bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and degeneration of retina in rds mutant mice: observations in chimaeras of heterozygous mutant and normal genotype
Authors: Sanyal S. , Dees C. , Zeilmaker G. H.
Published: April 2021

In homozygous rds mutant mice the photoreceptor cells lack outer segment discs and slowly degenerate. In the heterozygotes the receptor cells develop abnormal outer segments and show altered disc shedding properties as revealed by the pigment epithelial phagosome content. The receptor cells also degenerate at a slower rate than in the homozygotes. The nature of the interaction resulting in dilution of the retinal lesion in the heterozygous retina was analysed in a series of chimaeras consisting of rds/ + and +/+ genotypes, which also differed in colour genes.

In 64% of the chimaeras (18 out of 28) presence of both rds/+ and +/+ types of photoreceptors could be detected by electron microscopy. The relative proportion and patch size of the two components varied greatly between individuals but the location of the two types of photoreceptors was not related to the genotypes of the overlying pigment epithelial cells. Frequent occurrence of abnormally large phagosomes, resembling the rds/+ phenotype, was noted regularly in both rds/ + and +/+ types of pigment epithelial cells located above rds/+ types of receptors, but not in the cells of either genotype located above normal receptors. In the eyes examined at 12–18 months, localized and partial depletion of the perikaryal population in the outer nuclear layer was observed, and the location of such areas was also unrelated to the genotypes of the pigment epithelial cells. These findings confirm that the rds gene acts within the neural retina and possibly within the receptor cells and further show that the genetic interaction between the rds gene and its normal allele in the retina of the heterozygous mice takes place within the receptor cells.
Development of neural tube basal lamina during neurulation and neural crest cell emigration in the trunk of the mouse embryo
Authors: Martins-Green M. , Erickson C. A.
Published: April 2021

In the trunk of higher vertebrates, the neural crest (NC) cells remain temporarily within the dorsal portion of the neural tube after fusion of the neural folds; shortly thereafter they emigrate, invading surrounding spaces and tissues. One of the factors postulated to be important in the initiation of migration of NC cells is the disruption of the basal lamina (BL) over the dorsal portion of the neural tube. It has been assumed by many that the BL must be discontinuous in order that the NC cells can leave the neural tube; and indeed, experiments performed in our laboratory, and by others, have shown that NC cells cannot penetrate an intact BL. Therefore, we have undertaken a systematic ultrastructural study to evaluate the condition of the BL during neural fold elevation and NC cell emigration. Our results show that: (i) BL surrounding the neural epithelium (NE) becomes progressively more extensive from neural fold to migratory stages. It first forms on the lateral portion of the neuroepithelium of the neural folds and then extends ventrally into the region adjacent to the notochord; (ii) BL becomes continuous beneath the epidermal ectoderm (EE) that overlies the NC cell region only during the terminal stages of NC cell emigration; (iii) BL does not form over the dorsal portion of the neural tube until NC emigration is terminated; and (iv) the morphology of the BL changes as development proceeds. We conclude that absence of a BL over the premigratory NC cell population in the trunk of mouse embryos is a necessary but not a sufficient condition for emigration to take place.
An in vitro assay for neural crest cell migration through the somites
Authors: Guillory Georgia , Bronner-Fraser Marianne
Published: April 2021

Neural crest cells in the trunk of the avian embryo come into contact with the somites and neural tube during the course of their migration. However, the relationship between the somites and the early migratory routes followed by these cells is not yet completely understood. Here, we use a tissue culture assay to examine if avian neural crest cells migrate through the somites. Cultures of quail somites were prepared from four adjacent regions along the neural axis in the trunk. Each region had four pairs of consecutive somites with region I being most anterior and region IV containing the last four segments. Within each region, the somites were separated from other tissues by enzymatic digestion and plated onto collagen-coated dishes. Immunocytochemical techniques were used to confirm that no neural crest cells, recognized by the HNK-1 antibody, were present on the surface of the somites at the time of explantation. After several days in culture, the explanted somites were screened to identify pigment cells. Because neural crest cells give rise to all of the melanocytes in the trunk, the presence of pigment cells indicated that neural crest precursors were contained within the initial explant. After 5–11 days in vitro, the percentage of somite cultures containing pigment cells in regions I through IV, respectively, was 36%, 51%, 31% and 1 %. These results suggest that neural crest cells migrate through the somitic mesenchyme and first enter the somites between 5 to 9 segments rostral to the most recently formed somite.
Binding of zona pellucida proteins to a boar sperm polypeptide ofMr53000 and identification of zona moieties involved
Authors: Brown C. R. , Jones R.
Published: April 2021

Experiments have been carried out to identify proteins on boar spermatozoa that bind to components of the zona pellucida. Polypeptides in sodium deoxycholate extracts of boar spermatozoa and in whole seminal plasma have been separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose sheet by electroblotting and probed with 125I-labelled heat-solubilized zona pellucida from pig oocytes or ovulated eggs. Zona proteins bound avidly and consistently to a polypeptide of Mr 53 000 on blots of capacitated and noncapacitated sperm and weakly to polypeptides of Mr 67 000, 38 000 and 18000. On blots of seminal plasma the 125I-labelled probes bound to two polypeptides of Mr 65 000 and 19-24 000. Identification of the zona proteins that were binding to the aforementioned proteins on blots showed that all the major zona pellucida glycoproteins were involved, including those acquired from oviduct secretions. Binding of 125I-ovulated zona pellucida to the polypeptide of Mr 53 000 also occurred in extracts of testicular and epididymal boar spermatozoa. The results are discussed in relation to sperm-egg recognition in the pig.
The spatial and temporal pattern of collagens I and II and keratan sulphate in the developing chick metatarsophalangeal joint
Authors: Craig Fiona M. , Bentley George , Archer Charles W.
Published: April 2021

Both intrinsic and extrinsic factors are known to be involved in the morphogenesis of diarthrodial joints. The use of specific antibodies to collagens I and II and keratan-sulphate-containing proteoglycans (KSPG) has enabled the distributions of these macromolecules to be followed during the development of the third metatarsophalangeal joint in the chicken embryo.

Our study shows that cartilage differentiation occurs as a continuous rod, which is then subsequently divided into separate elements. Further development also reveals that, unlike the matrix of the cartilaginous elements, there is a differential distribution of collagen (type II) and KSPG in the presumptive joint region. It is proposed that a decrease in KSPG in the presumptive joint region at stages 28/30 may be involved in the mechanism for the flattening of cells in formation of the interzone. Whereas, a decrease in collagen across the joint interzone region may provide an area of weakness, which might allow forces produced by the developing musculature to cause cavitation.
Rescue of developmental lens abnormalities in chimaeras of noncataractous and congenital cataractous mice
Authors: Muggleton-Harris A. L. , Hardy K. , Higbee N.
Published: April 2021

In the study of the lens of a congenital cataractous mouse mutant (CAT), it has been shown that a loss of growth regulation at the cellular level causes gross lens abnormalities. The phenotypic characteristics of the cataractous mouse lens are similar to those seen in human congenital cataract and thus serves as a model system for medical research.

In this present investigation, we have demonstrated that the abnormalities of the congenital cataractous lens can be rescued by forming chimaeras between DBA/2 (a noncataractous strain of mouse) and the CAT mutant. This report describes the histological, cellular and biochemical analysis of the resultant chimaeric eyes, and discusses possible mechanisms by which these results were achieved.
A molecular analysis of mouse development from 8 to 10 dayspost coitumdetects changes only in embryonic globin expression
Authors: Wilkinson David G. , Bailes Juliet A. , Champion Janet E. , Mcmahon Andrew P.
Published: April 2021

The pattern of protein synthesis in 8-, 9- and 10-day post coitum (p.c.) mouse embryos was examined by 2-D gel electrophoresis of [35S]methionine-labelled proteins. Of the 600-800 polypeptides detected only one, a 14×103Mr (14 K) protein, was found to accumulate over this period. To isolate cDNA clones that potentially encode this protein, 32P-labelled cDNA was synthesized from 9 and 10 days p.c. embryo poly(A)+RNA, and used for the differential screening of an 8·5-day p.c. mouse embryo cDNA library cloned in λgt10. Six clones that hybridized strongly to the 10-day probe were purified and their inserts subcloned into plasmid vectors. Cross hybridization and restriction mapping of these inserts indicate that they fall into four distinct groups. Each of these hybridize with transcripts of approximately 600 nucleotides, which accumulate in the embryo from 9 to 10 days p.c. Expression was barely detectable in adult tissues and restricted to liver and spleen. Expression of one of these clones, 10.1, was examined by in situ hybridization of 35S-labelled RNA probes to 8·5-12·5 day p.c. embryo sections. Strong hybridization was observed in yolk sac blood islands, fetal liver and embryonic erythrocytes, suggesting that 10.1A encodes an erythrocyte-specific protein. DNA sequence analysis indicates that the four classes of cDNA were derived from transcripts of the α 1, ζ, βh1 and ϵ globin genes. Labelling of 10-day p.c. erythrocyte proteins with [35S]methionine, followed by 2-D gel electrophoresis, clearly demonstrates that the most abundant polypeptide migrates to the same position as the 14 K protein which accumulates from 8 to 10 days p.c. Thus the only abundant transcripts and corresponding proteins that change over a period of profound morphogenetic change correspond to globins of the newly established blood system.
The specificity of motor innervation of the chick wing does not depend upon the segmental origin of muscles
Authors: Keynes R. J. , Stirling R. V. , Stern C. D. , Summerbell D.
Published: April 2021

In vertebrate embryos, motor axons originating from a particular craniocaudal position in the neural tube innervate limb muscles derived from myoblasts of the same segmental level. We have investigated whether this relationship is important for the formation of specific nerve-muscle connections, by altering the segmental origin of muscles and examining their resulting innervation. First, by grafting quail wing somites to a new craniocaudal position opposite the chick wing, we established that the segmental origin of a muscle can be altered: presumptive muscle cells migrated according to their new, rather than their original, somitic level, colonizing a different subset of muscles. However, after reversal of a length of brachial somitic mesoderm along the craniocaudal axis, or exchange or shift of brachial somites, the craniocaudal position of wing muscle motoneurone pools within the spinal cord was undisturbed, despite the new segmental origin of the muscles themselves. While not excluding the possibility that muscles and their motor nerves are labelled segmentally, we conclude that specific motor axon guidance in the wing does not depend upon the existence of such labels.
Observations on scale production in prymnesium parvum
Authors: Manton Irene
Published: April 2021

Information is provided regarding certain structural and developmental aspects of the single Golgi body present in the brown flagellate, Prymnesium parvum. Probably the most important new observation is the demonstration of scale production within certain Golgi cisternae. Different developmental states from immature to mature can be found in successive cisternae on one side of the Golgi centre, the scales themselves being all similarly oriented with respect to the inner and outer faces of the cisternae in which they are formed. A deep pit found in a relatively constant position attached to the plasmalemma beside each flagellar base is interpreted as being possibly the site of scale liberation. These findings are discussed in a preliminary way.
Characterizing titin’s I-band Ig domain region as an entropic spring
Authors: Linke Wolfgang A. , Stockmeier Marc R. , Ivemeyer Marc , Hosser Hiltraud , Mundel Peter
Published: April 2021

The poly-immunoglobulin domain region of titin, located within the elastic section of this giant muscle protein, determines the extensibility of relaxed myofibrils mainly at shorter physiological lengths. To elucidate this region’s contribution to titin elasticity, we measured the elastic properties of the N-terminal I-band Ig region by using immunofluorescence/immunoelectron microscopy and myofibril mechanics and tried to simulate the results with a model of entropic polymer elasticity. Rat psoas myofibrils were stained with titin-specific antibodies flanking the Ig region at the N terminus and C terminus, respectively, to record the extension behaviour of that titin segment. The segment’s end-to-end length increased mainly at small stretch, reaching ∼90% of the native contour length of the Ig region at a sarcomere length of 2.8 µm. At this extension, the average force per single titin molecule, deduced from the steady-state passive length-tension relation of myofibrils, was ∼ 5 or 2.5 pN, depending on whether we assumed a number of 3 or 6 titins per half thick filament. When the force-extension curve constructed for the Ig region was simulated by the wormlike chain model, best fits were obtained for a persistence length, a measure of the chain’s bending rigidity, of 21 or 42 nm (for 3 or 6 titins/half thick filament), which correctly reproduced the curve for sarcomere lengths up to 3.4 µm. Systematic deviations between data and fits above that length indicated that forces of >30 pN per titin strand may induce unfolding of Ig modules. We conclude that stretches of at least 5-6 Ig domains, perhaps coinciding with known super repeat patterns of these titin modules in the I-band, may represent the unitary lengths of the wormlike chain. The poly-Ig regions might thus act as compliant entropic springs that determine the minute levels of passive tension at low extensions of a muscle fiber.
Transient ER retention as stress response: Conformational repair of heat damaged proteins to secretion-competent structures
Authors: Saris Nina , Makarow Marja
Published: April 2021

Mechanisms to acquire tolerance against heat, an important environmental stress condition, have evolved in all organisms, but are largely unknown. When Saccharomyces cerevisiae cells are pre-conditioned at 37°C, they survive an otherwise lethal exposure to 48-50°C, and form colonies at 24°C. We show here that incubation of yeast cells at 48-50°C, after pre-conditioning at 37°C, resulted in inactivation of exocytosis, and in conformational damage and loss of transport competence of proteins residing in the endoplasmic reticulum (ER). Soon after return of the cells to 24°C, membrane traffic was resumed, but cell wall invertase, vacuolar carboxypeptidase Y and a secretory β-lactamase fusion protein remained in the ER for different times. Thereafter their transport competence was resumed very slowly with widely varying kinetics. While the proteins were undergoing conformational repair in the ER, their native counterparts, synthesized after shift of the cells to 24°C, folded normally, by-passed the heat- affected copies and exited rapidly the ER. The Hsp70 homolog Lhs1p was required for acquisition of secretion competence of heat-damaged proteins. ER retention and refolding of heat-denatured glycoproteins appear to be part of the cellular stress response.
GTPγS-induced actin polymerisation in vitro: ATP- and phosphoinositide independent signalling via Rho-family proteins and a plasma membrane- associated guanine nucleotide exchange factor
Authors: Katanaev Vladimir L. , Wymann Matthias P.
Published: April 2021

In a cell-free system from neutrophil cytosol GTPγS can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTPγS stimulation was susceptible to an excess of GDP, but not Bordetellapertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPγS·xGiα2 or Gβ1γ2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPγS. When recombinant, constitutively activated and GTPγS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations >100 times the endogenous, however, they were ineffective. Purified Cdc42/Racinteractive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPγS. The action of GTPγS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B- sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPγS under physiologic conditions.

The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPγS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPγS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.
High Proliferative Potential-Quiescent cells: a working model to study primitive quiescent hematopoietic cells
Authors: Fortunel Nicolas , Batard Pascal , Hatzfeld Antoinette , Monier Marie-Noëlle , Panterne Béatrice , Lebkowski Jane , Hatzfeld Jacques
Published: April 2021

Human adult hematopoietic stem cells are mostly quiescent or slow cycling. We have previously demonstrated that blocking of transforming growth factor-β1 (TGF-β1) is able to activate, in the presence of cytokines, primitive quiescent hematopoietic multipotent progenitors which could not grow in a two week semi-solid culture assay (short term culture). We have also shown that anti-TGF-β1 can up-modulate c-KIT, the receptor of the stem cell factor (steel factor). To elucidate whether TGF-β1 plays a central role in controlling the quiescence of hematopoietic primitive cells, it was necessary to demonstrate, as detailed in this study, that: (1) whatever the cytokine combination tested, addition of anti-TGF-β1 releases from quiescence multipotent progenitors with a significantly higher hematopoietic potential than those activated by cytokines alone. (2) Other important cytokine receptors controlling the most primitive hematopoietic cells such as FLT3 and the IL6 receptor (IL6-R) are down-modulated by TGF-β1 but rapidly up-modulated by anti-TGF-β1. (3) Anti-TGF-β1-sensitive multipotent and high proliferative potential progenitors express these cytokine receptors at a low level (FLT3low and IL6-Rlow).

According to these results, we propose the working model of ‘High Proliferative Potential-Quiescent cells’ to refer to these primitive hematopoietic multipotent progenitors that are highly sensitive to the growth inhibitory effect of TGF-β1. This model could be valid not only to study the human hematopoietic quiescent progenitors but also for other somatic stem cell systems.
Molecular organization of the desmoglein-plakoglobin complex
Authors: Chitaev Nikolai A. , Averbakh Alexander Z. , Troyanovsky Regina B. , Troyanovsky Sergey M.
Published: April 2021

Different epithelial intercellular junctions contain distinct complexes incorporating plakoglobin. In adherens junctions, plakoglobin interacts with two molecules, the transmembrane adhesion protein of the cadherin family (e.g. E-cadherin) and α-catenin. The latter is thought to anchor the cadherin-plakoglobin complex to the cortical actin cytoskeleton. In desmosomes, plakoglobin forms a complex with desmosomal cadherins, either desmoglein (Dsg) or desmocollin (Dsc), but not with α-catenin. To further understand the structure and assembly of the plakoglobin-cadherin complexes we analyzed amino acid residues involved in plakoglobin-Dsg interactions using alanine scanning mutagenesis. Previously, we have shown that plakoglobin interacts with a 72 amino acid-long cytoplasmic domain (C-domain) that is conserved among desmosomal and classic cadherins. In this paper, we show that a row of the large hydrophobic residues located at the C-terminal portion of the Dsg C-domain is indispensable for interaction with plakoglobin. To study a reciprocal site we expressed plakoglobin (MPg) or its mutants tagged by 6 myc epitope in epithelial A-431 cells. Using sucrose gradient centrifugation and subsequent coimmunoprecipitation, MPg was found to be efficiently incorporated into the same type of complexes as endogenous plakoglobin. A major pool of Dsg-plakoglobin complexes sedimented at 8S and exhibited a 1:1 stoichiometry. Using alanine scanning mutagenesis and the co-immunoprecipitation assay we identified nine hydrophobic amino acids within the arm repeats 1-3 of plakoglobin, that are required for binding to Dsg and Dsc. Eight of these amino acids also participate in the interaction with α-catenin. No mutations were found to reduce the affinity of plakoglobin binding to E-cadherin. These data provide direct evidence that the same hydrophobic plakoglobin surface is essential for mutually exclusive interaction with distinct proteins such as α-catenin and desmosomal cadherins.
PDGF induces reorganization of vimentin filaments
Authors: Valgeirsdóttir Sigrĺdur , Claesson-Welsh Lena , Bongcam-Rudloff Erik , Hellman Ulf , Westermark Bengt , Heldin Carl-Henrik
Published: April 2021

In this study we demonstrate that stimulation with platelet-derived growth factor (PDGF) leads to a marked reorganization of the vimentin filaments in porcine aortic endothelial (PAE) cells ectopically expressing the PDGF β-receptor. Within 20 minutes after stimulation, the well-spread fine fibrillar vimentin was reorganized as the filaments aggregated into a dense coil around the nucleus. The solubility of vimentin upon Nonidet-P40-extraction of cells decreased considerably after PDGF stimulation, indicating that PDGF caused a redistribution of vimentin to a less soluble compartment. In addition, an increased tyrosine phosphorylation of vimentin was observed. The redistribution of vimentin was not a direct consequence of its tyrosine phosphorylation, since treatment of cells with an inhibitor for the cytoplasmic tyrosine kinase Src, attenuated phosphorylation but not redistribution of vimentin. These changes in the distribution of vimentin occurred in conjunction with reorganization of actin filaments. In PAE cells expressing a Y740/751F mutant receptor that is unable to bind and activate phosphatidylinositol 3′-kinase (PI3-kinase), the distribution of vimentin was virtually unaffected by PDGF stimulation. Thus, PI3-kinase is important for vimentin reorganization, in addition to its previously demonstrated role in actin reorganization. The small GTPase Rac has previously been shown to be involved downstream of PI3-kinase in the reorganization of actin filaments. In PAE cells overexpressing dominant negative Rac1 (N17Rac1), no change in the fine fibrillar vimentin network was seen after PDGF-BB stimulation, whereas in PAE cells overexpressing constitutively active Rac1 (V12Rac1), there was a dramatic change in vimentin filament organization independent of PDGF stimulation. These data indicate that PDGF causes a reorganization of microfilaments as well as intermediate filaments in its target cells and suggest an important role for Rac downstream of PI3-kinase in the PDGF stimulated reorganization of both actin and vimentin filaments.
cyk-1: a C. elegans FH gene required for a late step in embryonic cytokinesis
Authors: Swan Kathryn A. , Severson Aaron F. , Carter J. Clayton , Martin Paula R. , Schnabel Heinke , Schnabel Ralf , Bowerman Bruce
Published: April 2021

A maternally expressed Caenorhabditis elegans gene called cyk-1 is required for polar body extrusion during meiosis and for a late step in cytokinesis during embryonic mitosis. Other microfilament- and microtubule-dependent processes appear normal in cyk-1 mutant embryos, indicating that cyk-1 regulates a specific subset of cytoskeletal functions. Because cytokinesis initiates normally and cleavage furrows ingress extensively in cyk-1 mutant embryos, we propose that the wild-type cyk-1 gene is required for a late step in cytokinesis. Cleavage furrows regress after completion of mitosis in cyk-1 mutants, leaving multiple nuclei in a single cell. Positional cloning and sequence analysis of the cyk-1 gene reveal that it encodes an FH protein, a newly defined family of proteins that appear to interact with the cytoskeleton during cytokinesis and in the regulation of cell polarity. Consistent with cyk-1 function being required for a late step in embryonic cytokinesis, we show that the CYK-1 protein co-localizes with actin microfilaments as a ring at the leading edge of the cleavage furrow, but only after extensive furrow ingression. We discuss our findings in the context of other studies suggesting that FH genes in yeast and insects function early in cytokinesis to assemble a cleavage furrow.
Effector caspases are dispensable for the early nuclear morphological changes during chemical-induced apoptosis
Authors: Johnson V. L. , Ko S. C. W. , Holmstrom T. H. , Eriksson J. E. , Chow S. C.
Published: April 2021

Nuclear morphological changes during apoptosis are very distinct and effector caspases have been implicated to play a central role in these processes. To investigate this in greater detail we examined the effect of blocking caspase activity and its activation on the nuclear morphological change in Jurkat T cells undergoing apoptosis after staurosporine treatment. In the presence of caspase inhibitors, like benzyloxycarbonyl-Val-Ala-Asp fluoro-methylketone (z-VAD-FMK), N-acetyl Tyr-Val-Ala-Asp chloromethylketone (Ac-YVAD-CMK) and benzyloxy-carbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-FMK), staurosporine-treated Jurkat cells displayed a nuclear morphological change distinct from that of normal and apoptotic cells. This nuclear morphological change is an early event, characterised by convoluted nuclei with cavitations, and clumps of chromatin abutting to inner regions of the nuclear envelope between the nuclear pores. Both the nuclear envelope and endoplasmic reticulum were grossly dilated. This pre-apoptotic nuclear change precedes the externalisation of phosphatidylserine, chromatin condensation and DNA laddering, and can be dissociated from the formation of high molecular weight DNA fragments and cell shrinkage. Although cytochrome c efflux from the mitochondria and the processing of caspase-3 were observed in Jurkat cells with pre-apoptotic nuclear morphology, caspase-2, -6, -7 and -8 were not activated. In the presence of z-DEVD-FMK or Ac-YVAD-CMK, caspase-3 was processed to both the p17 and p20 fragments in staurosporine-treated cells, but only to p20 fragment in the presence of z-VAD-FMK. However, the caspase-3 substrate, poly(ADP ribose) polymerase was not cleaved in the presence of z-VAD-FMK, despite >70% of the cells have pre-apoptotic nuclei. In addition, caspase-3 null MCF-7 cells also undergo pre-apoptotic nuclear change when treated with staurosporine in the presence of caspase inhibitors, indicating that caspase-3 is not required for the early nuclear morphological change in cells undergoing apoptosis. Although cell death in staurosporine-treated Jurkat cells was markedly delayed, they eventually die without discernible downstream apoptotic features. Other apoptotic stimuli like etoposide and the heavy metal chelator, N,N,N’,N’-tetrakis (2-pyridylmethyl) ethylenediamine also induced this nuclear morphological change in Jurkat cells in the presence of z-VAD-FMK. In summary, the effector caspases are not involved in early nuclear morphological change, which precedes the conventional hallmark morphological changes associated with chemical-induced apoptosis.
Insect Cardioactive Peptides: II. Neurohormonal Control of Heart Activity by Two Cardioacceleratory Peptides In the Tobacco Hawkmoth, Manduca Sexta
Authors: Tublitz Nathan J. , Truman James W.
Published: April 2021

The physiological characteristics of two cardioacceleratory peptides (CAPs) were analysed in the tobacco hawkmoth, Manduca sexta, to determine if either CAP functioned as a cardioregulatory neurohormone.

In vivo heart recordings from pharate and newly emerged adults revealed a dramatic increase in heart rate associated with wing-spreading behaviour. Bioassay of whole blood taken from wing-spreading (WS) animals indicated the presence of a stage-specific, blood-borne cardioacceleratory factor(s). Gel filtration of WS blood identified two cardioacceleratory factors which co-eluted with the two CAPs.

A depletion of the ventral nerve cord levels of both CAPs was observed during WS behaviour. Measurements of blood CAP levels showed that the peak CAP titres were coincident with the initiation of WS behaviour. Experimental manipulations that delayed the onset of WS behaviour also prevented CAP release. High potassium incubation evoked the release of both CAPs in a calcium-dependent manner. In vivo injections of CAPi or CAP2 caused a dose-dependent increase in heart rate.

These results confirm the hypothesis that both CAPs function as cardioregulatory neurohormones during wing-spreading behaviour in Manduca sexta.
Habituation of Swimming Activity in the Medicinal Leech
Authors: Debski Elizabeth A. , Friesen W. Otto
Published: April 2021

Tactile stimulation (light stroking) of a body wall flap attached to the ventral nerve cord of the medicinal leech evokes episodes of swimming activity. This swimming response undergoes habituation, involving changes in swim initiation and swim maintenance.

Repeated stimulation of the body wall flap evoked swimming activity between three and 39 times before this response failed. During repetitive stimulation, the length of swim episodes decreased by about 50%. The number of swim episodes which could be elicited was not correlated with swim episode length. Following habituation, swim initiation showed significant spontaneous recovery, but swim episode length returned only to 60% of control values. In preparations where spontaneous recovery was followed by rehabituation, the number of swim episodes elicited declined with each habituation-recovery sequence. Additional stimulation immediately following habituation trials had a dual effect: recovery of the swimming response was delayed, but the lengths of swim episodes following spontaneous recovery were increased. Pinching the body wall flap immediately restored the swimming response in an habituated preparation. Swim initiation habituated more rapidly during stimulation of anterior body wall flaps than during stimulation of mid-body or posterior flaps. However, swim length was independent of this regional variation in swim responsiveness. The number of swim episodes elicited by stimulation of body wall flaps attached to posterior or anterior segments depended upon whether this segment was stimulated before or after other flaps. In contrast, in mid-body segments there was no evidence for such stimulus generalization. The lengths of swim episodes elicited during sequential stimulation of several body wall flaps were independent of the stimulation sequence. We propose that separate processes control swim initiation and swim maintenance. These processes must be repeated in most, if not all, of the segmental ganglia of the leech ventral nerve cord.
Swimming In The Pteropod Mollusc, Clione limacina II. Physiology*
Authors: Satterlie Richard A. , Spencer Andrew N.
Published: April 2021

The central pattern generator (CPG) for swimming in Clione limacina was localized in cutting experiments. A separate pattern generator for each wing is located in the ipsilateral pedal ganglion. The CPGs are tightly coupled but can be isolated by severing the pedal commissure. Removal of the cerebral ganglia results in a decrease in swim frequency and regularity suggesting descending modulation of the CPGs.

Two classes of pedal neurones display firing patterns in phase with swimming movements. One class, swim motor neurones, are further divided into depressor and elevator groups. Motor neurone recordings show complex subthreshold activity consisting of alternate depolarizations and hyperpolarizations. The complex activity is in antiphase in antagonistic motor neurones. Significant motor neurone-motor neurone interactions do not occur centrally as neither electrical coupling nor chemical synaptic interactions could be demonstrated. Injected currents do not alter the motor neurone firing rhythm or the swimming rhythm.

Motor neurone cell bodies are located in the lateral region of the ipsilateral pedal ganglion, near the emergence of the wing nerve. Each motor neurone provides a single axon to the wing nerve which branches repeatedly in the wing. Each motor neurone has an extremely large innervation field, some covering up to half of the wing area.

The second class of pedal neurones that display firing patterns in phase with either wing upswing or downswing are pedal-pedal inter neurones. Each swim interneurone provides axon branches in both pedal ganglia and the axon runs in the pedal commissure. Interneurone axon branches occur in the lateral neuropile of each pedal ganglion, in the region of motor neurone branching. The swim interneurones presumably play a major role in bilateral coordination of the wings and are involved in pattern generation since injected currents were found to accelerate or slow the firing rhythms of interneurones and motor neurones, and wing movements.
Partitioning of Body Fluids and Cardiovascular Responses to Circulatory Hypovolaemia in the Turtle, Pseudemys Scripta Elegans
Authors: Smits Allan W. , Kozubowski Mark M.
Published: April 2021

Investigations were conducted (1) to measure the steady state compartmentation of body fluids and (2) to assess the efficacy of blood volume and pressure maintenance during haemorrhage-induced hypovolaemia in the pond turtle, Pseudemys scripta elegans. The pre-haemorrhage blood volume, as determined by tracer dilution of 51Cr-labelled erythrocytes, averaged 6·89 ±0·33% of the body mass, and was part of comparatively large extracellular (40·2 ± 0·70%) and total body fluid volumes (75·25 ± 1·48%). Turtles exhibited progressive reductions in systemic arterial pressure throughout a cumulative haemorrhage of −48% of their original blood volume, despite dramatic increases in heart rate and comparatively large magnitudes of transcapillary fluid transfer from interstitial to intravascular spaces. Arterial blood pressure returned to pre-haemorrhage values 2h after experimental haemorrhage ceased, concomitant with the restoration of the original blood volume. Our results support arguments made in previous studies that the resistance to fluid movement between vascular and extravascular locations in reptiles is comparatively low. Furthermore, the haemodynamic responses of turtles to experimental hypovolaemia suggest that barostasis through adjustments in vascular tone is less effective than that observed in other reptiles.
Serotonin Modulates the Central Pattern Generator for Locomotion in the Isolated Lamprey Spinal Cord
Authors: Harris-Warrick Ronald M. , Cohen Avis H.
Published: April 2021

The central pattern generator for locomotion in the spinal cord of the lamprey can be activated in vitro by the addition of D-glutamate to the bathing saline. Serotonin has no effects when bath-applied alone, but it modulates the D-glutamate-activated swimming pattern. Three major effects are observed: (1) a dose-dependent reduction in the frequency of rhythmic ventral root burst discharge; (2) enhancement of the intensity of burst discharge, due in part to the recruitment of previously inactive motoneurones; (3) prolongation of the intersegmental phase lag. Motoneurone activation appears to result from enhanced synaptic drive from the central pattern generator; no direct effects of serotonin on the motoneurones themselves (resting potential, input resistance or threshold for action potential generation) were observed. Theoretical and experimental studies suggest that the prolongation of the intersegmental phase lag results at least in part from differential effects of serotonin on segmental oscillators in different parts of the spinal cord. Isolated caudal pieces of the cord were more strongly affected by serotonin than isolated rostral pieces. We propose that serotonin may be an endogenous modulator of the central pattern generator for locomotion in the lamprey. It may have a role in the generation of a family of related undulatory movements (swimming, crawling, burrowing) by a single central pattern generator.
Strand Receptors Associated with the Femoral Chordotonal Organs of Locust Legs
Authors: Bräunig Peter
Published: April 2021

The so-called flexor strand of the femoral chordotonal organ of the locust jumping leg was found to be innervated by a single sensory neurone with its cell body in the metathoracic ganglion. The strand is therefore a new mechanoreceptor of the strand receptor class. It is sensitive to extension of the femoro-tibial joint. The innervation is rather unusual in the hind leg as well as in the fore and middle legs.
Effect of Limb Mass and its Distribution on the Energetic Cost of Running
Authors: Myers M. J. , Steudel K.
Published: April 2021

Functional morphologists have traditionally regarded cost of locomotion as an important influence on the design of locomotor structures. If cost of locomotion is an important constraint in the natural selection of these structures, it should be possible to show that animals differing in limb morphology also differ in their locomotor costs. In previous experiments on three species of cursorial mammals differing considerably in limb structure, no such differences were detected. Since the factors that determine the rate of energy consumption of a running animal are not well understood, we felt that the effect of limb morphology on cost could best be examined in a system in which only the inertial properties of limbs were varied while other factors remained constant. Consequently, we have measured changes in the rate of energy consumption of running human subjects produced by artificial alterations in limb inertial properties. Other variables that might influence cost have been controlled. We found that the cost of adding a given mass to the limbs is significantly greater than adding it to the centre of mass and that this effect becomes more pronounced as the limb loads are moved distally. Thus a clear effect of limb mass and its distribution on cost of locomotion has been demonstrated.
Water Transport Across the Short-Circuited Midgut of the American Silkworm
Authors: Zerahn Karl
Published: April 2021

The midgut of the American silkworm transports potassium actively from the blood-side to the lumen (Harvey & Nedergaard, 1964) and when the midgut is short-circuited, the short-circuit current (ISC) is approximately equivalent to the active K transport. The rate of movement of water has been considered to be low. It has been measured as 9 ±70μl h−1 (N=3) from blood-side to lumen in an open system (Nedergaard, 1972). In the closed system described by Harvey, Haskell & Zerahn (1967) the water flow could be monitored from the shape and size of the midgut and it was obvious that the movement of water was small.
Determination of Pure Voltage-Dependent Ca2+ Current In Paramecium Caudatum and Its Inhibition By Divalent Cations
Authors: Wehner Frank , Hildebrand Eilo
Published: April 2021

Voltage-dependent Ca2+ currents in Paramecium caudatum were studied under voltage clamp conditions. To separate Caz+ inward currents from concomitant K+ outward currents, the voltage-dependent Ca2+ conductance was temporarily inactivated by a preceding depolarization. The remaining currents were then subtracted from the overall currents measured in the absence of a prepulse. In this way pure Ca2+ currents could be obtained up to a depolarization of 100 mV, which is about 50 mV below the theoretical Ca2+ equilibrium potential (ECa). Ca2+ currents were maximal at a depolarization of 35 mV and declined with further approach to ECa, but they did not reverse sign in the voltage range tested.

In the presence of Mg2+, Co2+, Mr2+ or Ni2+, the Ca2+ inward currents decreased to a different extent. From experiments where these cations were added at different concentrations and from measurements at different Ca2+ concentrations in the absence of other divalent cations the following ratio of apparent dissociation constants could be derived: kNi: kCo: kCa: kMn: kMg = 1:3:4·3–4·7: 5:6·5. With a confidence of 95% the absolute value of kCa lies between 40 and 130μmoll−1. These results indicate that Ca2+ and other divalent cations compete for binding sites at the Ca-channel and thus determine excitability. Indirect effects due to changes of the surface potential are discussed.
Evidence for High-Affinity Ca2+-ATPase Activity And Atp-Driven Ca2+-Transport in Membrane Preparations of the Gill Epithelium of the Cichlid Fish Oreochromis Mossambicus
Authors: Flik Gert , Van Rijs Jeanne H. , Wendelaar Bonga Sjoerd E.
Published: April 2021

A high-affinity Ca2+-ATPase activity was demonstrated among the phosphatase activities in plasma membranes of tilapia branchial epithelium: its characteristics (K0·5 = 0·063 μmoll−1 Ca2+, Vmax = 6·02μmnol P,h −1 mg−protein at 37°C) resemble those of Ca2+-translocating enzymes present in mammalian erythrocytes or enterocytes. The ratio of this Ca2+-ATPase activity to Na+/K+-ATPase activity was 1:20·4. Radioimmunoassayable calmodulin was demonstrated in the Ca2+-ATPase-containing membrane fraction.

ATP-dependent Ca2+-transport was demonstrated in tight-vesicle preparations of the branchial cell membranes; 30% of the vesicles in the preparation were inside-out, 44% were right-side-out and 26% were leaky. The characteristics of the active Ca2+-transport activity are consistent with a Ca2+-extrusion mechanism involving high-affinity Ca2+-ATPase activity.

The branchial Ca2+-transport activity per fish, as calculated on the basis of the transport activity determined for the vesicle preparation, is of the order of the branchial Ca2+-influx rates observed in vivo. The data provide the first biochemical evidence for active Ca2+-transport in plasma mem-branes of branchial epithelium. A model is presented for the mechanism of active transepithelial Ca2+-transport in fish gills.
Intracellular Recordings of Neck Muscle Motoneurones During Eye Cleaning Behaviour of the Cricket
Authors: Hensler Klaus
Published: April 2021

Intracellular recordings were made from prothoracic neurones of crickets which were free to move in a quite normal way. During the head roll component of eye cleaning, the motoneurones (MNs) to the driving muscles, dorso-ventral neck muscles 55, 56 and 60, received excitatory and/or inhibitory input from several spiking neurones. The discharge patterns of these MNs were basically identical to those in intact animals.

Lesion of connectives showed that the motor pattern of eye cleaning is generated by a complex neural network in the prothoracic and the suboesophageal ganglia, in which both neck connectives are involved.

Single electric shocks to the axons of the interommatidial mechanoreceptors through which eye cleaning is elicited evoked an EPSP followed by an IPSP with a latency of about 10 ms in all MNs of the three muscles on both sides. During repeated stimulation of the receptors this input is predominantly inhibitory.
The Relationship Between Lung Volume, Respiratory Drive and Breathing Pattern in the Turtle, Chrysemys Picta
Authors: Milsom W. K. , Chan P.
Published: April 2021

Induced changes in resting lung volume (VLR) in the turtle Chrysemys picta (Schneider) had no effect on resting levels of minute ventilation in animals breathing room air but did change their breathing pattern. Increasing VLR caused an increase in the number of breaths in each episode (burst) of breathing but a reduction in the incidence of such breathing bursts and thus an increase in the length of periods of breath-holding. The data indicate that these effects were largely the consequence of changes in lung volume per se rather than changes in lung gas stores.

Although both hypoxia and hypercapnia stimulated ventilation via increases in tidal volume and breathing frequency, they produced distinct changes in breathing pattern. While hypoxia (3% O2) caused an increase in the number of bursts of breathing (B/min) and reduced the number of breaths (b) in each burst (b/B), hypercapnia (5% CO2) increased both B/min and b/B. These data suggest that the size and incidence of bursts of breathing must be under separate control. One consequence of the different effects of hypoxia and hypercapnia on breaths per burst (b/B) was that hypoxic-hypercapnic gas mixtures (3% O2+5% CO2) failed to stimulate ventilation as much as hypercapnia alone.

Administration of hypoxic, hypercapnic and hypoxic-hypercapnic gas mixtures to elevate respiratory drive eliminated the effects of changes in VLR on breathing pattern. Thus, although changes in VLR are important in the control of breathholding in animals breathing air, their effect decreases as respiratory drive increases.
Binocular Spatial Localization in the Praying Mantis
Authors: Rossel Samuel
Published: April 2021

The way in which mantids localize the direction and the distance of their prey has been analysed, to clarify the importance of binocular interactions. The results suggest the following major points: Binocular interactions coordinate monocular directional information, such that the direction in which a mantid fixates its prey is intermediate between the image directions in the two eyes (Figs 3, 4).Binocular interactions elaborate the disparity between the monocular images, thus providing the mantid with a means of estimating the distance of its prey (Fig. 5 ; Tables 1, 2).

The above findings, together with the known visual recognition tasks performed by the mantid, finally allow the formulation of a simple neuronal model of binocular vision.