Complex between a Multicrossover DNA Nanostructure, PX-DNA, and T7 Endonuclease I.

Paranemic crossover DNA (PX-DNA) is a four-stranded multicrossover structure that has been implicated in recombination-independent recognition of homology. Although existing evidence has suggested that PX is the DNA motif in homologous pairing (HP), this conclusion remains ambiguous. Further investigation is needed but will require development of new tools. Here, we report characterization of the complex between PX-DNA and T7 endonuclease I (T7endoI), a junction-resolving protein that could serve as the prototype of an anti-PX ligand (a critical prerequisite for the future development of such tools). Specifically, nuclease-inactive T7endoI was produced and its ability to bind to PX-DNA was analyzed using a gel retardation assay. The molar ratio of PX to T7endoI was determined using gel electrophoresis and confirmed by the Hill equation. Hydroxyl radical footprinting of T7endoI on PX-DNA is used to verify the positive interaction between PX and T7endoI and to provide insight into the binding region. Cleavage of PX-DNA by wild-type T7endoI produces DNA fragments, which were used to identify the interacting sites on PX for T7endoI and led to a computational model of their interaction. Altogether, this study has identified a stable complex of PX-DNA and T7endoI and lays the foundation for engineering an anti-PX ligand, which can potentially assist in the study of molecular mechanisms for HP at an advanced level.

Fast design of arbitrary length loops in proteins using InteractiveRosetta.

BACKGROUND: With increasing interest in ab initio protein design, there is a desire to be able to fully explore the design space of insertions and deletions. Nature inserts and deletes residues to optimize energy and function, but allowing variable length indels in the context of an interactive protein design session presents challenges with regard to speed and accuracy. RESULTS: Here we present a new module (INDEL) for InteractiveRosetta which allows the user to specify a range of lengths for a desired indel, and which returns a set of low energy backbones in a matter of seconds. To make the loop search fast, loop anchor points are geometrically hashed using C α-C α and C ß-C ß distances, and the hash is mapped to start and end points in a pre-compiled random access file of non-redundant, protein backbone coordinates. Loops with superposable anchors are filtered for collisions and returned to InteractiveRosetta as poly-alanine for display and selective incorporation into the design template. Sidechains can then be added using RosettaDesign tools. CONCLUSIONS: INDEL was able to find viable loops in 100% of 500 attempts for all lengths from 3 to 20 residues. INDEL has been applied to the task of designing a domain-swapping loop for T7-endonuclease I, changing its specificity from Holliday junctions to paranemic crossover (PX) DNA.

Probing the impact of GFP tagging on Robo1-heparin interaction.

Green fluorescent proteins (GFPs) and their derivatives are widely used as markers to visualize cells, protein localizations in in vitro and in vivo studies. The use of GFP fusion protein for visualization is generally thought to have negligible effects on cellular function. However, a number of reports suggest that the use of GFP may impact the biological activity of these proteins. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins mediating diverse patho-physiological processes. In the heparin-based interactome studies, heparin-binding proteins are often prepared as GFP fusion proteins. In this report, we use surface plasmon resonance (SPR) spectroscopy to study the impact of the GFP tagging on the binding interaction between heparin and a heparin-binding protein, the Roundabout homolog 1 (Robo1). SPR reveals that heparin binds with higher affinity to Robo1 than GFP-tagged Robo1 and through a different kinetic mechanism. A conformational change is observed in the heparin-Robo1 interaction, but not in the heparin-Robo1-GFP interaction. Furthermore the GFP-tagged Robo1 requires a shorter (hexasaccharide) than the tag-free Robo1 (octadecasaccharide). These data demonstrate that GFP tagging can reduce the binding affinity of Robo1 to heparin and hinder heparin binding-induced Robo1 conformation change.

Characterization of the interaction between Robo1 and heparin and other glycosaminoglycans.

Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2-Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand-receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ~650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1-heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1-heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.

Structural studies of the interaction of Crataeva tapia bark protein with heparin and other glycosaminoglycans.

CrataBL, a protein isolated from Crataeva tapia bark, which is both a serine protease inhibitor and a lectin, has been previously shown to exhibit a number of interesting biological properties, including anti-inflammatory, analgesic, antitumor, and insecticidal activities. Using a glycan array, we have now shown that only sulfated carbohydrates are effectively bound by CrataBL. Because this protein was recently shown to delay clot formation by impairing the intrinsic pathway of the coagulation cascade, we considered that its natural ligand might be heparin. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins, including thrombin and antithrombin III, which have a critical, essential pharmacological role in regulating blood coagulation. We have thus employed surface plasmon resonance to improve our understanding of the binding interaction between the heparin polysaccharide and CrataBL. Kinetic analysis shows that CrataBL displays strong heparin binding affinity (KD = 49 nM). Competition studies using different size heparin-derived oligosaccharides showed that the binding of CrataBL to heparin is chain length-dependent. Full chain heparin with 40 saccharides or large oligosaccharides, having 16-18 saccharide residues, show strong binding affinity for CrataBL. Heparin-derived disaccharides through tetradecasaccharides show considerably lower binding affinity. Other highly sulfated GAGs, including chondroitin sulfate E and dermatan 4,6-disulfate, showed CrataBL binding affinity comparable to that of heparin. Less highly sulfated GAGs, heparan sulfate, chondroitin sulfate A and C, and dermatan sulfate displayed modest binding affinity as did chondroitin sulfate D. Studies using chemically modified heparin show that N-sulfo and 6-O-sulfo groups on heparin are essential for CrataBL-heparin interaction.

The alpha1- and beta1-adrenergic modulation of lacrimal gland function in the mouse.

PURPOSE: To determine the expression patterns of alpha(1)- and beta(1)-adrenergic receptors in the mouse exorbital lacrimal gland (LG). An alpha- and beta-receptor agonist and antagonist were used to elucidate the receptors' relevance to protein secretion. METHODS: Mouse LGs were processed for single- and double-labeled indirect immunofluorescence studies and examined with confocal scanning microscopy. Protein secretion was measured from gland fragments in response to adrenergic agonists. RESULTS: Extensive alpha(1)-immunoreactivity (IR) was found on the surface and cytoplasm of acinar cells and much more alpha(1)-IR in the interstitial areas. In contrast, more beta(1)-IR was found in the LG, and most beta(1)-IR appeared to concentrate in the cytoplasm of acinar cells, with almost no beta(1)-IR in the interstitial areas. The protein secretion in response to phenylephrine and isoproterenol showed that direct stimulation of either the alpha(1)- or beta(1)-receptor could induce significant protein secretion from LGs. The specificity of this stimulation was further indicated by the effects of adrenergic antagonists. No synergism was observed between alpha(1)- and beta-receptor-mediated protein secretions. CONCLUSIONS: The results support the notion that there is extensive adrenergic control in the mouse LG. The adrenergic receptors may be a better choice of markers, compared with tyrosine hydroxylase and dopamine beta-hydroxylase, to reflect the extent of adrenergic control because circulating norepinephrine in the bloodstream should be taken into consideration. Both confocal microscopy observations and protein secretion data suggest the presence of alpha(1)- and beta(1)-mediated pathways in the mouse LG.

Fluid secretion and the Na+-K+-2Cl- cotransporter in mouse exorbital lacrimal gland.

We have previously suggested that fluid flow in the mouse exorbital lacrimal gland is driven by the opening of apical Cl- and K+ channels. These ions move into the lumen of the gland and water follows by osmosis. In many tissues, the Na+-K+-2Cl- cotransporter (NKCC1) replaces the Cl- and K+ ions that move into the lumen. We hypothesize that mouse exorbital lacrimal glands would have NKCC1 co-transporters and that they would be important in fluid transport by this gland. We used immunocytochemistry to localize NKCC1-like immunoreactivity to the membranes of the acinar cells as well as to the basolateral membranes of the duct cells. We developed a method to measure tear flow and its composition from mouse glands in situ. Stimulation with the acetylcholine agonist carbachol produced a peak flow followed by a plateau. Ion concentration measurements of this stimulated fluid showed it was high in K+ and Cl-. Treatment of the gland with furosemide, a blocker of the NKCC1 cotransporter, reduced the plateau phase of fluid flow by approximately 30%. Isolated cells exposed to a hypertonic shock shrank by approximately 20% and then showed a regulatory volume increase (RVI). Both the RVI and swelling were blocked by treatment with furosemide. Cells isolated from these glands shrink by approximately 10% in the presence of carbachol. Blocking NKCC1 with furosemide reduced the amount of shrinkage by approximately 50%. These data suggest that NKCC1 plays an important role in fluid secretion by the exorbital gland of mice.

Human mesenchymal stem cells make cardiac connexins and form functional gap junctions.

Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell-to-cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell-to-cell coupling to each other, to HeLa cells transfected with Cx40, Cx43 and Cx45 and to acutely isolated canine ventricular myocytes. The junctional currents (I(j)) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage (V(j)) dependence. I(j) records from hMSC-HeLaCx43 and hMSC-HeLaCx40 cell pairs also showed symmetrical and asymmetrical V(j) dependence, while hMSC-HeLaCx45 pairs always produced asymmetrical I(j) with pronounced V(j) gating when the Cx45 side was negative. Symmetrical I(j) suggests that the dominant functional channel is homotypic, while the asymmetrical I(j) suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (gamma(j)) of 30-80 pS. The macroscopic I(j) obtained from hMSC-cardiac myocyte cell pairs exhibited asymmetrical V(j) dependence, while single channel events revealed gamma(j) of the size range 40-100 pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.

Sympathetic neural control of the mouse lacrimal gland.

PURPOSE: To explore the sympathetic innervation pattern and the role of sympathetic nervous system control of protein secretion in the exorbital lacrimal glands of normal mice. METHODS: Mouse lacrimal glands were processed for single- and double-label indirect immunofluorescence studies to show their innervation patterns. The sucrose-potassium phosphate-glyoxylic acid method was also used to visualize the adrenergic innervation. The effects of adrenergic and cholinergic agonists on protein secretion were evaluated. RESULTS: The mouse lacrimal gland can be divided into two different areas based on the innervation density and distribution pattern. One area, approximately 10% to 30% of the gland, exhibited much higher innervation density, both parasympathetic and sympathetic, than the rest of the gland. The adrenergic agonists norepinephrine and phenylephrine induced increases in protein secretion that were of a magnitude similar to the increases induced by the cholinergic agonist carbachol at 10(-6) to 10(-4) M. Isoproterenol, the beta-adrenergic agonist, also elicited protein secretion at 10(-5) to 10(-4) M. CONCLUSIONS: The data indicate that there is extensive sympathetic innervation of the mouse lacrimal gland and that sympathetic input can modulate protein secretion. The division of the lacrimal gland into two areas suggests that the mouse lacrimal gland is a mixed gland and that these two areas may play different roles in secreting tears of different compositions in various situations. These data appear to support the notion that differential secretion is accomplished by activating different populations of secretory cells that are differentially innervated.

Role of gap junctions in fluid secretion of lacrimal glands.

In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.

Calcium dependence of exocytosis in lacrimal gland acinar cells.

Simultaneous measurements of membrane capacitance and intracellular calcium concentration were used to examine the calcium dependence of exocytosis in single acinar cells from mouse lacrimal gland and to establish the quantitative relation between calcium concentration and rate of exocytosis. Application of adrenergic or muscarinic agonists elevated intracellular calcium and evoked exocytosis, as indicated by an increase in membrane capacitance of single cells. The capacitance response to agonist stimulation was eliminated by internal dialysis with the calcium buffer EGTA, which demonstrated that the increase in intracellular calcium was necessary for agonist-evoked exocytosis. When internal calcium was elevated by application of the calcium ionophore ionomycin, exocytosis was evoked in the absence of agonist stimulation. Thus an increase in intracellular calcium was necessary and sufficient for exocytosis in single acinar cells. The rate of change of membrane capacitance increased as approximately the third power of the calcium concentration, which is similar to the dependence of exocytosis rate on calcium concentration in other secretory cells.

Differences in stimulus induced calcium increases in lacrimal gland acinar cells from normal and NZB/NZW F1 female mice.

PURPOSE: To compare the changes in intracellular free calcium in response to both cholinergic and adrenergic agonists in cells isolated from exorbital lacrimal glands of NZB/NZW F1 female mice (NZB/W) and normal Swiss Webster mice (SW). METHODS: We have loaded cells with Fura-2 and measured total intracellular calcium using ratiomicrofluorometric methods. Isolated cells were also patch-clamped using perforated patch. RESULTS: In all cells, both carbachol (CCh) and phenylephrine (PE) increased intracellular calcium. The calcium increase to both CCh and PE was less in cells from NZB/W animals than in cells from SW animals. In cells from young animals, the baseline calcium levels were the same, but in cells from older (6 months) NZB/W animals, the baseline levels of calcium were 50% higher than those seen in SW animals. CONCLUSION: These data suggest that cells from the NZB/W mice have different calcium dynamics, which could contribute to their compromised fluid secretion.

The Lacrimal Gland and Its Veil of Tears.

The secretory cells of the lacrimal gland produce a highly complex product of water, ions and proteins. At least five neurotransmitter receptors and three different second message systems are involved in controlling the different secretory processes of this highly sophisticated secretory epithelium.