Subunit-specific monoclonal antibodies to tarantula hemocyanin,and a common epitope shared with calliphorin

Summary Three murine hybridoma cell lines secreting IgG₁ antibodies to 4×6 tarantula (Eurypelma californicum) hemocyanin were isolated, and the monoclonal antibodies Ec-7, Ec-8 and Ec-24 characterized by immunoblotting, immunoelectrophoresis and ELISA. WholeEurypelma hemocyanin, and the isolated subunitsa tog served as probes. For the subunits a novel, quick purification scheme on FPLC combined with immuno-affinity chromatography was established.Additionally, two cell lines secreting IgM antibodies were isolated. These antibodies showed irrelevant cross reactivities.Ec-7 strongly reacts with subunitd and weakly withb. Ec-8 and Ec-24 are specifically directed againstEurypelma subunitsa ande, respectively. The epitopes of Ec-7 and Ec-8 are sequence-dependent, whereas the Ec-24 epitope is conformation-dependent. Ec-8 and Ec-24 are specific forEurypelma hemocyanin. Ec-7 is not reactive to crustacean, centipede or gastropod hemocyanins, but binds to scorpion hemocyanin and to the immunological correlates of subunitsd andf in the hemocyanins of the spiderCupiennius salei and the xiphosuranLimulus polyphemus.In immunoblots with different polyclonal antisera,Eurypelma andAstacus hemocyanin cross-reacted with calliphorin, a larval serum protein from the blowflyCalliphora vicina. Calliphorin and chelicerate hemocyanins share the Ec-7 epitope. Sedimentation coefficients, pH stability regions, subunit size, and electron microscopical appearance of calliphorin are indiscernable from a typical 1×6 arthropod hemocyanin. This relationship is discussed in the context of hemocyanin evolution.Abbreviations FPLC fast performance liquid chromatography - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate A preliminary account of this work was presented in June 1987 at the annual meeting of the Deutsche Zoologische Gesellschaft at Ulm (Markl 1987a)     

Postural dynamics of walking in humans

The dynamics of postural control in human biped locomotion were studied using(1) a model, and(2) experimentally applied impulsive force disturbances. The model was planar, and contained five rigid segments, articulating at frictionless pin joints. The model was used to identify joint torque combinations which would successfully correct for an impulsive force disturbance applied at different points in the walking cycle. The simulation results suggested that(1) early responses (within 80ms) can be effective in compensating for impulsive disturbances,(2) the same strategies which successfully counteract similar disturbances during quiet standing are also effective in certain phases of the walking cycle,(3) modifications in the response strategies are needed to accomodate differences in the dynamics over the stride cycle, and(4) the swing leg is ineffective in compensating for disturbances in the short term. These model predictions were tested experimentally. Subject responses to an impulsive force disturbance applied during walking were studied. The electromyographic results generally support the model predictions.     

Biomechanics of below-knee amputee gait

Sagittal plane biomechanical and EMG analyses from eight below knee (B/K) amputee trials demonstrate considerably modified motor patterns from the residual muscles at the hip and knee. Five SACH fittings, two Uniaxial and one Gressinger prostheses were analysed. Moments of force and mechanical power were analysed on all eight trials and EMG profiles are reported for three of the amputees fitted with SACH prostheses. The findings can be summarized as follows: 1. All eight trials had similar internal moment of force patterns at the ankle. A dorsiflexor moment commenced at heel contact and continued for the first third of stance. The prostheses generated a plantarflexor moment for the balance of stance which increased in late stance to about 2/3 that seen in normals. 2. The two Uniaxial prostheses showed a 20% recovery of stored energy which was returned at push-off. The recovery by the Gressinger fitting was 30%. 3. For all but the Gressinger prosthesis the knee moment of force was negligible during early stance (when normals have an extensor moment), below normal in late stance and fairly normal during swing. The amputee wearing the Gressinger prosthesis had a normal but slightly reduced pattern of moments of force over the entire stride. 4. All eight trials had hyperactive hip extensors during early and mid-stance which resulted in above-normal energy generation by these concentrically contracting muscles. This compensation makes up for the loss of the major energy generation by the plantarflexors at push-off. 5. The moment of force and power patterns at the hip for all eight trials during late stance and swing were fairly normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve growth factor (NGF) regulates adult rat cultured dorsal root ganglion neuron responses to the excitotoxin capsaicin

An overlap between subpopulations of nerve growth factor (NGF)-responsive and capsaicin-sensitive dorsal root ganglion (DRG) sensory neurons has been suggested from a number of in vivo studies. To examine this apparent link in more detail, we compared the effects of capsaicin on adult rat DRG neurons cultured in the presence or absence of NGF. Capsaicin sensitivity was assessed histochemically by a cobalt staining method, by measuring capsaicin-induced 45Ca2+ uptake, and by electrophysiological recording of capsaicin-evoked membrane currents. When cultured with NGF, approximately 50% of these adult DRG neurons were capsaicin-sensitive, whereas adult sympathetic neurons or ganglionic nonneuronal cells were insensitive. DRG cultures grown in the absence of NGF, however, were essentially unresponsive to capsaicin. Capsaicin sensitivity could be regained fully within 4-6 days of replacement of NGF. These results indicate that, at least in vitro, NGF can modify the capsaicin sensitivity of adult DRG neurons.     

Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae.

A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.     

Structural features and sites of expression of a new murine 65 kD and 48 kD hair-related keratin pair, associated with a special type of parakeratotic epithelial differentiation

In the course of studies on local keratin phenotypes in the epidermis of the adult mouse, we have identified a new 65 kD and 48 kD keratin pair. In mouse skin, this keratin pair is only expressed in suprabasal cells of adult mouse tail scale epidermis which is characterized by the complete absence of a granular layer and the formation of a remarkably compact stratum corneum. A second site in which the 65 kD and 48 kD keratin pair is suprabasally expressed and whose morphology corresponds to that of tail scale epidermis is found in the posterior unit of the complex filiform papillae of mouse tongue. The causal relationship of the expression of the 65 kD and 48 kD keratins with this particular type of a non-pathological epithelial parakeratosis is emphasized by the suppression of the mRNA synthesis of the two keratins during retinoic acid mediated orthokeratotic conversion of tail scale epidermis. Apart from tail scale epidermis and the posterior unit of the filiform papillae, the 65 kD and 48 kD keratin pair is, however, also coexpressed with "hard" alpha keratins in suprabulbar cells of hair follicles and in suprabasal cells of the central core unit of the lingual filiform papillae. The non alpha-helical domains of the two new keratins are rich in cysteine and proline residues and lack the typical subdomains into which epithelial keratins of both types can be divided. This structural resemblance of the 65 kD and 48 kD keratins to "hard" alpha keratins is supported by comparative flexibility predictions for their non alpha-helical domains. Phylogenetic investigations then show that the 65 kD and 48 kD keratin pair has evolved together with hair keratins, but has diverged from these during evolution to constitute an independent branch of a pair of hair-related keratins. In view of this exceptional position of the 65 kD and 48 kD keratins within the keratin multigene family, their expression has apparently been adopted by rare anatomical sites in which an orthokeratinized stratum corneum would be too soft and a hard keratinized structure would be too rigid to meet the functional requirement of the respective epithelia.     

Dry matter production and photosynthetic capacity in Gossypium hirsutum L. under conditions of slightly suboptimum leaf temperatures and high levels of irradiance

Summary Gossypium hirsutum L. var. Delta Pine 61 was cultivated in controlled-environment chambers at 1000–1100 mol photosynthetically active photons m^-2 s^-1 (medium photon flux density) and at 1800–2000 mol photons m^-2 s^-1 (high photon flux density), respectively. Air temperatures ranged from 20° to 34°C during 12-h light periods, whereas during dark periods temperature was 25° C in all experiments. As the leaf temperature decreased from about 33° to 27° C, marked reductions in dry matter production, leaf chlorophyll content and photosynthetic capacity occurred in plants growing under high light conditions, to values far below those in plants growing at 27° C and medium photon flux densities. The results show that slightly suboptimum temperatures, well above the so-called chilling range (0–12° C), greatly reduce dry matter production in cotton when combined with high photon flux densities equivalent to full sunlight.Abbreviations DW dry weight - F _v variable fluorescence yield - F _M maximum fluorescence yield - PFD photon flux density (400–700 nm)