saporin

Staudt MD, Truitt WA, McKenna KE, de Oliveira CV, Lehman MN, Coolen LM (2012) A pivotal role of lumbar spinothalamic cells in the regulation of ejaculation via intraspinal connections. J Sex Med 9(9):2256-2265. doi: 10.1111/j.1743-6109.2011.02574.x

Summary: The authors examined the hypothesis that specific lumbar spinothalamic (LSt) cells control ejaculation through intraspinal connections. Rats received six bilateral injections of SSP-SAP (Cat. #IT-11) into the spinal cord, 48 ng in total. Saporin (Cat. #PR-01) was used as a control. It was found that while erectile function and emission were not affected, the usual rhythmic contractions of the bulbocaveronosus muscle during ejaculation were prevented.

Related Products: SSP-SAP (Cat. #IT-11), Saporin (Cat. #PR-01)

Read the featured article in Targeting Trends.

Talman WT, Nitschke Dragon D, Jones S, Moore SA, Lin L-H (2011) Cardiovascular dysfunction and cardiac injury result from selective glial damage in the nucleus tractus solitarii. Neuroscience 2011 Abstracts 664.14. Society for Neuroscience, Washington, DC.

Summary: In man, extensive CNS dysfunction as may occur after subarachnoid hemorrhage may lead to cardiac damage and cardiac arrhythmias. We have shown that highly selective and restricted lesions of the nucleus tractus solitarii (NTS) may lead to similar cardiac and cardiovascular compromise. For example, using conjugates including the cytotoxin saporin (SAP) to selectively damage NTS neurons that express NK1 receptors or those that express tyrosine hydroxylase (TH) leads to cardiac dysfunction and associated lability of arterial pressure. In continuing efforts to better characterize cellular changes produced by introducing into the NTS conjugates containing SAP, we have studied the effect of anti-dopamine-beta-hydroxylase (anti-DBH)-SAP, stabilized substance P (SSP)-SAP, SAP (unconjugated), blank-SAP (non-targeted peptide conjugate), IgG-SAP (non-targeted immunoglobulin conjugate), and 6-hydoxydopamine (6-OHDA) as a control without SAP injected into NTS. We assessed effects of the injected agents both on cellular markers [NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), gamma-aminobutyric acid (GABA) receptor type a and b, neuronal nitirc oxide synthase (nNOS), TH, vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), glial fibrillary acidic protein (GFAP), connexin 43 (Cx43), DBH and protein gene product 9.5 (PGP 9.5)] and on cardiovascular and cardiac function. We have found that each compound containing SAP (including blank-SAP, IgG-SAP, unconjugated SAP) led to loss of GFAP and Cx43 immunofluorescent labeling in the NTS as well as lability of arterial pressure, cardiac arrhythmias, and cardiac myocytolysis. Those outcomes occurred despite neuronal specificity for each of the SAP conjugates. For example, anti-DBH-SAP led to a decrease in TH and DBH staining as well as a profound loss in GFAP and Cx43. In contrast, SSP-SAP led to loss of NK1 as well as GFAP, Cx43, and glutamate receptor markers but did not lead to loss of DBH or GABA. SSP-SAP also caused a loss in PGP9.5 which was not observed in all other agents. SAP and blank-SAP, on the other hand, led to loss of GFAP and Cx43 while 6-OHDA led to loss of TH and DBH, increased GFAP and decreased Cx-43. We are still investigating the effects of 6-OHDA on lability of arterial pressure and cardiac events but preliminary data suggest that, in doses used, it led to loss of TH and DBH but did not lead to either lability or cardiac events that were seen with each of the conjugates containing an SAP moiety. This study suggests that glial dysfunction may alone interefere with cardiovascular control through the NTS and may lead to cardiac damage and cardiovascular dysfunction.

Related Products: Anti-DBH-SAP (Cat. #IT-03), SSP-SAP (Cat. #IT-11), Mouse IgG-SAP (Cat. #IT-18), Blank-SAP (Cat. #IT-21), Saporin (Cat. #PR-01)

Ritter S, Li A-J, Wang Q, Dinh TT (2011) Basal metabolic substrate utilization is altered by lesion of hindbrain catecholamine neurons that innervate the medial hypothalamus and substrate selection during glucoprivation is impaired. Neuroscience 2011 Abstracts 88.05. Society for Neuroscience, Washington, DC.

Summary: Central injection of the targeted immunotoxin, anti-dopamine beta hydroxylase (DBH)-saporin (DSAP), retrogradely and selectively lesions norepinephrine (NE) and epinephrine (E) neurons with projections to the injection site. Previous work has shown that DSAP injections targeting the hypothalamic paraventricular nucleus eliminate key counterregulatory responses to acute glucose deficit, including feeding and corticosterone secretion. To examine the role of these NE an E neurons in metabolic control under basal conditions, we injected rats in the PVH with DSAP or control unconjugated saporin (SAP) and analyzed their metabolic profiles using metabolic chambers (Columbus Instruments). Rats were maintained on a standard pelleted rodent diet. We found that the respiratory exchange ratio (RER) was consistently elevated in DSAP rats across the entire circadian cycle under basal conditions, compared to the RER of SAP controls, indicating increased dependence on carbohydrate utilization. Metabolic rate and activity did not differ between groups. This result suggests a chronic enhancement of glucose mobilization or an impairment of the ability to mobilize fatty acids in the DSAP rats. We also found that when challenged by 2-deoxy-D-glucose induced glucoprivation, SAP controls exhibited a rapid decrease in RER, indicating a switch to fat metabolism, whereas DSAP rats did not exhibit this response. Together these results favor the possibility that a central mechanism for fat mobilization is impaired in DSAP rats and that this impairment is reflected under both basal and glucoprivic conditions. The previously reported observation that PVH DSAP-injected rats exhibit a slowly-developing obesity also supports this possibility. Additional findings suggest that this impairment may be due to the loss of NE/E control of corticosterone secretion in the DSAP rats.

Related Products: Anti-DBH-SAP (Cat. #IT-03), , Saporin (Cat. #PR-01)

I’anson H, Jethwa PH, Warner A, Ebling FJ (2011) Histaminergic regulation of seasonal metabolic rhythms in Siberian hamsters. Physiol Behav 103(3-4):268-278. doi: 10.1016/j.physbeh.2011.02.035

Summary: The role of central histaminergic mechanisms on seasonal catabolic state was investigated in hamsters. Siberian hamsters received bilateral 3.8-ng injections of orexin-SAP (Cat. #IT-20) into the tuberomammillary posterior hypothalamic region. Saporin (Cat. #PR-01) was used as a control. During long days, lesioned animals displayed higher locomotor activity, greater oxygen intake, and no net weight gain. During shorter days (hibernation) with less activity, lesioned animals did not lose weight. The data indicate that histaminergic neurons are involved in body weight regulation.

Related Products: Orexin-B-SAP (Cat. #IT-20), Saporin (Cat. #PR-01)

Hutchins BM, Kazane SA, Staflin K, Forsyth JS, Felding-Habermann B, Smider VV, Schultz PG (2011) Selective formation of covalent protein heterodimers with an unnatural amino acid. Chem Biol 18(3):299-303. doi: 10.1016/j.chembiol.2011.01.006 PMID: 21439474

Summary: This work demonstrates the creation of a variety of constructs containing specific defined conjugation sites. One use for these molecules is to create homogenous antibody conjugates‚ meaning the properties of these conjugates can be quantitatively evaluated. Having greater control of such conjugations is essential if these types of constructs are to move toward use as therapeutics. The authors created an anti-Her2 Fab-saporin molecule and tested it in vitro. Analysis by western used anti-SAP-HRP (Cat. #AB-15-HRP) to detect the conjugated molecule.

Related Products: Saporin Goat Polyclonal, HRP-labeled (Cat. #AB-15HRP)

Li AJ, Wang Q, Ritter S (2011) Participation of hindbrain AMP-activated protein kinase in glucoprivic feeding. Diabetes 60(2):436-442. doi: 10.2337/db10-0352

Summary: Catecholamine neurons innervating the medial hypothalamus are involved in the control of glucoprivic feeding as well as other responses to glucose deficit. Rats received bilateral 82-ng injections of anti-DBH-SAP (Cat. #IT-03) into the paraventricular hypothalamic nucleus. Saporin (Cat. #PR-01) was used as a control. Lesioned animals did not respond to the administration of a competitive glucose inhibitor, nor did they display phosphorylation of pAMPKα, suggesting that AMPK may be part of a glucose- sensing mechanism.

Related Products: Anti-DBH-SAP (Cat. #IT-03), Saporin (Cat. #PR-01)

Marshall RS, D’Avila F, Di Cola A, Traini R, Spano L, Fabbrini MS, Ceriotti A (2011) Signal peptide-regulated toxicity of a plant ribosome-inactivating protein during cell stress. Plant J 65(2):218-29. doi: 10.1111/j.1365-313X.2010.04413.x PMID: 21223387

Summary: Type I ribosome inactivating proteins (RIPs) are thought to have a role in defending plants against viral or fungal infections. Most type I RIPs have signal peptides for insertion into the endoplasmic reticulum, followed by transportation to a vacuole or the cell wall. The authors examined signal peptide regulation under stress in tobacco plants transfected with saporin. One method of analysis was western blots using anti-saporin (Cat. #AB-15).

Related Products: Saporin Goat Polyclonal (Cat. #AB-15)

Carvalho AF, Reyes AR, Sterling RC, Unterwald E, Van Bockstaele EJ (2010) Contribution of limbic norepinephrine to cannabinoid-induced aversion. Psychopharmacology (Berl) 211(4):479-491. doi: 10.1007/s00213-010-1923-7

Summary: The authors used bilateral injections of anti-DBH-SAP (Cat. #IT-03) into the nucleus accumbens and the bed nucleus of the stria terminalis to investigate the role of neuroepinephrine in cannabinoid-induced aversion and anxiety. Lesioned animals received bilateral 52.5 ng-injections of anti-DBH-SAP into the nucleus accumbens or 63 ng into the bed nucleus of the stria terminalis. Saporin (Cat. #PR-01) was used as a control. Lesioned animals displayed reversed aversive behavior, but no change in anxiety-like behavior.

Related Products: Anti-DBH-SAP (Cat. #IT-03), Saporin (Cat. #PR-01)

Emanuel AJ, Ritter S (2010) Hindbrain catecholamine neurons modulate the growth hormone but not the feeding response to ghrelin. Endocrinology 151(7):3237-3246. doi: 10.1210/en.2010-0219

Summary: In this work the authors investigated the role of hindbrain catecholamine neurons in the response to a gastrointestinal peptide, ghrelin. Rats received 42 ng injections of anti-DBH-SAP (Cat. #IT-03) into the paraventricular nucleus of the hypothalamus. Saporin (Cat. #PR-01) was used as a control. Lesioned animals had a prolonged growth hormone (GH) response to ghrelin administration as compared to controls, but the feeding response was unchanged. The results indicate that ghrelin or GH may be involved with a negative feedback response controlling GH levels.

Related Products: Anti-DBH-SAP (Cat. #IT-03), Saporin (Cat. #PR-01)

Q: Saporin (Cat. #PR-01) has been shown to enzymatically inhibit the function of the ribosome, which follows that protein synthesis is then inhibited. Inhibition of protein synthesis brings about “cell death” to my knowledge. To detect “cell death” usually does not take a longer time to detect than “growth inhibition,” I suppose. So what I would like to ask you is: “at least” how many hours will it take to detect “cell death” caused by saporin. In your protocol, the recommended duration of assay is 72 hours. Does that duration contain much allowance? Of course, the duration must be dependent on the speed (or efficiency) of internalization of saporin, I understand. But once saporin is internalized, how many hours (or minutes) will it take to kill the target cell?

A: 72 hours is for the great majority of cell lines, but there are a very few that require 48 hours and a very few that require 96 hours (maybe 1 of each of the 100 or so that we’ve tried). The variation in time from 72 hours is not much on the shorter side, but is only limited by the few living cells proliferating on the longer side.

It is easy to see dead cells in the microscope, so you may want to visually check your cells at different times to verify that 72 hours is correct.

How many hours will it take after internalization to kill a cell? Quite a few, because there are several processes that need to occur: the enzyme must inactivate a sufficient number of ribosomes to inhibit protein synthesis, and then the cell has to stop living because of the turnover and loss of those proteins. That takes time.

Related: Saporin (Cat. #PR-01)