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Paranemic crossover DNA (PX-DNA) is a four-stranded multicross-over 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.

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 β-C β 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.

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.

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.

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 (Ij) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage (Vj) dependence. Ij records from hMSC–HeLaCx43 and hMSC–HeLaCx40 cell pairs also showed symmetrical and asymmetrical Vj dependence, while hMSC–HeLaCx45 pairs always produced asymmetrical Ij with pronounced Vj gating when the Cx45 side was negative. Symmetrical Ij suggests that the dominant functional channel is homotypic, while the asymmetrical Ij suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (γj) of 30–80pS. The macroscopic Ij obtained from hMSC–cardiac myocyte cell pairs exhibited asymmetrical Vj dependence, while single channel events revealed γj of the size range 40–100pS. 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.

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.

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.

The fluid secretory impairment of lacrimal and salivary glands in Sjögren's syndrome (SS) is thought to be related to the extent of lymphocytic infiltration (LI) and subsequent loss of glandular tissue. In this study, we examine the correlation between the extent of tear flow reduction and the extent of LI of lacrimal glands in the NZB/W mouse, a model of SS.We stimulated tear production by topical application of carbachol onto the gland while fluid was collected from the lacrimal duct. The lacrimal glands were removed after fluid collection for histology.Fluid secretion in response to carbachol was less in the majority of young NZB/W females compared to C57 control animals and none of the glands showed LI. Fluid secretion was also impaired in the majority of old NZB/W females, and the extent of LI was highly variable. Some of the old SW females also showed blunted fluid secretory responses and some degree of focal LI. Young SW females showed no LI and most animals exhibited normal flow responses. Analysis of paired flow and LI measurements showed no correlation between LI and flow impairment in any of the groups or in the pooled data. Carbachol-stimulated protein secretion from lacrimal gland slices in vitro were similar in young and old SW and NZB/W mice.These results suggest that LI alone is not sufficient to explain the secretory dysfunction in the NZB/W mouse model of Sjögren's syndrome.

Objective: To develop a new method for viewing adrenergic innervation along renal preglomerular vessels; to assess nerve densities and vascular lesions along arcuate arteries (ArcA), arcuate arterial branches (ArcB), and interlobular arteries (ILA) in spontaneously hypertensive rats (SHR) and in angiotensin II (AngII) and in NG-nitro-l-arginine methyl ester (l-NAME) hypertensive rats. Methods: Preglomerular vasculatures were isolated after HCl maceration and were immunostained against synaptophysin, a membrane protein of synaptic vesicles. Lesions were stained with Sudan black. Longitudinal nerve densities and relative frequencies of ArcA, ArcB, and ILA endowed with sudanophilic lesions were assessed separately. Results: Synaptophysin immunostaining revealed the vascular neural plexus. Nerves were adrenergic, as the plexus was destroyed by treatment with 6-hydroxy dopamine. Vascular lesions were not seen in SHR, and increased nerve density was observed along ArcA and ILA. In l-NAME- and AngII-hypertensive rats, vascular lesions affected predominantly ArcB and ILA, and nerve density was reduced by 12% and 28% (ArcA), 37% and 31% (ArcB), and by 55% and 34% (ILA), respectively, versus normotensive controls. Endothelin-1 receptor blockade did not affect AngII-induced hypertension but prevented both lesion development and reduction of density of the vascular neural plexus. Conclusions: The method we have devised provides a direct en face view of the vascular adrenergic innervation of isolated preglomerular vasculature. Measurements in hypertensive rat models suggest a link between vascular lesions and reduction in nerve density in hypertension. Endothelin-1 likely plays a key role in mediating both vascular injury and altered vascular nerve density in hypertension.

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 existence of a functional connection between the nervous and immune systems has long been argued. To determine if such a link exists in the secretory immune system, we have examined the avian lacrimal gland (Harderian gland) which contains large numbers of plasma cells. We have shown that these plasma cells bind an antibody to muscarinic acetylcholine receptor and that carbachol, an acetylcholine agonist, increases the secretion rate of IgG by these cells above a constitutive baseline level. This neurotransmitter-dependent increase of immunoglobulin secretion requires an influx of Ca2+, whereas the constitutive baseline secretion is apparently less dependent on such a flux. Furthermore, the Ca2+ flux appears to be mediated by voltage-dependent calcium channels. These data support the hypothesis that plasma cells can respond to neurotransmitters and, in the case of acetylcholine, increase immunoglobulin secretion.

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.

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 cotransporters 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 ∼30%. Isolated cells exposed to a hypertonic shock shrank by ∼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 ∼10% in the presence of carbachol. Blocking NKCC1 with furosemide reduced the amount of shrinkage by ∼50%. These data suggest that NKCC1 plays an important role in fluid secretion by the exorbital gland of 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.

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.

The mammalian lacrimal gland is densely innervated by parasympathetic and sympathetic nerves, as well as fibers that contain neuropeptides.1 The parasympathetic nerve fibers in the lacrimal gland arise from neurons in the pterygopalatine ganglion (PPG), a cranial parasympathetic ganglion that receives synaptic input from preganglionic parasympathetic neurons in the brainstem. PPG postganglionic axons innervate a variety of structures including the lacrimal gland, nasal and palatine mucosae, and cranial vasculature.2,3

One of the major constituents of the tear film is an aqueous fluid. It has been estimated that the cornea and conjunctiva produce up to about 20% of this fluid and that the main and accessory lacrimal glands supply the rest. The details of the mechanisms that move water across the secretory epithelium of the lacrimal glands are not well understood and neither are the ways by which these mechanisms are controlled. In this paper, we have attempted to pull together what is known about lacrimal gland fluid flow and to present a model, derived in part from our work on the cornea that suggests mechanisms responsible for water movement across the epithelium.

Water movement in the lacrimal gland is a consequence of ion channel and transporter activity. The production of tears is a consequence of acetylcholine (Ach) stimulation of acinar and duct cells, which produce a transepithelial flux of ions resulting in fluid movement. Ach acts through phospholipase C via Gq/11 and subsequently results in elevation of intracellular IP3 and DAG. The former raises intracellular calcium while the latter activates many PKC isoforms (Walcott, 1998; Dartt et al., 1998). In lacrimal gland K+, chloride and cation channels are activated by elevated intracellular calcium (Marty et al., 1984; Begenisich and Melvin, 1998).

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.

Intercellular conduction in the working myocardium of the mammalian heart is mediated by gap junctions composed of connexin43 or 45. Recently, it has been shown that myocardial connexin expression is malleable and may be altered with disease. To better understand myocardial conduction in left ventricular hypertrophy resulting from volume overload, we used indirect immunofluorescence microscopy to examine cardiac connexin43 expression in 10 New Zealand white rabbits with surgically induced aortic regurgitation (AR) and in 10 age-matched sham-operated controls. Animals were sacrificed at approximately 1 month oror =2.5 years after operation. All AR animals developed eccentric hypertrophy; none evidenced heart failure. The heart-to-body weight ratios for the 1 month AR and control groups were 2.9+/-0.8 vs 1.8+/-0.2 g/kg (por = 0.01) while ratios for theor =2.5 year AR and control groups were 2.4+/-0.3 vs 1.9+/-0.3 (por = 0.05). No significant differences in posterior wall thickness were found among any of the groups. Although the overall pattern of connexin43-like immunoreactivity was similar for all four groups, staining in the I month AR animals tended to be less than that of age-matched controls; staining was increased in theor =2.5 year AR animals and was greater than control (p0.05), in which staining did not change with animal age. This disease duration-related increase differs from the long-term decrease in connexin43 expression associated with other forms of heart disease and suggests that alterations in connexin expression may play a role in the rhythm abnormalities commonly seen in AR.

One manifestation of dry eye is a reduction in tear volume. In mammals, much of the tear volume is thought to arise from the acinar secretory and duct cells of the lacrimal gland. The subjacent tissue of the gland contains reticular-like connective tissue with scattered lymphocytes. Plasma cells within the connective tissue produce IgA, which is sequestered intracellularly by acinar cells and processed and finally secreted into the lumen of the gland.1 Many other secretory products are generated by the acinar cells. Secretagogue-triggered vesicle release is the process used by acinar cells to secrete proteins.

In many dry eye conditions including Sjogren’s syndrome, significant loss of secretory ability can be present even though areas of the lacrimal gland appear morphologically not affected by the disease process.1 This suggests an interruption(s) in the stimulation/secretion process that has affected the remaining acini, reducing the amount of protein and water they can secrete. The stimulation/secretion process begins with neurons releasing neurotransmitters, at least two of which, acetylcholine and vasoactive intestinal polypeptide (VIP), bind to receptors on the acinar cell surface and activate G protein pathways, increasing cAMP and IP3 levels.2 The IP3 will induce an increase in intracellular free Ca++, which, together with the increased levels of cAMP, will stimulate the secretion of protein. These same second message pathways may also affect anion and cation membrane channels that can alter the movement of water as well as ions such as Ca++ across the secretory epithelium into the lumen of the gland.3,4 We have examined this stimulus/secretion process in the extraorbital lacrimal glands from two mouse models for Sjogren’s syndrome,5 the MRL/MR-lpr-lpr [hereafter called MRL] and the (NZB/NZW) F1 [hereafter called NZB] strains, at different ages, and thus different stages of the disease, and compared them to those of a normal strain of mouse, the Swiss Webster [hereafter called SW] strain, at comparable ages. We have determined the innervation pattern and density in these glands and measured their ability to secrete protein in response to direct exposure to carbachol, an acetylcholine agonist. Our results strongly suggest that, in the NZB strain at least, there are important changes in the stimulus/secretion process associated with the disease that occur before significant lymphocytic infiltration that could account for the reduced tear output seen in lacrimal glands from these animals.

The rapid spread of locally restricted neural and hormonal signals among the vast array of largely inexcitable corporal smooth muscle cells is an absolute prerequisite to normal erectile function. And yet the mechanism(s) responsible for this phenomenon is not well understood. As a first step toward a more integrative understanding of erectile physiology and/or dysfunction, an 8- to 12-wk period of experimental diabetes was induced in 2-mo-old male Fischer 344 rats by either intraperitoneal streptozotocin (STZ) injection (35 mg/kg; n = 22) or subtotal pancreatectomy (n = 11). Fourteen age-matched control animals received injection of vehicle only while nine others served as sham-operated control animals. Eight STZ-diabetic animals received insulin replacement. Erectile function was assessed by evaluation of penile reflexes and monitoring of intracavernous pressure responses to both electrical stimulation of the cavernous nerve and intracorporal papaverine or nitroglycerin injection. Intracavernous pressure responses to neurostimulation were significantly attenuated in both STZ-diabetic and subtotal pancreatectomy animals compared with age-matched control animals (P < 0.05). Penile reflexes were also significantly diminished (P < 0.05). Regression analysis revealed that diabetes-related decreases in neurostimulated intracavernous pressure responses were strongly correlated with diminished synaptophysin immunoreactivity in the corpora (P < 0.001; r = 0.88). However, there were no detectable diabetes-related differences in pharmacological erections induced by intracavernous papaverine or nitroglycerin injection. Northern analysis revealed a marked diabetes-related increase in the amount of connexin 43 mRNA measured in frozen corporal tissue. Insulin replacement partially restored (attenuated the loss of) synaptophysin immunoreactivity and maintained neurostimulated intracavernous pressure responses to control levels while having no effect on penile reflexes. These observations may have important implications to the understanding of erectile physiology as well as the etiology of diabetes-related erectile dysfunction.

The chicken Harderian (lacrimal) gland contains an abundance of plasma cells in the interstitium of the gland that secrete IgG, IgM, and IgA. In in vitro preparations of this gland, the cholinergic agonist carbachol causes a transient increase in the secretion rate of IgG above a basal level of secretion. We have investigated the effects of the cyclic mononucleotides cAMP and cGMP on this secretagogue response. Pretreatment with 20 μM forskolin or 1 mM dibutyryl cAMP abolished the carbachol-induced secretory response. When the gland was isolated in normal media and then treated with either forskolin or dibutyryl cAMP, there was no change in the baseline secretion rate. cGMP at either 10 μM or 1.0 mM did not affect the baseline secretion rate, nor did it have an effect on the carbachol response. We postulate that muscarinic receptor activation leads to a calcium influx that in turn leads to an increased secretion rate of IgG. The opposing effects of elevating cAMP and cGMP are discussed in the context of this model of cholinergic activation of avian plasma cells.

The comparative anatomic and physiological study of primary lacrimal glands raises a number of important questions the answers to which can provide insight into lacrimal gland function and dysfunction.

Avian tears originate from two sources: a relatively small lacrimal gland located at the lateral canthus of the eye, and a much larger Harderian gland located medial to the eyeball within the orbit. In the chicken, the Harderian gland is the dominant orbital gland1, producing a mucoid secretion2,3 that aids in corneal lubrication.

One of the more ubiquitous cell types within the parenchyma of the lacrimal gland is the plasma cell. It is solely responsible for the production of immunoglobulins which are secreted into tears. While it is true that immunoglobulins such as IgA are modified by epithelial cells, it is still the plasma cell which acts as the source. In the case of IgG there is no involvement of the epithelium other than to act as a passive filter via the lateral interstitial space1. In terminally differentiated B-lymphocytes or plasma cells the secretion of immunoglobulin has been thought of as a constitutive-like process. The plasma cells simply secrete at a constant rate2. The processes which cause differentiation into plasma cells and hence increased output of immunoglobulin have been major focal points. It is thought that alteration in immunoglobulin output is affected more by changes in cell number rather than dynamic changes within individual cells’ secretory ability.

Lacrimal glands are extensively innervated by both the parasympathetic and sympathetic divisions of the autonomic nervous system. The neurotransmitters acetylcholine and norepinephrine are present in nerve fibers distributed among the secretory acini. Physiological studies using glands from a number of different species suggest that muscarinic acetylcholine receptors and both alpha and beta adrenergic receptors are present on the secretory acinar cells. There are no data, however, on the anatomic distribution of any neurotransmitter receptor in lacrimal glands. Given that the innervation density is sufficiently low so that it is unlikely that each acinar cell is directly innervated (see Walcott et al, this volume), it becomes even more important to determine the distribution of receptors in the glands in order to understand the control of lacrimal gland secretion.

Whole-cells, excised outside-in and outside-out membrane patches were employed to study the electrophysiological properties of plasma cells isolated from the Harderian (lacrimal) gland of chicken. The study revealed that the whole-cell currents are dominated by outward rectifying currents which display slow inactivation times of the order of seconds. Records from excised outside-in and outside-out patches consistently revealed one channel type, a maxi-K channel. These maxi-K channels were shown to be both voltage and calcium sensitive. The single channel conductance of the maxi-K channel, with KCl solutions on both sides of the patch, ranged from 200-265 pS (n = 26). Both whole-cell currents and single channel activity (outside-out) were reduced by the introduction of 10 mM TEA in the bath. The ease with which a large number of plasma cells can be isolated free of undifferentiated B-lymphocytes makes this preparation ideal for studying the relationship between the electrophysiological properties and immunoglobulin secretion in plasma cells.

One of the major constituents of the tear film is an aqueous fluid. It has been estimated that the cornea and conjunctiva produce up to about 20% of this fluid and that the main and accessory lacrimal glands supply the rest. The details of the mechanisms that move water across the secretory epithelium of the lacrimal glands are not well understood and neither are the ways by which these mechanisms are controlled. In this paper, we have attempted to pull together what is known about lacrimal gland fluid flow and to present a model, derived in part from our work on the cornea that suggests mechanisms responsible for water movement across the epithelium.

Particulate iron was found within the trophocytes of the fat body of the adult honey-bee. These iron granules differed in their structure and composition from iron granules found in other biological systems. The granules had an average diameter of 0·32 ± 0·07 μm and were composed of iron, calcium and phosphorus in a noncrystalline arrangement. The granules were apparently randomly distributed within the cytoplasm of the cells, and were not associated with any particular cellular organelle. Electron microscopy revealed the presence of cell junctions between the trophocytes. In tissues treated with colloidal lanthanum, 20-nm gaps were seen between the outer leaflets of the cells forming the cell junction. Physiological studies showed that these cells are electrically coupled, but the coupling ratio is low, as a result of extensive coupling to many cells.

Electrical synapses provide a basis for efficient signal transmission in a wide variety of nervous systems1. These synapses are composed of specialized cell-to-cell contacts known as nexuses2 or gap junctions3 which mediate the direct transfer of ions and small molecules between adjacent cell interiors1,4 by way of intercellular channels embedded in the junctional membrane5. The crayfish giant motor synapse (CMS) was the first cell-to-cell junction clearly demonstrated to operate by an electrical mechanism6. Current applied to the presynaptic lateral giant (LG) axon or to the neurite of the postsynaptic giant flexor motoneurone (MoG) spreads passively through the synapse into the adjacent neurone6,7. Each GMS behaves like an electrical rectifier: its conductance is high when LG is positive with respect to MoG, and decreases dramatically when the sign of the trans-synaptic voltage is reversed6,7. We have now examined GMS conductance and dye permeability at thoracic and abdominal levels of the crayfish nerve cord. At both levels, values of GMS chord conductance fit a simple Boltzmann model in which the conductance of individual synaptic channels is assumed to be voltage dependent. Moreover, thoracic synapses display higher limiting conductances than do those at an abdominal level, apparently as a result of their larger size. We also find that synapses at both locations are permeable to the fluorescent dye Lucifer yellow, even in conditions where electrical conductance is low. These results provide a framework for understanding the operation and functional limits of rectifying electrical synapses, and illustrate that dye permeability can be associated even with their relatively low conductance condition.

By quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, paramyosin:myosin heavy chain molecular ratios were calculated for three molluscan muscles:Aequipecten striated adductor, Mercenaria opaque adductor, and Mytilus anterior byssus retractor; and four arthropodan muscles:Limulus telson, Homarus slow claw. Balanus scutal depressor, and Lethocerus air tube retractor. These ratios correlate positively with both thick filament dimensions and maximum active tension development in these tissues. The role of paramyosin in these muscles is discussed with respect to the following characteristics: force development, "catch," and extreme reversible changes in length.

Antibody specific for Limulus paramyosin stains the A bands of Lethocerus flight muscle sarcomeres. SDS-polyacrylamide gels of both Limulus telson and Lethocerus flight myofibrillar preparations show bands with mobility similar to that of purified Limulus paramyosin.

1. Intracellular recordings were made from the retinula units of the crayfish (Cherax destructor) compound eyes in different states of light/dark adaptation. 2. 10 dark-adapted units had acceptance angles of 24° ±8 SD (Fig. 1). 3. 10 light-adapted units had acceptance angles of 4° ±2° SD (Fig. 2). 4. There was no correlation between the relative sensitivity of a unit to a point source of light and its acceptance angle (Fig 4). 5. There was a good correlation between the number of facets accepting light for a unit and its acceptance angle: The larger the acceptance angle of a unit, the more facets that were accepting the light (Fig. 5).

We show that the "escape response" (running) of the cockroach Periplaneta americana is interrupted when the insect runs through a shadow. Shadows were cast by holding an opaque plastic sheet so that the cockroach ran beneath it. In control experiments, a similarly positioned clear plastic sheet was ineffective. Removal of the shadow stimulus allowed running to continue. The likelihood that cockroaches would resume running after removal of the shadow stimulus decreased with the time they were allowed to remain in shadow.