Swelling activation of transport pathways in erythrocytes: effects of Cl-, ionic strength, and volume changes

If swelling of acell is induced by a decrease in external medium tonicity, theregulatory response is more complex than if swelling of similarmagnitude is due to salt uptake. The present results provide anexplanation. In fish erythrocytes, two distinct transport pathways wereswelling activated: a channel of broad specificity and aK⁺-Clcotransporter. Each was activated by a specific signal: the channel bya decrease in intracellular ionic strength and theK⁺-Clcotransporter by cell enlargement. A decrease in ionic strength alsoaffectedK⁺-Clcotransport activity, but by acting as a negative modulator of thecotransport. Thus cells swollen by salt accumulation respond byactivating exclusively theK⁺-Clcotransport, leading to aCl-dependentK⁺ loss. By contrast, cellsswollen by electrolyte dilution respond by activating both pathways,leading to a reduced loss of electrolytes and a large loss of taurine.Thus two swelling-sensitive pathways, differently regulated, wouldallow control of the ionic composition of a cell exposed to differentvolume perturbations.

     

An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.     

Fibroblasts form a body-wide cellular network

Loose connective tissue forms a network extending throughout the body including subcutaneous and interstitial connective tissues. The existence of a cellular network of fibroblasts within loose connective tissue may have considerable significance as it may support yet unknown body-wide cellular signaling systems. We used a combination of histochemistry, immunohistochemistry, confocal scanning laser microscopy (confocal microscopy), and electron microscopy to investigate the extent and nature of cell-to-cell connections within mouse subcutaneous connective tissue. We found that fibroblasts formed a reticular web throughout the tissue. With confocal microscopy, 30% of fibroblasts processes could be followed continuously from one cell to another. Connexin 43 immunoreactivity was present at apparent points of cell-to-cell contact. Electron microscopy revealed that processes from adjacent cells were in close apposition to one another, but gap junctions were not observed. Our findings indicate that soft tissue fibroblasts form an extensively interconnected cellular network, suggesting they may have important and so far unsuspected integrative functions at the level of the whole body.     

Phloem import and storage metabolism are highly coordinated by the low oxygen concentrations within developing wheat seeds

We studied the influence of the internal oxygen concentration in seeds of wheat (Triticum aestivum) on storage metabolism and its relation to phloem import of nutrients. Wheat seeds that were developing at ambient oxygen (21%) were found to be hypoxic (2.1%). Altering the oxygen supply by decreasing or increasing the external oxygen concentration induced parallel changes in the internal oxygen tension. However, the decrease in internal concentration was proportionally less than the reduction in external oxygen. This indicates that decreasing the oxygen supply induces short-term adaptive responses to reduce oxygen consumption of the seeds. When external oxygen was decreased to 8%, internal oxygen decreased to approximately 0.5% leading to a decrease in energy production via respiration. Conversely, increasing the external oxygen concentration above ambient levels increased the oxygen content as well as the energy status of the seeds, indicating that under normal conditions the oxygen supply is strongly limiting for energy metabolism in developing wheat seeds. The intermediate metabolites of seed storage metabolism were not substantially affected when oxygen was either increased or decreased. However, at subambient external oxygen concentrations (8%) the metabolic flux of carbon into starch and protein, measured by injecting (14)C-Suc into the seeds, was reduced by 17% and 32%, respectively, whereas no significant effect was observed at superambient (40%) oxygen. The observed decrease in biosynthetic fluxes to storage compounds is suggested to be part of an adaptive response to reduce energy consumption preventing excessive oxygen consumption when oxygen supply is limited. Phloem transport toward ears exposed to low (8%) oxygen was significantly reduced within 1 h, whereas exposing ears to elevated oxygen (40%) had no significant effect. This contrasts with the situation where the distribution of assimilates has been modified by removing the lower source leaves from the plant, resulting in less assimilates transported to the ear in favor of transport to the lower parts of the plant. Under these conditions, with two strongly competing sinks, elevated oxygen (40%) did lead to a strong increase in phloem transport to the ear. The results show that sink metabolism is affected by the prevailing low oxygen concentrations in developing wheat seeds, determining the import rate of assimilates via the phloem.     

Microscopy images as interactive tools in cell modeling and cell biology education

The advent of genomics, proteomics, and microarray technology has brought much excitement to science, both in teaching and in learning. The public is eager to know about the processes of life. In the present context of the explosive growth of scientific information, a major challenge of modern cell biology is to popularize basic concepts of structures and functions of living cells, to introduce people to the scientific method, to stimulate inquiry, and to analyze and synthesize concepts and paradigms. In this essay we present our experience in mixing science and education in Brazil. For two decades we have developed activities for the science education of teachers and undergraduate students, using microscopy images generated by our work as cell biologists. We describe open-air outreach education activities, games, cell modeling, and other practical and innovative activities presented in public squares and favelas. Especially in developing countries, science education is important, since it may lead to an improvement in quality of life while advancing understanding of traditional scientific ideas. We show that teaching and research can be mutually beneficial rather than competing pursuits in advancing these goals.     

Cleavage of the matricellular protein SPARC by matrix metalloproteinase 3 produces polypeptides that influence angiogenesis

SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu2+-binding sequence KHGK) exhibited a biphasic effect on [3H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca2+-binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu2+-binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.     

A family of leukemia inhibitory factor-binding peptides that can act as antagonists when conjugated to poly(ethylene glycol)

A panel of six na?ve 14-residue random peptide libraries displayed polyvalently on M13 phage was pooled and sorted against human leukemia inhibitory factor (LIF). After four rounds of selection, a single large family of peptides with the consensus sequence XCXXXXG(A/S)(D/E)(W/F)WXCF was found to bind specifically to LIF. Peptides within this family did not bind related members of the interleukin-6 family of cytokines, nor to murine LIF that has 80% sequence identity with human LIF. A representative peptide from this family was synthesized and found to bind to LIF with an affinity of approximately 300 nM. The phage-displayed form of this peptide was able to compete with the LIF receptor alpha chain (LIFR) for binding to LIF; however, the free synthetic peptide was unable to inhibit LIF-LIFR binding or inhibit LIF bioactivity in vitro. Using a panel of human/murine chimeric LIF molecules, the peptide-binding site on LIF was mapped to a groove located between the B and the C helices of the LIF structure, which is distinct from the surfaces involved in binding to receptor. To mimic the effect of the phage particle and convert the free peptide into an antagonist of LIFR binding, a 40 kDa poly(ethylene glycol) (PEG) moiety was conjugated to the synthetic LIF-binding peptide. This PEG-peptide conjugate was found to be both an antagonist of LIF-LIFR binding and of LIF signaling in engineered Ba/F3 cells expressing LIFR and the gp130 coreceptor.     

Purification and characterization of a receptor for human parathyroid hormone and parathyroid hormone-related peptide

The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 microg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr(23), consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (K(d) = 16.5 +/- 1.3 versus 11.9 +/- 1.9 nm), and the purified receptors bound rat [Nle(8,21),Tyr(34)]PTH-(1-34)-NH(2) (PTH-(1-34)), and rat [Ile(5),Trp(23),Tyr(36)]PTHrP-(5-36)-NH(2) with indistinguishable affinity. Maximal displacement of (125)I-PTH-(1-34) binding by rat [alpha-aminoisobutyric acid (Aib)(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH-(1-21)-NH(2) and rat [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]PTH-(1-14)-NH(2) of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [(35)S]GTP gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.