycoproteins occurs exclusively in the endomeninx. Generally, ependymins share characteristics with proteins mediating cell-contact phenomena. Here, we show that the endomeninx of the rainbow trout is composed of three different layers, viz. an outer layer, an arachnoid-like intermediate barrier layer and an inner layer. This structure is in agreement with a meningeal barrier concept separating the PMF from the CSF. Furthermore, by immuno-electron microscopy, we have localized the majority of intracellular ependymins to the rough endoplasmic reticulum of fibroblast-like cells of the inner layer and to cells to the intermediate barrier layer. This pattern is compatible with the observed distribution of ependymins in both the PMF and CSF. In addition to their intracellular localization, an extracellular association of ependymins with bundles of collagen fibrils is demonstrated; this is particularly pronounced around all blood vessels of the brain.