Identification of genetic factors contributing to heterosis in a hybrid from two elite maize inbred lines using molecular markers.

The use of molecular markers to identify quantitative trait loci (QTLs) affecting agriculturally important traits has become a key approach in plant genetics-both for understanding the genetic basis of these traits and to help design novel plant improvement programs. In the study reported here, we mapped QTLs (and evaluated their phenotypic effects) associated with seven major traits (including grain yield) in a cross between two widely used elite maize inbred lines, B73 and Mo17, in order to explore two important phenomena in maize genetics-heterosis (hybrid vigor) and genotype-by-environment (G x E) interaction. We also compared two analytical approaches for identifying QTLs, the traditional single-marker method and the more recently described interval-mapping method. Phenotypic evaluations were made on 3168 plots (nearly 100,000 plants) grown in three states. Using 76 markers that represented 90-95% of the maize genome, both analytical methods showed virtually the same results in detecting QTLs affecting grain yield throughout the genome, except on chromosome 6. Fewer QTLs were detected for other quantitative traits measured. Whenever a QTL for grain yield was detected, the heterozygote had a higher phenotype than the respective homozygote (with only one exception) suggesting not only overdominance (or pseudooverdominance) but also that these detected QTLs play a significant role in heterosis. This conclusion was reinforced by a high correlation between grain yield and proportion of heterozygous markers. Although plant materials were grown and measured in six diverse environments (North Carolina, Iowa and Illinois) there was little evidence for G x E interaction for most QTLs.

In vitro and in vivo inhibition of complement activity by a single-chain Fv fragment recognizing human C5.

Complement activation has been implicated in the pathogenesis of several human diseases. Recently, a monoclonal antibody, (N19-8) that recognizes the human complement protein C5 has been shown to effectively block the cleavage of C5 into C5a and C5b, thereby blocking terminal complement activation. In this study, a recombinant N19-8 scFv antibody fragment was constructed from the N19-8 variable regions, and produced in both mammalian and bacterial cells. The N19-8 scFv bound human C5 and was as potent as the N19-8 monoclonal antibody at inhibiting human C5b-9-mediated hemolysis of chicken erythrocytes. In contrast, the N19-8 scFv only partially retained the ability of the N19-8 monoclonal antibody to inhibit C5a generation. To investigate the ability of the N19-8 scFv to inhibit complement-mediated tissue damage, complement-dependent myocardial injury was induced in isolated mouse hearts by perfusion with Krebs-Henseleit buffer containing 6% human plasma. The perfused hearts sustained extensive deposition of human C3 and C5b-9, resulting in increased coronary artery perfusion pressure, end-diastolic pressure, and a decrease in heart rate until the hearts ceased beating approximately 10 min after addition of plasma. Hearts treated with human plasma supplemented with either the N19-8 monoclonal antibody or the N19-8 scFv did not show any detectable changes in cardiac performance for at least 1 hr following the addition of plasma. Hearts treated with human plasma alone showed extensive deposition of C3 and C5b-9, while hearts treated with human plasma containing N19-8 scFv showed extensive deposition of C3, but no detectable deposition of C5b-9. Administration of a 100 mg bolus dose of N19-8 scFv to rhesus monkeys inhibited the serum hemolytic activity by at least 50% for up to 2 hr. Pharmacokinetic analysis of N19-8 scFv serum levels suggested a two-compartment model with a T1/2 alpha of 27 min. Together, these data suggest the recombinant N19-8 scFv is a potent inhibitor of the terminal complement cascade and may have potential in vivo applications where short duration inhibition of terminal complement activity is desirable.

Sympathetic nervous system mediation of acute cardiovascular actions of gamma 2-melanocyte-stimulating hormone.

Peptides of the pro-opiocortin class produce pronounced cardiovascular and natriuretic actions. We have investigated the acute cardiovascular effects of one of the most potent members of this class, gamma 2-melanocyte stimulating hormone (gamma 2-MSH), in rats. Pressor actions of gamma 2-MSH administered systemically were eliminated by ganglionic blockade with chlorisondamine. Peripheral cholinergic blockade failed to affect either the pressor or cardioaccelerator responses to gamma 2-MSH. Administration of gamma 2-MSH (2.0-10.0 micrograms) produced vasoconstriction primarily in the mesenteric and hindlimb vascular beds, while the renal bed showed little response. Infusions of phenylephrine produced pressor responses similar to those found with gamma 2-MSH, which were accompanied by a decrease in heart rate and vasoconstriction in the mesenteric and renal vascular beds. Hemodynamic changes produced by gamma 2-MSH and phenylephrine were blocked or attenuated by alpha 1-adrenergic receptor blockade with prazosin. Direct injection of gamma 2-MSH into the renal artery produced an acute renal vasoconstriction that was not attenuated by alpha 1-adrenergic or ganglionic blockade. These findings and the results of previous publications are consistent with the hypothesis that gamma 2-MSH may produce a centrally mediated activation of the sympathetic nervous system, have direct vasoconstriction actions on the renal vasculature, and inhibit baroreceptor function to produce an increase in blood pressure without an accompanying bradycardia.

Rapid expression of an anti-human C5 chimeric Fab utilizing a vector that replicates in COS and 293 cells.

Inhibition of complement system activation requires the development of soluble nonimmunogenic inhibitors with good tissue penetrating abilities that are themselves unable to activate complement. Chimeric mouse/human Fabs capable of blocking the activity of complement proteins are likely to fulfill these criteria. Several monoclonal antibodies that inhibit the activation of the human complement system have recently been developed. To examine the properties of chimeric Fab derived from these monoclonal antibodies, we have developed an expression system which allows the rapid production of milligram quantities of chimeric Fab. Both the chimeric light chain and the chimeric Fd were co-expressed from the same vector, pAPEX-3P. This vector contains the SV40 origin of replication, which allows the rapid production of chimeric Fab in COS cells for preliminary characterization. Additionally, pAPEX-3P contains the Epstein-Barr virus origin of replication and a puromycin selectable marker for maintenance as a stable episome in human cell lines. A production system consisting of transfected 293-EBNA cells cultured in serum free medium followed by protein G-Sepharose chromatography of the conditioned medium was found to be sufficient for the rapid production of purified chimeric Fab. Here we have utilized this expression system to demonstrate that an anti-human C5 chimeric Fab was a potent inhibitor of complement activation in both in vitro activation assays and an ex vivo model of complement-mediated tissue damage.

Monoclonal antibodies directed against human C5 and C8 block complement-mediated damage of xenogeneic cells and organs.

The hyperacute rejection (HAR) of xenotransplanted organs is initiated by the deposition of natural antibodies on donor endothelium followed by the activation of the recipient complement system, which rapidly destroys the graft. Studies of the role of activated complement in HAR have suggested that natural antibody as well as early (C3a, C3b) and late (C5a, C5b-9) activated complement components may contribute to cell activation and damage. Attenuation of HAR has been achieved by blockade of C3 activation with soluble CR1 or consumptive depletion of complement with cobra venom factor; however, similar studies using specific inhibitors of terminal complement components have not been described. To address the contribution of C5a and the membrane attack complex (C5b-9, MAC) to complement-mediated xenogeneic cell and organ damage, we utilized functionally blocking monoclonal antibodies directed against the human terminal complement components C5 and C8. Our data show that both anti-C5 and anti-C8 mAbs protect porcine aortic endothelial cells from membrane damage mediated by human C5b-9. Additionally, both the anti-C5 and anti-C8 mAbs blocked complement-mediated generation of membrane prothrombinase activity on porcine aortic endothelial cells challenged with human serum. To test the ability of these antibodies to attenuate antibody and complement-mediated damage of xenogeneic organs, an ex vivo model was developed wherein isolated rat hearts were perfused with human serum in the presence or absence of the anti-C5 and anti-C8 mAbs. Our data demonstrate that mAbs directed against human C5 and C8 prevented organ damage by human serum complement and suggest that these molecules may serve as potent inhibitors of HAR.

Renal alpha 1- and alpha 2-adrenoceptor mediated vasoconstriction in dogs.

Clonidine (C) and guanabenz (G) are both alpha 2-agonists. Whereas C has been reported to cause anti-natriuresis proportional to renal vasoconstriction with acute infusion, G increases sodium and water excretion with little change in renal blood flow (RBF). C-mediated vasoconstriction may involve alpha 1-receptors. RBF in anaesthetized dogs was measured electromagnetically. Boluses of phenylephrine (P), C and G were injected into the renal artery via a perfusion cannula. Responses were expressed as delta RBF. Then prazosin or yohimbine were administered intra-arterially and dose-response curves repeated. The shift in dose ratio (delta ED50 after prazosin/yohimbine) for P was 52.9, for C was 4.7 and for G was 0.62. G was a 10-fold weaker vasoconstrictor than C, but G was still effective when alpha 1-receptors were blocked. Ten-fold differences were found between P, C and G for dependence upon alpha 1-adrenoceptors. Denervation did not significantly shift the curves. Thus, postsynaptic alpha 2-receptors can contribute to renal vasoconstriction, but the receptors are either less numerous or less efficiently coupled to contractile elements than in other beds. Verapamil markedly attenuated responses to C and G. Since tubular alpha 1-receptors have also been implicated in sodium transport, C may be more prone than G to cause anti-natriuresis both through stimulated tubular reabsorption and renal vasoconstriction.

Mitochondrial dynamics regulates migration and invasion of breast cancer cells.

Mitochondria are highly dynamic and undergo constant fusion and fission that are essential for maintaining physiological functions of cells. Although dysfunction of mitochondria has been implicated in tumorigenesis, little is known about the roles of mitochondrial dynamics in metastasis, the major cause of cancer death. In the present study, we found a marked upregulation of mitochondrial fission protein dynamin-related protein 1 (Drp1) expression in human invasive breast carcinoma and metastases to lymph nodes. Compared with non-metastatic breast cancer cells, mitochondria also were more fragmented in metastatic breast cancer cells that express higher levels of total and active Drp1 and less mitochondrial fusion protein 1 (Mfn1). Silencing Drp1 or overexpression of Mfn1 resulted in mitochondria elongation or clusters, respectively, and significantly suppressed metastatic abilities of breast cancer cells. In contrast, silencing Mfn proteins led to mitochondrial fragmentation and enhanced metastatic abilities of breast cancer cells. Interestingly, these manipulations of mitochondrial dynamics altered the subcellular distribution of mitochondria in breast cancer cells. For example, silencing Drp1 or overexpression of Mfn1 inhibited lamellipodia formation, a key step for cancer metastasis, and suppressed chemoattractant-induced recruitment of mitochondria to lamellipodial regions. Conversely, silencing Mfn proteins resulted in more cell spreading and lamellipodia formation, causing accumulation of more mitochondria in lamellipodia regions. More importantly, treatment with a mitochondrial uncoupling agent or adenosine triphosphate synthesis inhibitor reduced lamellipodia formation and decreased breast cancer cell migration and invasion, suggesting a functional importance of mitochondria in breast cancer metastasis. Together, our findings show a new role and mechanism for regulation of cancer cell migration and invasion by mitochondrial dynamics. Thus targeting dysregulated Drp1-dependent mitochondrial fission may provide a novel strategy for suppressing breast cancer metastasis.

Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis.

Excessive activation of G-protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) pathways has been linked to prostate cancer metastasis. Rac activation by guanine nucleotide exchange factors (GEFs) plays an important role in directional cell migration, a critical step of tumor metastasis cascades. We found that the upregulation of P-Rex1, a Rac-selective GEF synergistically activated by Gbetagamma freed during GPCR signaling, and PIP3, generated during either RTK or GPCR signaling, strongly correlates with metastatic phenotypes in both prostate cancer cell lines and human prostate cancer specimens. Silencing endogenous P-Rex1 in metastatic prostate cancer PC-3 cells selectively inhibited Rac activity and reduced cell migration and invasion in response to ligands of both epidermal growth factor receptor and G-protein-coupled CXC chemokine receptor 4. Conversely, expression of recombinant P-Rex1, but not its 'GEF-dead' mutant, in non-metastatic prostate cancer cells, such as CWR22Rv1, increased cell migration and invasion through Rac-dependent lamellipodia formation. More importantly, using a mouse xenograft model, we showed that the expression of P-Rex1, but not its mutant, induced lymph node metastasis of CWR22Rv1 cells without an effect on primary tumor growth. Thus, by functioning as a coincidence detector of chemotactic signals from both GPCRs and RTKs, P-Rex1-dependent activation of Rac promotes prostate cancer metastasis.

Renal alpha 1- and alpha 2-adrenoceptor mediated vasoconstriction in dogs: comparison of phenylephrine, clonidine, and guanabenz.

The alpha 2-agonist, guanabenz, increases Na and water excretion with little change in renal blood flow, while clonidine has been reported to cause antinatriuresis proportional to renal vasoconstriction. We hypothesized that clonidine-mediated renal vasoconstriction involves alpha 1-receptors. Dose-response curves were constructed correlating decreases in renal blood flow with doses of phenylephrine, clonidine, and guanabenz injected as boluses into renal arteries of anesthetized dogs. Changes in renal blood flow before and after prazosin, yohimbine or verapamil were recorded. Guanabenz was a 10-fold weaker vasoconstrictor than clonidine, but was still effective when alpha 1-receptors were blocked. Tenfold differences were found between phenylephrine, clonidine, and guanabenz for dependence on alpha 1-receptors. Denervation did not significantly shift the curves. Verapamil markedly attenuated only clonidine and guanabenz, which supported their dependence on calcium channels for vascular smooth muscle contraction. Thus, postsynaptic alpha 2-adrenoceptors can contribute to renal vasoconstriction in the dog but the receptors are either less numerous on the vasculature or are less efficiently coupled to contractile elements than are alpha 1-receptors. Clonidine constricts the renal vasculature through both adrenoceptor subtypes.

Subtype-selective alpha-1 adrenoceptor alkylation in the rat kidney and its effect on the vascular pressor response.

Separate genes for alpha-1A and alpha-1B adrenoceptors have now been identified. Whereas alpha-1 adrenoceptors are known to mediate rat renal vasoconstriction, the relative importance of these alpha-1 adrenoceptor subtypes was unknown. We cannulated the right suprarenal artery of anesthetized male Sprague-Dawley rats to permit administration of the alpha-1A and alpha-1B alkylating antagonists, SZL-49 (SZL) and chloroethylclonidine (CEC), respectively, directly into the right kidney. Treated kidneys were homogenized to identify the doses of SZL and CEC that caused the maximum reductions in Bmax for [3H]prazosin, the relatively nonselective alpha-1 adrenoceptor antagonist. In other rats, a Doppler flow probe was placed around the right renal artery, and dose-peak response curves for boluses of the alpha-1 adrenoceptor agonist phenylephrine (PHE) were generated before and after supramaximal dosages of SZL or CEC. Renal vasoconstriction to PHE was nearly obliterated by SZL. In contrast, CEC caused only a modest rightward shift in the PHE DRC. SZL also abolished the renal vascular response to two other alpha-1 adrenoceptor agonists, cirazoline and methoxamine. Our data support the conclusion that the alpha-1 adrenoceptors at the level of the rat renal resistance vessels are predominantly alpha-1A adrenoceptors.

Unmasking sensitive alpha 2-adrenoceptor-mediated renal vasoconstriction in conscious rats.

Renal vascular reactivity to intrarenal arterial boluses of norepinephrine (NE), phenylephrine (PHE; alpha 1-agonist), and guanabenz (GBZ; alpha 2-agonist) was assessed in conscious, freely moving, chronically instrumented, normotensive Wistar rats. Dose-response curves (DRCs) were obtained in the absence and cumulative presence of propranolol (PROP; beta-antagonist), corynanthine (CORY; alpha 1-antagonist) and idazoxan (IDX; alpha 2-antagonist) to estimate effective dosages (ED) required for 15 and 75% peak reductions in renal blood flow. The PHE DRC had a short shallow ED15 region, but was primarily linear with a steep slope. The GBZ DRC had a shallow slope. The NE DRC had a prolonged shallow phase in the ED15 region and a steep slope in the ED75 region. PROP had no effect on the DRCs. CORY caused a parallel rightward shift of the PHE DRC and had no effect on the GBZ DRC. After CORY, the shallow ED15 portion of the NE DRC was even more pronounced with a slope now identical to that of the GBZ DRC, whereas the ED75 region of the NE DRC was shifted rightward like the PHE DRC. IDX preferentially antagonized the ED15 regions of the GBZ and NE DRCs. In a second group of rats, the alpha 2-adrenoceptor antagonist, rauwolscine, was administered following base-line DRCs to demonstrate rightward shifts of the NE and GBZ DRCs when PHE DRCs remained unaffected. Therefore, when boluses of NE are injected into the rat kidney, the vasoconstrictive responses are a result of the activation of both alpha 1- and alpha 2-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo assessment of rat renal alpha adrenoceptors.

Despite a preponderance of alpha-2 adrenoceptors in homogenates of rat renal cortex, alpha-2 adrenoceptor agonists do not vasoconstrict the isolated buffer-perfused rat kidney. Both alpha-1 and alpha-2 adrenoceptor agonists can constrict the kidneys of dogs, cats and rabbits in vivo. Because alpha-2 adrenoceptor-mediated vasoconstriction is often difficult to demonstrate in vitro, and both subtypes of alpha agonists cause large increases in peripheral resistance in pithed rats, we tested the hypothesis that both alpha-1 and alpha-2 agonists would also constrict the rat kidney in vivo. Cannulation of the suprarenal artery and utilization of a high pressure liquid chromatography valve enabled random and reproducible intrarenal arterial bolus injections of agonists, and renal blood flow was monitored using Doppler flowmetry. Cirazoline, phenylephrine and norepinephrine bitartrate caused large renal vasopressor responses with minimal systemic effects. Although administered in a dosage range 100 to 1000 times that of alpha-1 agonists, the alpha-2 agonists (B-HT 920, UK 14,304 and guanabenz) produced only minimal renal vasoconstriction before systemic pressor effects. The low potency and efficacy of alpha-2 agonists could not be attributed to concomitant vasodilatory effects of these agents. Rat renal resistance vessels were less responsive to alpha-2 agonists than other species that have been examined. These studies are consistent with conclusions from in vitro examinations that only alpha-1 adrenoceptors mediate changes in renal vascular resistance and also autoradiographic studies reporting the localization of alpha-2 binding sites to rat renal tubules.

Improved separation method for rat proximal and distal renal tubules.

We modified and improved enzyme digestion and density gradient separation procedures to obtain fractions of proximal and distal renal tubules with high yield and viability. Kidneys from two anesthetized adult Wistar rats were flushed with Krebs-Henseleit buffer (KHB) and then perfused in situ with recirculated KHB containing collagenase and hyaluronidase at 125 mmHg. Cortices were excised, minced, and incubated in KHB containing enzymes for 35 min at 37 degrees C. Dissociated tubules were removed at 10-min intervals, rinsed, and placed in KHB containing 10% calf serum, vitamins, and amino acids at 4 degrees C. Separation was achieved by suspending the tissue in 45% isosmotic Percoll layered over an undiluted Percoll cushion and centrifuging. Proximal tubules sedimented near the cushion. Distal segments were isolated in the uppermost bands of a second 35% Percoll separation. Viability was greater than 95% as measured by lactate dehydrogenase leakage and quantitated by oxygen consumption and ATP content. Basal oxygen consumption was greater than 33 nmol O2 X min-1 X mg protein-1 in all fractions and was stimulated by succinate and inhibited by amiloride and ouabain. Basal ATP content averaged 9.7 nmol/mg ATP. An average 3.3-fold separation for the proximal fraction and 24.5-fold separation for the distal fraction was assessed by the enrichment of six specific enzyme markers, with several of the markers indicating separations up to 32-fold. Isolated tubules also displayed functional responses to parathyroid hormone and vasopressin. Distal, but not proximal, segments demonstrated significantly increased adenosine 3',5'-cyclic monophosphate formation with vasopressin.

Cosegregation of the renin gene with an increase in mean arterial blood pressure in the F2 rats of SHR-WKY cross.

Using the restriction endonuclease, BgI I, Samani et al. found a restriction fragment length polymorphism (RFLP) for the renin gene in spontaneously hypertensive rats (SHR) and its normotensive control Wistar-Kyoto (WKY) rats. This RFLP was confirmed in our laboratory in SHR and WKY rats using a rat renin cDNA probe. The correlation of blood pressure and the renin RFLP was examined in 106 F2 rats produced from F1 rats, the offspring of a cross between SHR males and WKY females. Systolic blood pressure was measured by the tail cuff method at 12 weeks of age. Mean arterial blood pressure of anesthetized rats was measured by cannulation of the femoral artery prior to sacrifice. The frequency of renin genotype showed a typical 1:2:1 Mendelian ratio in F2 rats of SHR and WKY cross. The mean arterial blood pressure of F2 rats homozygous with the SHR allele was significantly higher than F2 rats that were heterozygous or homozygous for the WKY allele. No significant difference in systolic blood pressure was observed in these F2 rats. Thus, the renin gene RFLP cosegregates with an increase in mean arterial blood pressure in the F2 rats of SHR and WKY cross.

Cosegregation of the renin gene with an increase in mean arterial blood pressure in the F2 rats of SHR-WKY cross.

Using the restriction endonuclease, Bgl I, Samani et al. found a restriction fragment length polymorphism (RFLP) for the renin gene in spontaneously hypertensive rats (SHR) and its normotensive control Wistar-Kyoto (WKY) rats (1). This RFLP was confirmed in our laboratory in SHR and WKY rats using a rat renin cDNA probe. The correlation of blood pressure and the renin RFLP was examined in 106 F2 rats produced from F1 rats, the offspring of a cross between SHR males and WKY females. Systolic blood pressure was measured by the tail cuff method at 12 weeks of age. Mean arterial blood pressure of anesthetized rats was measured by cannulation of the femoral artery prior to sacrifice. The frequency of renin genotype showed a typical 1:2:1 Mendelian ratio in F2 rats of SHR and WKY cross. The mean arterial blood pressure of F2 rats homozygous with the SHR allele was significantly higher than F2 rats that were heterozygous or homozygous for the WKY allele. No significant difference in systolic blood pressure was observed in F2 rats with different genotypes. Thus, the renin gene RFLP cosegregates with an increase in mean arterial blood pressure in the F2 rats of SHR and WKY cross.

Development and utility of cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLPs) linked to the Fom-2 fusarium wilt resistance gene in melon (Cucumis melo L.).

Fusarium wilt, caused by Fusarium oxysporum Schlecht f. sp. melonis Snyder & Hans, is a worldwide soil-borne disease of melon (Cucumis melo L.). Resistance to races 0 and 1 of Fusarium wilt is conditioned by the dominant gene Fom-2. To facilitate marker-assisted backcrossing with selection for Fusarium wilt resistance, we developed cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) markers by converting RAPD markers E07 (a 1.25-kb band) and G17 (a 1.05-kb band), respectively. The RAPD-PCR polymorphic fragments from the susceptible line 'Vedrantais' were cloned and sequenced in order to construct primers that would amplify only the target fragment. The derived primers, E07SCAR-1/E07SCAR-2 from E07 and G17SCAR-1/G17SCAR-2 from G17, yielded a single 1.25-kb fragment (designated SCE07) and a 1.05-kb fragment (designated SCG17) (the same as RAPD markers E07 and G17), respectively, from both resistant and susceptible melon lines, thus demonstrating locus-specific associated primers. Potential CAPS markers were first revealed by comparing sequence data between fragments amplified from resistant (PI 161375) and susceptible ('Vedrantais') lines and were then confirmed by electrophoresis of restriction endonuclease digestion products. Twelve restriction endonucleases were evaluated for their potential use as CAPS markers within the SCE07 fragment. Three (BclI, MspI, and BssSI) yielded ideal CAPS markers and were subsequently subjected to extensive testing using an additional 88 diverse melon cultigens, 93 and 119 F(2) individuals from crosses of 'Vedrantais' x PI 161375 and 'Ananas Yokneam'×MR-1 respectively, and 17 families from a backcross BC(1)S(1) population derived from the breeding line 'MD8654' as a resistance source. BclI- and MspI-CAPS are susceptible-linked markers, whereas the BssSI-CAPS is a resistant-linked marker. The CAPS markers that resulted from double digestion by BclI and BssSI are co-dominant. Results from BclI- and MspI-CAPS showed over 90% accuracy in the melon cultigens, and nearly 100% accuracy in the F(2) individuals and BC(1)S(1) families tested. This is the first report of PCR-based CAPS markers linked to resistance/susceptibility for Fusarium wilt in melon. The RFLP markers resulting from probing with a clone-derived 1.05-kb SCG17 PCR fragment showed 85% correct matches to the disease phenotype. Both the CAPS and RFLP markers were co-dominant, easier to score, and more accurate and consistent in predicting the melon phenotype than the RAPD markers from which they were derived.

Distribution of alpha1-adrenergic receptor mRNA species in rat heart.

Radioligand binding studies have detected alpha1A- and alpha1B-adrenergic receptors (AR) in rat heart, but the ligands available for these studies lack the sensitivity and specificity needed to map possible differences in alpha1-AR subtype expression. We therefore used competitive reverse transcriptase-polymerase chain reaction (RT-PCR) techniques to measure steady-state amounts of alpha1-AR messenger RNA (mRNA) subtypes in tissue dissected from several regions of rat heart. We detected mRNA for alpha1A-, alpha1B-, and alpha1D-AR in each region. Irrespective of the alphaAR subtype, the total number of alpha1-AR transcripts has the following regional rank order: left ventricular papillary muscle > left ventricle > left atrium > apex > right ventricle > ventricular septum > right atria. Among the regions, the fractional contribution of alpha1A-, alpha1B-, and alpha1D-AR mRNA to the total amount of alpha1-AR displays considerable variability. The alpha1B-AR mRNA accounts for >50% of the total alpha1-AR mRNA in all regions except the ventricular septum. There are also significant percentages of alpha1A-AR in each region, especially in the papillary muscle (48%) and ventricular septum (48%). The alpha1D-AR mRNA transcripts are found in comparatively low numbers; their highest levels (18% of total) were found in the right ventricle. These differences in alpha1-AR mRNA expression may contribute to the observed regional differences in myocardial responses to alpha1-AR agonists and antagonists.

Heterogeneous expression of alpha 1-adrenoceptor subtypes among rat nephron segments.

alpha 1-Adrenoceptor subtypes mediate many of the actions of the renal nerve, but their locations along the nephron are unknown. We investigated the distribution of alpha 1-adrenoceptor subtype mRNA and protein in rat proximal tubules and medullary thick ascending limbs (MTAL) using reverse transcription combined with polymerase chain reaction (PCR) and radioligand binding methods. Complementary primers were designed to span cDNA sequences in each of the third intracellular loops of the rat alpha 1B- and alpha 1D-adrenoceptors. Expression of the mRNA of alpha 1B- and alpha 1D-adrenoceptors was first detected in total RNA from whole rat kidney, and the PCR product identity was confirmed by sequencing. Endogenous expression of alpha 1B- and/or alpha 1D-adrenoceptor mRNA was then investigated in microdissected segments of the rat proximal convoluted tubule (S2 segments) and the MTAL. mRNA was reverse-transcribed directly from permeabilized microdissected segments and the resulting cDNA was subjected to PCR with the alpha 1-adrenoceptor primers. In proximal convoluted tubules, amplification of both alpha 1B- and alpha 1D-adrenoceptor mRNA was observed. In MTAL segments, only alpha 1D-adrenoceptor mRNA was detected. We also measured receptor protein using [3H]prazosin in saturation and competition binding experiments. Proximal tubular membranes contained 3.3-fold more alpha 1-adrenoceptor than did MTAL membranes (163 +/- 21 versus 49 +/- 3 fmol/mg of protein). When the alkylating agent chloroethylclonidine (CEC) (10 microM, 10 min) was used to define alpha 1-adrenoceptor subtypes, proximal tubules were found to contain primarily CEC-insensitive (alpha 1A) sites (68 +/- 4%) and MTAL primarily CEC-sensitive sites (75 +/- 3%). Most [3H]prazosin binding sites (72 +/- 2%) in MTAL segments were also sensitive to the alkylating agent SZL-49, consistent with their identification as alpha 1D-adrenoceptors. In competition studies with the antagonists WB4101, 5-methylurapidil, and (+)-niguldipine, both high and low affinity sites were observed in proximal tubules. WB4101 interacted with only one site in MTAL membranes, intermediate in affinity between those sites found in proximal tubules. We conclude that reverse transcription-PCR is a useful method for demonstrating the expression of alpha 1-adrenoceptor subtypes in small amounts of tissue. Results from our experiments suggest that alpha 1A-, alpha 1B-, and alpha 1D-adrenoceptors are all expressed in proximal tubules and that alpha 1D-adrenoceptors are the primary alpha 1-adrenoceptor subtype expressed in MTAL. The distinct anatomical distribution of each of these adrenoceptor subtypes suggests that they serve different functions in the kidney.