Role of soluble guanylyl cyclase in renal afferent and efferent arterioles.

Renal arteriolar tone depends considerably on the dilatory action of nitric oxide (NO) via activation of soluble guanylyl cyclase (sGC) and cGMP action. NO deficiency and hypoxia/reoxygenation are important pathophysiological factors in the development of acute kidney injury. It was hypothesized that the NO-sGC-cGMP system functions differently in renal afferent arterioles (AA) compared with efferent arterioles (EA) and that the sGC activator cinaciguat differentially dilates these arterioles. Experiments were performed in isolated, perfused mouse glomerular arterioles. Hypoxia (0.1% oxygen) was achieved by using a hypoxia chamber. Phosphodiesterase 5 (PDE5) and sGC subunits were considerably expressed on the mRNA level in AA. PDE5 inhibition with sildenafil, which blocks cGMP degradation, diminished the responses to ANG II bolus application in AA, but not significantly in EA. Vasodilation induced by sildenafil in ANG II-preconstricted vessels was stronger in EA than AA. Cinaciguat, an NO- and heme-independent sGC activator, dilated EA more strongly than AA after NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) treatment and preconstriction with ANG II. Cinaciguat-induced dilatation of l-NAME-pretreated and ANG II-preconstricted arterioles was similar to controls without l-NAME treatment. Cinaciguat also induced dilatation in iodinated contrast medium treated AA. Furthermore, it dilated EA, but not AA, after hypoxia/reoxygenation. The results reveal an important role of the NO-sGC-cGMP system for renal dilatation and that EA have a more potent sGC activated dilatory system. Furthermore, AA seem to be more sensitive to hypoxia/reoxygenation than EA under these experimental conditions.

Endothelial prostaglandin D2 opposes angiotensin II contractions in mouse isolated perfused intracerebral microarterioles.

HYPOTHESIS: A lack of contraction of cerebral microarterioles to Ang II ("resilience") depends on cyclooxygenase (COX) and lipocalin type prostaglandin D sythase L-PGDS producing PGD2 that activates prostaglandin D type 1 receptors (DP1Rs) and nitric oxide synthase (NOS). MATERIALS & METHODS: Contractions were assessed in isolated, perfused vessels and NO by fluorescence microscopy. RESULTS: The mRNAs of penetrating intraparenchymal cerebral microarterioles versus renal afferent arterioles were >3000-fold greater for L-PGDS and DP1R and 5-fold for NOS III and COX 2. Larger cerebral arteries contracted with Ang II. However, cerebral microarterioles were entirely unresponsive but contracted with endothelin 1 and perfusion pressure. Ang II contractions were evoked in cerebral microarterioles from COX1 -/- mice or after blockade of COX2, L-PGDS or NOS and in deendothelialized vessels but effects of deendothelialization were lost during COX blockade. NO generation with Ang II depended on COX and also was increased by DP1R activation. CONCLUSION: The resilience of cerebral arterioles to Ang II contractions is specific for intraparenchymal microarterioles and depends on endothelial COX1 and two products that are metabolized by L-PGDS to generate PGD2 that signals via DP1Rs and NO.

Extravasal albumin concentration modulates contractile responses of renal afferent arterioles.

AIM: Afferent arterioles (AA) hold a key position in the regulation of renal blood flow and glomerular filtration rate. Being the effector site of tubuloglomerular feedback, the afferent arteriole contributes to the renal handling of sodium and fluid. Dehydration goes along with increased renal interstitial protein concentration. Here, the hypothesis was tested that extravasal protein concentration directly modulates afferent arteriolar tone, a mechanism which may contribute to body fluid volume control. METHOD: The effect of increased extravasal albumin concentration on the vascular reactivity was investigated in renal AA and interlobar arteries of mice, in rat renal AA and in pancreatic islet arterioles. RESULTS: Albumin (2 and 4% in the bath solution) significantly potentiated the contractile response of renal afferent arterioles induced by angiotensin II and adenosine, as well as their combination, compared to the control situation (0.1% albumin). Albumin did not influence the contractility of larger renal vessels or pancreatic islet arterioles. Mimicking the increase in the osmolality induced by 4% albumin by applying mannitol to the bath solution also increased the response of renal arterioles to Ang II. However, the effect was smaller compared to that of albumin. The nitric oxide bioavailability, measured by DAF-FM fluorescence, was reduced in afferent arterioles exposed to 4% albumin. CONCLUSION: The protein-induced modulation of AA tone is mediated by the increased osmolality as well as by NO scavenging. The results suggest a possible contribution of these mechanisms to the control of extracellular fluid volume via adjustment of renal blood flow and glomerular filtration rate.

Myoglobin facilitates angiotensin II-induced constriction of renal afferent arterioles.

Vasoconstriction plays an important role in the development of acute kidney injury in rhabdomyolysis. We hypothesized that myoglobin enhances the angiotensin II (ANG II) response in afferent arterioles by increasing superoxide and reducing nitric oxide (NO) bioavailability. Afferent arterioles of C57Bl6 mice were isolated perfused, and vasoreactivity was analyzed using video microscopy. NO bioavailability, superoxide concentration in the vessel wall, and changes in cytosolic calcium were measured using fluorescence techniques. Myoglobin treatment (10-5 M) did not change the basal arteriolar diameter during a 20-min period compared with control conditions. NG-nitro-l-arginine methyl ester (l-NAME, 10-4 M) and l-NAME + myoglobin reduced diameters to 94.7 and 97.9% of the initial diameter, respectively. Myoglobin or l-NAME enhanced the ANG II-induced constriction of arterioles compared with control (36.6 and 34.2%, respectively, vs. 65.9%). Norepinephrine responses were not influenced by myoglobin. Combined application of myoglobin and l-NAME further facilitated the ANG II response (7.0%). Myoglobin or l-NAME decreased the NO-related fluorescence in arterioles similarly. Myoglobin enhanced the superoxide-related fluorescence, and tempol prevented this enhancement. Tempol also partly prevented the myoglobin effect on the ANG II response. Myoglobin increased the fura 2 fluorescence ratio (cytosolic calcium) during ANG II application (10-12 to 10-6 M). The results suggest that the enhanced afferent arteriolar reactivity to ANG II is mainly due to a myoglobin-induced increase in superoxide and associated reduction in the NO bioavailability. Signaling pathways for the augmented ANG II response include enhanced cytosolic calcium transients. In conclusion, myoglobin may contribute to the afferent arteriolar vasoconstriction in this rhabdomyolysis model.

c-Jun N-terminal Kinase mediates prostaglandin-induced sympathoexcitation in rats with chronic heart failure by reducing GAD1 and GABRA1 expression.

AIM: Prostaglandin E2 mediates sympathoexcitation in chronic heart failure (CHF) through EP3 receptors (PTGER3) in the paraventricular nucleus (PVN). The aim of this study was to investigate the role of c-Jun N-terminal kinase (JNK) in expressional regulation of gamma-aminobutyric acid signalling in PVN in CHF rats. METHODS: Chronic heart failure was induced by left coronary ligation in Wistar rats. Renal sympathetic nerve discharge (RSND) and mean arterial pressure (MAP) responses to the PVN infusion were determined in anaesthetized rats. Osmotic minipumps were used for chronic PVN infusion. PTGER3 expression was examined with immunofluorescence staining, quantitative real-time PCR and Western blot. RESULTS: Chronic heart failure rats had increased JNK activation and decreased glutamate decarboxylase 1 (GAD1) and GABAA receptor alpha 1 subunit (GABRA1) expression in the PVN. PVN infusion of the PTGER3 agonist SC-46275 caused sympathoexcitation in sham-operated control (Sham) rats and increased it further in CHF. The PTGER3 antagonist L798106 reduced sympathoexcitation and cardiac dysfunction in CHF. PVN infusion of EP1 receptor antagonist SC-19220, EP2 receptor antagonist AH6809 or EP4 receptor antagonist L-161982 had no effect on sympathoexcitation. The JNK inhibitor SP600125 normalized sympathoexcitation and GAD1 and GABRA1 expression in PVN in CHF rats. Both the p44/42 and p38 mitogen-activated protein kinase inhibitors PD98059 and SB203580 could not prevent the downregulation of GAD1 and GABRA1 expression in PVN in CHF. PTGER3 agonist activated JNK but downregulated GAD1 and GABRA1 expression in NG108 neuronal cells. CONCLUSION: Prostaglandin signalling through upregulated PTGER3 activates JNK which reduces GAD1 and GABRA1 expression in the PVN, and contributes to sympathoexcitation in CHF.

High-salt diet induces outward remodelling of efferent arterioles in mice with reduced renal mass.

AIM: The glomerular filtration rate (GFR) falls progressively in chronic kidney disease (CKD) which is caused by a reduction in the number of functional nephrons. The dysfunctional nephron exhibits a lower glomerular capillary pressure that is induced by an unbalance between afferent and efferent arteriole. Therefore, we tested the hypothesis that oxidative stress induced by CKD differentially impairs the structure or function of efferent vs. afferent arterioles. METHODS: C57BL/6 mice received sham operations (sham) or 5/6 nephrectomy (RRM) and three months of normal- or high-salt diet or tempol. GFR was assessed from the plasma inulin clearance, arteriolar remodelling from media/lumen area ratio, myogenic responses from changes in luminal diameter with increases in perfusion pressure and passive wall compliance from the wall stress/strain relationships. RESULTS: Mice with RRM fed a high salt (vs. sham) had a lower GFR (553 ± 25 vs. 758 ± 36 µL min-1  g-1 kidney, P < 0.01) and a larger efferent arteriolar diameter (9.6 ± 0.8 vs. 7.4 ± 0.7 µm, P < 0.05) resulting in a lower media/lumen area ratio (1.4 ± 0.1 vs. 2.4 ± 0.2, P < 0.01). These alterations were corrected by tempol. The myogenic responses of efferent arterioles were about one-half that of afferent arterioles and were unaffected by RRM or salt. Passive wall compliance was reduced by high salt in both afferent and efferent arterioles. CONCLUSION: A reduction in renal mass with a high-salt diet induces oxidative stress that leads to an outward eutrophic remodelling in efferent arterioles and reduced wall compliance in both afferent and efferent arterioles. This may contribute to the lower GFR in this model of CKD.

Increased hydrogen peroxide impairs angiotensin II contractions of afferent arterioles in mice after renal ischaemia-reperfusion injury.

AIM: Renal ischaemia-reperfusion injury (IRI) increases angiotensin II (Ang II) and reactive oxygen species (ROS) that are potent modulators of vascular function. However, the roles of individual ROS and their interaction with Ang II are not clear. Here we tested the hypothesis that IRI modulates renal afferent arteriolar responses to Ang II via increasing superoxide (O2-) or hydrogen peroxide (H2 O2 ). METHODS: Renal afferent arterioles were isolated and perfused from C57BL/6 mice 24 h after IRI or sham surgery. Responses to Ang II or noradrenaline were assessed by measuring arteriolar diameter. Production of H2 O2 and O2- was assessed in afferent arterioles and renal cortex. Activity of SOD and catalase, and mRNA expressions of Ang II receptors were assessed in pre-glomerular arterioles and renal cortex. RESULTS: Afferent arterioles from mice after IRI had a reduced maximal contraction to Ang II (-27±2 vs. -42±1%, P < 0.001), but retained a normal contraction to noradrenaline. Arterioles after IRI had a 38% increase in H2 O2 (P < 0.001) and a 45% decrease in catalase activity (P < 0.01). Contractions were reduced in normal arterioles after incubation with H2 O2 (-22±2 vs. -42±1%, P < 0.05) similar to the effects of IRI. However, the impaired contractions were normalized by incubation with PEG catalase despite a reduced AT1 R expression. CONCLUSIONS: Renal IRI in mice selectively impairs afferent arteriolar responses to Ang II because of H2 O2 accumulation that is caused by a reduced catalase activity. This could serve to buffer the effect of Ang II after IRI and may be a protective mechanism.

Oxidative status in the macula densa modulates tubuloglomerular feedback responsiveness in angiotensin II-induced hypertension.

AIM: Tubuloglomerular feedback (TGF) is an important mechanism in control of signal nephron glomerular filtration rate. The oxidative stress in the macula densa, primarily determined by the interactions between nitric oxide (NO) and superoxide (O2-), is essential in maintaining the TGF responsiveness. However, few studies examining the interactions between and amount of NO and O2- generated by the macula densa during normal and hypertensive states. METHODS: In this study, we used isolated perfused juxtaglomerular apparatus to directly measure the amount and also studied the interactions between NO and O2- in macula densa in both physiological and slow pressor Angiotensin II (Ang II)-induced hypertensive mice. RESULTS: We found that slow pressor Ang II at a dose of 600 ng kg(-1) min(-1) for two weeks increased mean arterial pressure by 26.1 ± 5.7 mmHg. TGF response increased from 3.4 ± 0.2 µm in control to 5.2 ± 0.2 µm in hypertensive mice. We first measured O2- generation by the macula densa and found it was undetectable in control mice. However, O2- generation by the macula densa increased to 21.4 ± 2.5 unit min(-1) in Ang II-induced hypertensive mice. We then measured NO generation and found that NO generation by the macula densa was 138.5 ± 9.3 unit min(-1) in control mice. The NO was undetectable in the macula densa in hypertensive mice infused with Ang II. CONCLUSIONS: Under physiological conditions, TGF response is mainly controlled by the NO generated in the macula densa; in Ang II induced hypertension, the TGF response is mainly controlled by the O2- generated by the macula densa.

Diadenosine pentaphosphate modulates glomerular arteriolar tone and glomerular filtration rate.

INTRODUCTION: Mechanisms and participating substances involved in the reduction of glomerular filtration (GFR) in contrast-induced acute kidney injury (CI-AKI) are still matter of debate. We hypothesized that diadenosine polyphosphates are released by the action of contrast media on tubular cells and may act on glomerular arterioles and reduce GFR. METHODS: Freshly isolated rat tubules were treated with the contrast medium iodixanol (47 mg iodine per mL) at 37 °C for 20 min. The content of Apn A (n = 3-6) in the supernatant of treated tubules and in the plasma of healthy persons and patients with AKI was analysed using reversed-phase chromatography, affinity chromatography and mass spectrometry. GFR was obtained in conscious mice by inulin clearance. Concentration response curves for Apn A (n = 3-6, 10(-12) -10(-5)  mol L(-1) ) were measured in isolated perfused glomerular arterioles. RESULTS: Iodixanol treatment of tubules significantly increased the concentration of Apn A (n = 3-5) in the supernatant. Ap6 A was below the detection limit. AKI patient shows higher concentrations of Apn A compared to healthy. Application of Ap5 A significantly reduced the GFR in conscious mice. Ap5 A reduced afferent arteriolar diameters, but did not influence efferent arterioles. The constrictor effect on afferent arterioles was strong immediately after application, but weakened with time. Then, non-selective P2 inhibitor suramin blocked the Ap5 A-induced constriction. CONCLUSION: The data suggest that Ap5 A plays a role in the pathophysiology of CI-AKI. We show a contrast media-induced release of Ap5 A from tubules, which might increase afferent arteriolar resistance and reduce the GFR.

Angiotensin II enhances the afferent arteriolar response to adenosine through increases in cytosolic calcium.

AIMS: Angiotensin II (Ang II) is a strong renal vasoconstrictor and modulates the tubuloglomerular feedback (TGF). We hypothesized that Ang II at low concentrations enhances the vasoconstrictor effect of adenosine (Ado), the mediator of TGF. METHODS: Afferent arterioles of mice were isolated and perfused, and both isotonic contractions and cytosolic calcium transients were measured. RESULTS: Bolus application of Ang II (10(-12) and 10(-10) M) induced negligible vasoconstrictions, while Ang II at 10(-8) m reduced diameters by 35%. Ang II at 10(-12), 10(-10) and 10(-8) m clearly enhanced the arteriolar response to cumulative applications of Ado (10(-11) to 10(-4) M). Ado application increased the cytosolic calcium concentrations in the vascular smooth muscle, which were higher at 10(-5) M than at 10(-8) M. Ang II (10(-11) to 10(-6) M) also induced concentration-dependent calcium transients, which were attenuated by AT(1) receptor inhibition. Simultaneously applied Ang II (10(-10) M) additively enhanced the calcium transients induced by 10(-8) and 10(-5) M Ado. The transients were partly inhibited by AT(1) or A(1) receptor antagonists, but not significantly by A(2) receptor antagonists. CONCLUSION: A low dose of Ang II enhances Ado-induced constrictions, partly via AT(1) receptor-mediated calcium increase. Ado increases intracellular calcium by acting on A(1) but not A(2) receptors. The potentiating effect of Ang II on Ado-induced arteriolar vasoconstrictions may involve calcium sensitization of the contractile machinery, as Ang II only additively increased cytosolic calcium concentrations, while its effect on the arteriolar constriction was more than additive. The potentiating effect of Ang II might contribute to the resetting of TGF.

Adenosine increases calcium sensitivity via receptor-independent activation of the p38/MK2 pathway in mesenteric arteries.

AIM: Adenosine (Ado) restores desensitized angiotensin II-induced contractions in the renal arterioles via an intracellular, receptor-independent mechanisms including the p38 mitogen-activated protein kinase (MAPK). In the present study we test the hypothesis that MAPK-activated protein kinase 2 (MK2) mediates the Ado effect downstream from p38 MAPK resulting in an increased phosphorylation of the regulatory unit of the myosin light chain (MLC(20)). METHODS AND RESULTS: Contraction experiments were performed in rings of mesenteric arteries under isometric conditions in C57BL6 and MK2 knock out mice (MK2-/-). Ado pretreatment (10(-5) mol L(-1)) strongly increased Ang II sensitivity, calcium sensitivity and the phosphorylation of MLC(20). Treatment with Ado (3 x 10(-6) or 10(-5) mol L(-1) in between successive Ang II applications) enhanced the desensitized Ang II responses (second to fifth application). Ca(2+) transients were not effected by Ado. Further, blockade of type 1 and type 2 Ado receptors during treatment did not influence the effect. Type 3 receptor activation by inosine instead of Ado had no effect. Conversely, inhibition of nitrobenzylthioinosine-sensitive Ado transporters prevented the effects of Ado. Inhibition of p38 MAPK as well as use of MK2-/- mice prevented contractile Ado effects on the mesenteric arteries and the phosphorylation of MLC(20). CONCLUSION: The study shows that Ado activates the p38 MAPK/MK2 pathway in vascular smooth muscle via an intracellular action, which results in an increased MLC(20) phosphorylation in concert with increased calcium sensitivity of the contractile apparatus. This mechanism can significantly contribute to the regulation of vascular tone, e.g. under post-ischaemic conditions.

Contribution of adenosine receptors in the control of arteriolar tone and adenosine-angiotensin II interaction.

Adenosine (Ado) mediates vasoconstriction via A(1)-Ado receptors and vasodilation via A(2)-Ado receptors in the kidney. It interacts with angiotensin II (Ang II), which is important for renal hemodynamics and tubuloglomerular feedback (TGF). The aim was to investigate the function of Ado receptors in the Ado-Ang II interaction in mouse microperfused, afferent arterioles. Ado (10(-11)-10(-4) mol/l) caused a biphasic response: arteriolar diameters were reduced (-7%) at Ado 10(-11)-10(-9) mol/l and returned to control values at higher concentrations. Treatment with Ang II (10(-10) mol/l) transformed the response into a concentration-dependent constriction. N(6)-cyclopentyladenosine (A(1)-Ado receptor agonist) reduced diameters (12% at 10(-6) mol/l). Application of CGS21680 (10(-12)-10(-4) mol/l, A(2A) receptor agonist) increased the diameter by 13%. Pretreatment with ZM241385 (A(2A)-Ado receptor antagonist) alone or in combination with MRS1706 (A(2B)-Ado receptor antagonist) resulted in a pure constriction upon Ado, whereas 8-cyclopentyltheophylline (CPT) (A(1)-Ado receptor antagonist) inhibited the constrictor response. Afferent arterioles of mice lacking A(1)-Ado receptor did not show constriction upon Ado. Treatment with Ado (10(-8) mol/l) increased the response upon Ang II, which was blocked by CPT. Ado (10(-5) mol/l) did not influence the Ang II response, but an additional blockade of A(2)-Ado receptors enhanced it. The action of Ado on constrictor A(1)-Ado receptors and dilatory A(2)-Ado receptors modulates the interaction with Ang II. Both directions of Ado-Ang II interaction, which predominantly leads to an amplification of the contractile response, are important for the operation of the TGF.

Nitric oxide counteracts angiotensin II induced contraction in efferent arterioles in mice.

AIM: Efferent arterioles (Ef) are one of the final control elements in glomerular haemodynamics. The influence of nitric oxide (NO) on Ef remains ambiguous. METHODS: To test the hypothesis that endothelial NO plays an important role in this context, afferent arterioles (Af) and Ef of wild-type mice (WT), and Ef of mice lacking the endothelial NO synthetase [eNOS(-/-)] were perfused. Perfusion was performed in Ef via Af (orthograde) as well as from the distal end of Ef (retrograde), which provides an estimate for the importance of substances derived from the glomerulus. Angiotensin II (Ang II) was added in doses ranging from 10(-12) to 10(-6) mol L(-1) to the bath solution. RESULTS: Ang II reduced the luminal diameter of Af to 68 +/- 7 and in Ef to 55 +/- 8% during orthograde, and to 35 +/- 6% during retrograde perfusion (10(-6) mol L(-1) Ang II) in WT. Pre-treatment with N(G)-Nitro-L-arginine-methylester (l-NAME) (10(-4) mol L(-1)) increased the Ang II sensitivity in retrograde (17 +/- 9%) and orthograde perfused Ef (19 +/- 9%). The Ang II sensitivity was enhanced in eNOS(-/-) mice compared with WT, too. Already at a dose of Ang II 10(-9) mol L(-1), luminal diameters diminished to 8 +/- 7 and 7 +/- 4%. CONCLUSION: The increased Ang II sensitivity during L-NAME pre-treatment and in eNOS(-/-) mice indicates a strong counteraction of endothelial derived NO on Ang II induced contraction in Ef. Moreover, Ef are similarly sensitive to Ang II during either retrograde or orthograde perfusion in the absence of NO effects, suggesting that NO mediates, at least in part, the action of potential vasodilatory substances from the glomerulus.

Centrin is synthesized and assembled into basal bodies during Naegleria differentiation.

During differentiation of Naegleria from vegetative amoebae to temporary flagellates, the microtubular cytoskeleton, including two basal bodies and flagella, is assembled de novo. Centrin is an integral component of these basal bodies [Levy et al., 1996, Cell Motil. Cytoskeleton 33: 298-323]. In many organisms, centrin appears to be a constitutive protein, but in Naegleria centrin gene expression occurs only during differentiation. Centrin mRNA, which has not been detected in amoebae, appears and disappears earlier in differentiation than a coordinately regulated set of differentiation-specific mRNAs encoding flagellar tubulin and calmodulin. Centrin antigen accumulates during differentiation, and then decreases in abundance as the flagellates mature and revert to amoebae. No localization of centrin has been detected in amoebae. During differentiation, centrin becomes localized to the basal bodies as soon as these structures are detected with anti-tubulin antibodies, first as a single dot and finally as two basal bodies. During reversion of flagellates to amoebae, centrin remains localized to the basal bodies for as long as they are present. When assembly of tubulin-containing structures during differentiation is prevented using oryzalin, centrin localization is prevented as well, yet inhibition of assembly does not affect accumulation of centrin antigen. Apparently in Naegleria, the role of centrin is primarily for a differentiation- or flagellate-specific function. The temporary presence of centrin is concurrent with the presence of centriolar basal bodies, which supports the conjecture that in Naegleria centrin may be needed only when these organelles are present.

Stable intermediates and holdpoints in the rapid differentiation of Naegleria.

The rapidity of the optional 90-min differentiation of Naegleria gruberi from amoebae to flagellates suggests the possibility of a free-running cascade of events from initiating stimulus through gene expression to organelle assembly and cell morphogenesis. Instead our experiments reveal two points early in the differentiation at which the strength of the inducing stimulus is reevaluated by the cells. Two new physical start signals for differentiation, temperature downshift (DeltaT) and mechanical agitation, are shown to regulate differentiation synergistically with each other and with previously defined signals. A DeltaT of -10 degrees C induces complete differentiation directly in the growth environment, whereas smaller DeltaTs initiate differentiation and allow it to progress for a short time, after which the cells "hold" for up to 4 h, awaiting a stimulus to continue differentiation. Our work defines two "holdpoints," optional points in development where progress can stop, awaiting a suitable signal, while cells retain whatever intermediates represent progress. We propose that such holdpoints, which can be detected in this system because of the temporal reproducibility of the differentiation, are likely to be found in other differentiating cells.

Centrin is a conserved protein that forms diverse associations with centrioles and MTOCs in Naegleria and other organisms.

Centrin, a approximately or equal to 20 kDa calcium-binding protein also known as caltractin, is a component of centrosome-associated algal flagellar roots capable of calcium-mediated contraction, and is also found in the centrosomes of vertebrate cells. Our analysis of a centrin gene from a protist, the amoeboflagellate Naegleria gruberi, reveals conserved features that distinguish centrins from calmodulin. Antibodies to bacterially expressed Naegleria centrin, which also recognize yeast Cdc31p, were employed to localize centrin immunoreactivity in selected organisms possessing specialized microtubule-organizing centers (MTOCs) or accessory structures. There is a striking morphological diversity of such structures. In the simplest associations, as found in Naegleria flagellates and vertebrates tracheal epithelium, centrin is intimately associated with the cylinder of the basal bodies. In cells with unfocused mitotic spindles, Naegleria amoebae and onion root tips, no localization of centrin was detected. In Dictyostelium discoideum and Saccharomyces cerevisiae, which lack centrioles, centrin immunoreactivity was observed as punctate cytoplasmic bodies but not associated with spindle pole MTOCs. In Paramecium multimicronucleatum, centrin immunoreactivity is localized to the infraciliary lattice, previously shown to exhibit calcium-mediated contraction. In Vorticella microstoma, known for the calcium-induced rapid contraction of its stalk, centrin immunoreactivity is localized to the contractile spasmoneme and myonemes. Similar antigens from Paramecium and Vorticella are detected by anti-centrin and anti-spasmin. The pattern of localization of centrin immunoreactivity supports the conjecture that a contractile system involving centrin, initially associated with centriolar structures, was recruited during evolution to build specialized organelles in different organisms and cell types.

A flagellar calmodulin gene of Naegleria, coexpressed during differentiation with flagellar tubulin genes, shares DNA, RNA, and encoded protein sequence elements.

Two calmodulins are synthesized during differentiation of Naegleria gruberi from amoebae to flagellates; one remains in the cell body and the other becomes localized in the flagella. The single, intronless, expressed gene for flagellar calmodulin has been cloned and sequenced. The encoded protein is a typical calmodulin with four putative calcium-binding domains, but it has an amino-terminal extension of 10 divergent amino acids preceding conserved calmodulin residue 4. The transcripts encoding flagellar calmodulin and flagellate cell body calmodulin are clearly divergent. Expression of the flagellar calmodulin gene is differentiation-specific; its mRNA appears and then disappears concurrently with those encoding flagellar alpha- and beta-tubulin. Three provocative sequence elements are shared among these unrelated coexpressed genes: (i) a palindromic DNA sequence element is found in duplicate or triplicate upstream to each transcribed region; (ii) a perfect 12-nucleotide match is found near the AUG start codon of flagellar calmodulin and alpha-tubulin; and (iii) the novel amino-terminal extension of flagellar calmodulin contains a 5-amino-acid element similar to the amino terminus of flagellar alpha-tubulin. These shared sequence elements are proposed to have roles in differentiation, possibly in regulation of transcription, mRNA stability, and localization of these proteins to flagella.

A calcineurin-B-encoding gene expressed during differentiation of the amoeboflagellate Naegleria gruberi contains two introns.

One of two similar genes in the unicellular eukaryote Naegleria gruberi is shown to encode calcineurin B (CnB), the regulatory subunit of calcium-calmodulin-regulated protein phosphatase 2B. Over a span of 156 amino acids, excluding divergent N-termini, the encoded sequence shows 62% identity with vertebrate CnB, and also shows sequence elements specific, among calcium-binding proteins, to CnB. In contrast, the sequence shows only 23% identity with N. gruberi flagellar calmodulin. CNB mRNA is readily detected in amoebae; its abundance increases fourfold during differentiation to flagellates, reaches a peak at 50-70 min, when flagella are forming, and then declines. A genomic clone matches an expressed cDNA, except that it is interrupted by two phase I introns. The position of one intron, which separates the divergent N-terminal domain from the four calcium-binding domains (EF hands), is shared with a yeast CNB gene; the other is located in the central helix between the two pairs of calcium-binding loops; features that support an ancient origin. These introns, the first found in protein-coding genes of Naegleria, are flanked by characteristic splice junction sequences. N. gruberi CnB also shares similarities with recoverins. The finding in a protist of a CNB gene that contains two introns separating functional domains, shares similarities to recoverins and shows increased expression during differentiation is provocative. If the phylogeny of major groups derived from ribosomal RNA is accepted, Naegleria is among the earliest branching eukaryotes known to contain canonical pre-mRNA introns.

[Continuing hospice care of cancer--a three-year experience].

The hospice at Mackay Memorial Hospital was established in February 1990. A group of team workers including physicians, nurses, social workers and the clergy were involved in this holistic care program for terminal cancer patients. Four hundred and seventy-nine patients were eligible for the program up to February 1993. Regarding duration of stay, 62.5% of patients resided for 14 days. Those surviving under 90 days constituted 75.5% of patients. Fifty-one point eight percent of patients died in the hospice and 18.2% died at home soon after being discharged from the hospice. Pain is the most common symptom among the patients. Treatment strategies vary according to the three-step-ladder protocol designed by WHO. Total pain relief was achieved in 80% of patients. Opportune private talking and family conferences formed the basis of the "peer model". Through this model, treatment decisions including physical, psychosocial and spiritual issues were made. Before the peer model, only 36 (10.3%) patients agreed with the idea of hospice care, while 257 (73.6%) patients agreed after the model was established. Awareness of dying was evident in 412 (86%) patients. Two hundred and eighty (68%) patients became aware of the prospect of death through guessing, while the other 132 (32%) patients were informed by medical staff. Problems encountered by the team workers included 1) needs in education and training, 2) psychological pressure, 3) management of loss and grief, 4) needs in supportive system and 5) troubles caused by families' lying to patients. The team workers were satisfied with the quality of care in 38.4% of patients and fairly satisfied with 30.7% of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

A beta-tubulin gene of Naegleria encodes a carboxy-terminal tyrosine. Aromatic amino acids are conserved at carboxy termini.

A gene that directs the programmed synthesis of flagellar beta-tubulin during the rapid differentiation of Naegleria gruberi from amoebae to flagellates has been cloned and sequenced. The intronless gene is one of 8 to 10 similar but non-identical genes that are dispersed in the genome. beta-Tubulin mRNA homologous to this gene family is expressed transiently during differentiation, and has not been detected in amoebae. The encoded beta-tubulin is strongly conserved, with features that closely resemble the beta-tubulins of diverse organisms, especially organisms that, like Naegleria, use tubulin to assemble flagellar axonemes. In most sequenced alpha-tubulins, the encoded carboxy-terminal amino acid is tyrosine, which undergoes post-translational removal and readdition, conserved processes of unknown function. In N. gruberi, unusually, the terminus of alpha-tubulin is encoded as glutamine while that of beta-tubulin is tyrosine. The presence of these divergent termini on subunits of a conserved tubulin provoked us to re-examine aromatic amino acids at the termini of alpha- and beta-tubulins. Although evolution has tinkered extensively with the carboxy-terminal domains of tubulin subunits, we find an unexpected conservation. In every organism or cell type for which both tubulin subunits have been sequenced, except the ciliate Stylonychia lemnae, at least one tubulin subunit of some or all tubulin heterodimers terminates in an aromatic amino acid, either tyrosine or phenylalanine. This remarkable conservation of carboxy-terminal aromatic amino acids suggests that these residues serve some crucial function.