Analogs of human epidermal growth factor which partially inhibit the growth factor-dependent protein-tyrosine kinase activity of the epidermal growth factor receptor

Three site-directed mutants of human epidermal growth factor, Leu-26----Gly, Leu-47----Ala, and Ile-23----Thr, were examined for their ability to stimulate the protein-tyrosine kinase activity of the epidermal growth factor receptor. The receptor binding affinities of the mutant growth factors were 20- to 50-fold lower, as compared to wild-type growth factor. At saturating concentrations of growth factor, the velocities of the phosphorylation of exogenously added substrate and receptor autophosphorylation were significantly lower with the mutant analogs, suggesting a partial 'uncoupling' of signal transduction. The mutant analogs were shown to compete directly with the binding of wild-type, resulting in a decrease in growth factor-stimulated kinase activity.     

Effects of cryoprotectants on actin filaments during the cryopreservation of one-cell rabbit embryos

A dynamic equilibrium between globular and filamentous actin plays a crucial role in cell structure and motility. Many factors such as pH, ionic strength, temperature, and divalent cations, are known to influence this equilibrium. Some organic solvents, such as those used for the cryopreservation of cells, may also alter the dynamic equilibrium of this system. Fluorescence staining with NBD-phallacidin permits polymerized actin to be visualized in embryos and provides evidence that propanediol depolymerizes actin, whereas dimethyl sulfoxide does not. This depolymerizing effect is reversible after propanediol removal. Biochemical techniques were used to study the influence of these solvents on rabbit skeletal actin. Results obtained by sedimentation, fluorescence, DNase inhibition, electron microscopy, and viscometry analysis demonstrate that propanediol has a dual effect on actin polymers in vitro: it decreases the proportion of filamentous actin and the remaining filaments appear shorter and aggregate to form bundles. In contrast, dimethyl sulfoxide does not alter dramatically the actin polymer integrity. Propanediol is shown to exert a good cryoprotective action on rabbit embryos, while dimethyl sulfoxide does not. We suggest that the depolymerization of actin filaments by propanediol prior to cooling may facilitate the cryopreservation of one-cell rabbit embryos.     

Seasonal changes in fish catch and environmental variables in a large Tropical Lake,Volta, Ghana

Fish species assemblage and selected environmental variables were monitored in Lake Volta from September 2014 to August 2016 to determine seasonal variability in species composition, catch and environmental variables that determine the structure of the fish community. A total of 1,557 individual fish belonging to 41 species, 25 genera and 13 families were recorded. The important fish species with respect to frequency of occurrence, abundance and weight, respectively, were as follows: Chrysichthys (100%; 43.03%; 17.93%), tilapias (100%; 28.97%; 17.86%), Alestes (100%; 14.13%; 32.10%) and Bagrus (91.67%; 5.65%; 12.80%) in that order. The composition of fish species and their temporal variation in experimental catches were similar to that of commercial catches. There were no significant differences (p > 0.05) in species abundance, weight and diversity indices between the dry and wet seasons. The modal class of length frequency distribution of the dominant species in the catch, Chrysichthys, reduced from 125 mm in 2006 to 95 mm in the current study indicating overfishing. Environmental variables considered showed little variation and within optimal ranges for fish survival and did not differ significantly between seasons. Canonical Correspondence Analysis showed that environmental variables explained 43.30% of the variation in species abundance with Lake water level, nitrite‐nitrogen and total dissolved solids being the main environmental factors influencing the structure of the fish community.     

Persistence length of human cardiac α-tropomyosin measured by single molecule direct probe microscopy

α-Tropomyosin (αTm) is the predominant tropomyosin isoform in adult human heart and constitutes a major component in Ca2?-regulated systolic contraction of cardiac muscle. We present here the first direct probe images of WT human cardiac αTm by atomic force microscopy, and quantify its mechanical flexibility with three independent analysis methods. Single molecules of bacterially-expressed human cardiac αTm were imaged on poly-lysine coated mica and their contours were analyzed. Analysis of tangent-angle (θ(s)) correlation along molecular contours, second moment of tangent angles (<θ2(s)>), and end-to-end length (L(e-e)) distributions respectively yielded values of persistence length (L(p)) of 41-46 nm, 40-45 nm, and 42-52 nm, corresponding to 1-1.3 molecular contour lengths (L(c)). We also demonstrate that a sufficiently large population, with at least 100 molecules, is required for a reliable L(p) measurement of αTm in single molecule studies. Our estimate that L(p) for αTm is only slightly longer than L(c) is consistent with a previous study showing there is little spread of cooperative activation into near-neighbor regulatory units of cardiac thin filaments. The L(p) determined here for human cardiac αTm perhaps represents an evolutionarily tuned optimum between Ca2? sensitivity and cooperativity in cardiac thin filaments and likely constitutes an essential parameter for normal function in the human heart.     

Convergent evolution of a parasite-encoded complement control protein-scaffold to mimic binding of mammalian TGF-β to its receptors,TβRI and TβRII

The mouse intestinal helminth Heligmosomoides polygyrus modulates host immune responses by secreting a transforming growth factor (TGF)-β mimic (TGM), to expand the population of Foxp3⁺ T_regs. TGM comprises five complement control protein (CCP)-like domains, designated D1-D5. Though lacking homology to TGF-β, TGM binds directly to the TGF-β receptors TβRI and TβRII and stimulates the differentiation of naïve T-cells into T_regs. However, the molecular determinants of binding are unclear. Here, we used surface plasmon resonance, isothermal calorimetry, NMR spectroscopy, and mutagenesis to investigate how TGM binds the TGF-β receptors. We demonstrate that binding is modular, with D1-D2 binding to TβRI and D3 binding to TβRII. D1-D2 and D3 were further shown to compete with TGF-β(TβRII)₂ and TGF-β for binding to TβRI and TβRII, respectively. The solution structure of TGM-D3 revealed that TGM adopts a CCP-like fold but is also modified to allow the C-terminal strand to diverge, leading to an expansion of the domain and opening potential interaction surfaces. TGM-D3 also incorporates a long structurally ordered hypervariable loop, adding further potential interaction sites. Through NMR shift perturbations and binding studies of TGM-D3 and TβRII variants, TGM-D3 was shown to occupy the same site of TβRII as bound by TGF-β using both a novel interaction surface and the hypervariable loop. These results, together with the identification of other secreted CCP-like proteins with immunomodulatory activity in H. polygyrus, suggest that TGM is part of a larger family of evolutionarily plastic parasite effector molecules that mediate novel interactions with their host.     

Ultrastructure of muscle satellite cells in hypersomatotropic rats

Female Wistar-Furth rats were injected at one week of age with cells from either the GH1 or GH3 rat pituitary cell lines. Controls were injected with vehicle. Rats were killed at 11 weeks of age and satellite cells in the soleus and extensor digitorum longus (EDL) muscles were examined using transmission electron microscopy. Satellite cells in both the soleus and EDL muscles of rats with tumours which secreted growth hormone generally appeared to be metabolically more active than those cells seen in the muscles of control rats. The source of pituitary cell line did not appear to influence satellite cell ultrastructure. In rare instances, myofibers of tumor-bearing rats appeared to extend cytoplasmic projections around satellite cells as if to engulf the latter. There was no evidence of a pathological condition. Since only one time frame was observed, the effects of prolonged exposure to elevated blood growth hormone levels on satellite cells are not known.     

Conversion of naive T cells to a memory-like phenotype in lymphopenic hosts is not related to a homeostatic mechanism that fills the peripheral naive T cell pool

To examine directly whether a limited number of naive T cells transferred to lymphopenic hosts can truly fill the peripheral naive T cell pool, we compared the expansion and phenotype of naive T cells transferred to three different hosts, namely recombination-activating gene-deficient mice, CD3epsilon-deficient mice, and irradiated normal mice. In all three recipients, the absolute number of recovered cells was much smaller than in normal mice. In addition, transferred naive T cells acquired a memory-like phenotype that remained stable with time. Finally, injected cells were rapidly replaced by host thymic migrants in irradiated normal mice. Only continuous output of naive T cells by the thymus can generate a full compartment of truly naive T cells. Thus, conversion of naive T cells to a memory-like phenotype in lymphopenic hosts is not related to a homeostatic mechanism that fills the peripheral naive T cell pool.     

Copy number variations involving the microtubule-associated protein tau in human diseases

Mutations of the MAPT (microtubule-associated protein tau) gene are associated with FTLD (frontotemporal lobar degeneration) with tau pathology. These mutations result in a decreased ability of tau to bind MTs (microtubules), an increased production of tau with four MT-binding repeats or enhanced tau aggregation. In two FTLD patients, we recently described CNVs (copy number variations) affecting the MAPT gene, consisting of a partial deletion and a complete duplication of the gene. The partial deletion resulted in a truncated protein lacking the first MT-binding domain, which had a dramatic decrease in the binding to MTs but acquired the ability to bind MAP (microtubule-associated protein) 1-B. In this case, tauopathy probably resulted from both a loss of normal function and a gain of function by which truncated tau would sequester another MAP. In the other FTLD patient, the complete duplication might result in the overexpression of tau, which in the mouse model induces axonopathy and tau aggregates reminiscent of FTLD-tau pathology. Interestingly, the same rearrangement was also described in several children with mental retardation, autism spectrum disorders and dysmorphic features, as well as in a schizophrenic patient. Finally, complete deletions of the MAPT gene have been associated with mental retardation, hypotonia and facial dysmorphism.     

Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy.

Partial denaturation mapping, restriction endonuclease digestion, and electron microscopy were used to determine which end of the linear duplex replicative-form (RF) DNA molecule contains the origin of RF replication for the parvovirus H-1. This origin was localized within approximately 300 base pairs of the arbitrarily designated right end of the RF DNA, in the EcoRI or HaeII-A fragment. Based on denaturation behavior in formamide, the right end was also found to have a relatively high guanine plus cytosine content, whereas the region adjacent to the left terminus of the RF DNA molecule was adenine plus thymine rich.