Changes in calmodulin level and cAMP-dependent protein kinase activity during epididymal maturation of ram spermatozoa

Calmodulin concentration and cAMP-dependent protein kinase activity were simultaneously determined on ram spermatozoa collected by cannulation of successive segments of the epididymal tubule. Epididymal transit was characterized on one hand by an overall decrease in the calmodulin level and on the other by a dramatic rise in the cAMP-dependent protein kinase activity. In contrast to the calmodulin level, the cAMP-dependent protein kinase activity was correlated with the acquisition of flagellar beat. No further alterations in the level of these two proteins could be detected as spermatozoa acquired progressive motility.     

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.     

Regression of blood vessels precedes cartilage differentiation during chick limb development

We have previously investigated distinct areas of vascular regression in the developing vascular system of the chick limb bud. Avascular areas appear in a characteristic spatial and temporal pattern, and are correlated with the position of developing cartilage. In the present study, we examined limb-bud sections which had been double labeled for endothelial cells and developing cartilage in order to determine the relationship between the appearance of cartilage and the disappearance of capillaries. Endothelial cells, which specifically take up acetylated low-density lipoprotein (acLDL), were labeled by intravenously injecting fluorescent acLDL (DiIacLDL) into chick embryos at Hamburger and Hamilton stages 26-30. Avascular zones, which correspond to the developing digits, were clearly visible within the fluorescently labeled distal vasculature. The same sections were labeled with monoclonal antibodies specific for cartilage. We found that progressing avascularity in the digital regions was followed by increased staining for cartilage antigens in the same areas. Zones of avascularity always developed earlier than morphologically and immunologically detectable cartilage in all planes of section and were always larger than the areas of cartilage. These results demonstrate that blood vessels disappear in predictable areas prior to the overt differentiation of cartilage.     

Genetic linkage of Beckwith-Wiedemann syndrome to 11p15.

Beckwith-Wiedemann syndrome (BWS), characterized by multiorgan developmental abnormalities and predisposition to cancer, usually occurs sporadically, but small apparently dominant pedigrees have been described. Since rare patients show varying karyotypic abnormalities on the short arm of chromosome 11, it has been suggested that BWS may be related to the Wilms tumor gene on 11p13 or, alternatively, to growth factor genes on 11p15. We performed genetic linkage analysis on two BWS kindreds, using RFLPs for loci on 11p. BWS was linked to the insulin gene (11p15.5), with an overall maximum lod score of 3.60 (recombination fraction = .00). Linkage to D11S16 (11p13) could be excluded for recombination fractions less than or equal to .03. These results suggest that BWS defines a tumor-predisposition gene on 11p15.     

Characterization of the recombinant Clostridium pasteurianum ferredoxin and comparison of its properties with those of the native protein

Ferredoxins (Fds) constitute an important class of nonheme iron-sulfur proteins. One of the most studied Fds is the [8Fe-8S] Fd from Clostridium pasteurianum. The gene for this Fd has previously been cloned and sequenced. We report the expression of this Fd in Escherichia coli, and the characterization and comparison of this recombinant protein to the native Fd. We have found that the purified recombinant protein has the same enzymatic, redox, magnetic and electronic properties as the native Fd isolated from C. pasteurianum, which indicates that the two [4Fe-4S] clusters present in the Fd were correctly formed in E. coli.     

Margaret Mead and Samoa: Coming of Age in Fact and Fiction

Derek Freeman's 1983 critique of Margaret Mead's Coming of Age in Samoa has prompted one of the most heated debates in recent anthropological memory. Yet even Mead's defenders have generally accepted the view that she saw Samoa as a romantic paradise. Here I argue that a careful reading of Coming of Age shows quite a different picture. In my reanalysis, Mead's work emerges as complex and somewhat enigmatic, with facile conclusions standing in contrast to much rich and sensitive ethnography. My goal in this article is to highlight what Mead actually said, pointing up the notable discrepancy between the popular perception and her actual account.     

Purification and properties of a low-redox-potential tetraheme cytochrome c3 from Shewanella putrefaciens.

Shewanella putrefaciens is a facultatively anaerobic bacterium in the gamma group of the proteobacteria, capable of utilizing a wide variety of anaerobic electron acceptors. An examination of its cytochrome content revealed the presence of a tetraheme, low-redox-potential (E'o = -233 mV), cytochrome c-type cytochrome with a molecular mass of 12,120 Da and a pI of 5.8. The electron spin resonance data indicate a bis-histidine coordination of heme groups. Reduction of ferric citrate was accompanied by oxidation of the cytochrome. The biochemical properties suggested that this protein was in the cytochrome c3 group, which is supported by N-terminal sequence data up to the first heme binding site.     

Lactoperoxidase-catalyzed iodination of horse cytochrome c:monoiodotyrosyl 74 cytochrome c.

Iodination of horse cytochrome c with the lactoperoxidase-hydrogen peroxide-iodide system results initially in the formation of the monoiodotyrosyl 74 derivative. This singly modified protein was obtained in pure form by ion exchange chromatography and preparative column electrophoresis. It shows an intact 695 nm absorption band, the midpoint potential of the native protein, a nuclear magnetic resonance spectrum which indicates an undisturbed heme crevice structure, a normal reaction with antibodies directed against native horse cytochrome c, and circular dichroic spectra in which the only changes from those of the native protein can be ascribed to the spectral properties of iodotyrosine itself. This conformationally intact derivative reacts with the succinate-cytochrome c reductase and the cytochrome c oxidase systems of beef mitochondrial particle preparations indistinguishably from the unmodified protein, showing that the region including tyrosine 74 is not involved in these enzymic electron transfer functions of the protein. The circular dichroic spectra of this derivative indicate that the minima observed at 288 and 282 nm in the spectrum of native ferricytochrome c originate from tyrosyl residue 74.     

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH₃CH(OH), CO₂, O₂, and e⁻_aq′ and the oxidation of the reduced cytochrome c derivatives by Fe(CN)³⁻₆ were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partitioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.