Search

Prior research in humans and animals suggest that exposure to chronic stress alters the response to drugs of abuse, increasing vulnerability to drug addiction. Chronic unpredictable stress (CUS) has been shown to augment the increase of dopamine in the striatum when challenged with high doses of methamphetamine immediately following stress exposure, however it is not known whether this neurochemical stress-sensitization continues after the cessation of the stressors or if behavioral sensitization is also present. Therefore, the current study examined the immediate and delayed effects of CUS on methamphetamine-induced behaviors and striatal dopamine levels. Male rats were exposed to 10 days of CUS and then tested in either an open field box to assess locomotion or underwent in vivo microdialysis to measure striatal dopamine levels immediately following CUS or after a 1-2 week delay. All rats exposed to CUS showed a potentiated locomotor response immediately following an acute injection of 7.5mg/kg methamphetamine compared to non-stressed control rats. Both groups of CUS rats also showed augmented dopamine release and rectal temperatures following methamphetamine with prolonged increases in the CUS rats tested after a delay. These results suggest that CUS increases the sensitivity of a rat to a single injection of methamphetamine and that the increased sensitivity persists for up to 2 weeks following the last stressor., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Chronic stress can influence behaviors associated with medial prefrontal cortex (mPFC) function, such as cognition and emotion regulation. Dopamine in the mPFC is responsive to stress and modulates its behavioral effects. The current study tested whether exposure to 10 days of chronic unpredictable stress (CUS) altered the effects of acute elevation stress on dopamine release in the mPFC and on spatial recognition memory. Male rats previously exposed to CUS or nonstressed controls were tested behaviorally, underwent microdialysis to assess mPFC dopamine levels or underwent blood sampling for corticosterone analysis. Dopamine in the mPFC significantly increased in both groups during acute elevation stress compared with baseline levels, but the level was attenuated in CUS rats compared with controls. Control rats exposed to elevation stress immediately before the T-maze test showed impaired performance, whereas CUS rats did not. No group differences were observed in general motor activity or plasma corticosterone levels following elevation stress. The present results indicate that prior exposure to this CUS procedure reduced dopamine release in the mPFC during acute elevation stress and prevented the impairment of performance on a spatial recognition test following an acute stressor. These findings may contribute to an understanding of the complex behavioral consequences of stress.

Purpose: The α2β1 integrin plays an important but complex role in angiogenesis and vasculopathies. Published GWAS studies established a correlation between genetic polymorphisms of the α2β1 integrin gene and incidence of diabetic retinopathy. Recent studies indicated that α2-null mice demonstrate superior vascularization in both the wound and diabetic microenvironments. The goal of this study was to determine whether the vasculoprotective effects of α2-integrin deficiency extended to the retina, using the oxygen-induced retinopathy (OIR) model for retinopathy of prematurity (ROP)., Methods: In the OIR model, wild-type (WT) and α2-null mice were exposed to 75% oxygen for 5 days (postnatal day [P] 7 to P12) and subsequently returned to room air for 6 days (P12-P18). Retinas were collected at postnatal day 7, day 13, and day 18 and examined via hematoxylin and eosin and Lectin staining. Retinas were analyzed for retinal vascular area, neovascularization, VEGF expression, and Müller cell activation. Primary Müller cell cultures from WT and α2-null mice were isolated and analyzed for hypoxia-induced VEGF-A expression., Results: In the retina, the α2β1 integrin was minimally expressed in endothelial cells and strongly expressed in activated Müller cells. Isolated α2-null primary Müller cells demonstrated decreased hypoxia-induced VEGF-A expression. In the OIR model, α2-null mice displayed reduced hyperoxia-induced vaso-attenuation, reduced pathological retinal neovascularization, and decreased VEGF expression as compared to WT counterparts., Conclusions: Our data suggest that the α2β1 integrin contributes to the pathogenesis of retinopathy. We describe a newly identified role for α2β1 integrin in mediating hypoxia-induced Müller cell VEGF-A production., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Background: Parental consanguinity is a risk factor for congenital heart disease (CHD) worldwide, suggesting that a recessive inheritance model may contribute substantially to CHD. In Bangalore, India, uncle-niece and first cousin marriages are common, presenting the opportunity for an international study involving consanguinity mapping of structural CHD. We sought to explore the recessive model of CHD by conducting a genome-wide linkage analysis utilizing high-density oligonucleotide microarrays and enrolling 83 CHD probands born to unaffected consanguineous parents., Methodology/principal Findings: In this linkage scan involving single nucleotide polymorphism (SNP) markers, the threshold for genome-wide statistical significance was set at the standard log-of-odds (LOD) score threshold of 3.3, corresponding to 1995ratio1 odds in favor of linkage. We identified a maximal single-point LOD score of 3.76 (5754ratio1 odds) implicating linkage of CHD with the major allele (G) of rs1055061 on chromosome 14 in the HOMEZ gene, a ubiquitously expressed transcription factor containing leucine zipper as well as zinc finger motifs. Re-sequencing of HOMEZ exons did not reveal causative mutations in Indian probands. In addition, genotyping of the linked allele (G) in 325 U.S. CHD cases revealed neither genotypic nor allele frequency differences in varied CHD cases compared to 605 non-CHD controls., Conclusions/significance: Despite the statistical power of the consanguinity mapping approach, no single gene of major effect could be convincingly identified in a clinically heterogeneous sample of Indian CHD cases born to consanguineous parents. However, we are unable to exclude the possibility that noncoding regions of HOMEZ may harbor recessive mutations leading to CHD in the Indian population. Further research involving large multinational cohorts of patients with specific subtypes of CHD is needed to attempt replication of the observed linkage peak on chromosome 14. In addition, we anticipate that a targeted re-sequencing approach may complement linkage analysis in future studies of recessive mutation detection in CHD.

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 comprises 11 serpin genes, many of which are expressed specifically in hepatic cells. Previous studies identified a locus control region (LCR) upstream of the human alpha1-antitrypsin (alpha1AT) gene that is required for gene activation, chromatin remodeling, and histone acetylation throughout the proximal serpin subcluster. Here we show that the LCR interacts with multiple liver-specific transcription factors, including hepatocyte nuclear factor 3beta (HNF-3beta), HNF-6alpha, CCAAT/enhancer binding protein alpha (C/EBPalpha), and C/EBPbeta. RNA polymerase II is also recruited to the locus through the LCR. Nongenic transcription at both the LCR and an upstream regulatory region was detected, but the deletion of the LCR abolished transcription at both sites. The deletion of HNF-3 and HNF-6 binding sites within the LCR reduced histone acetylation at both the LCR and the upstream regulatory region and decreased the transcription of the alpha1AT, corticosteroid binding globulin, and protein Z-dependent protease inhibitor genes. These results suggest that the LCR activates genes in the proximal serpin subcluster by recruiting liver-specific transcription factors and components of the general transcription machinery to regulatory regions upstream of the alpha1AT gene.

Previous research has found that exposure to unpredictable stress can augment anxiety in humans and animals. The appearance of anxiety symptoms in humans frequently develop after stress exposure has terminated, but few rodent studies have systematically examined the delayed anxiogenic effects of unpredictable stress. Therefore, the current study investigated whether anxiety-like behaviors in rats would increase at several time intervals following exposure to chronic unpredictable stress (CUS). Unconditioned and conditioned response tasks were used to assess anxiety in male rats 1, 7 or 14 days following exposure to 10 days of a variety of stressors. Rats exposed to CUS showed increased burying behaviors and immobility during the defensive burying test, a conditioned anxiety test. The effects on burying behavior were apparent 7 and 14 days after the termination of the unpredictable stress procedure, but not when tested 1 day after CUS. Total time immobile in the defensive burying test also increased 14 days after termination of the last stressor. In contrast, there were no significant effects of CUS on behavioral measures in the unconditioned response tasks, the elevated plus-maze or light-dark box, at any time point following exposure to CUS. The current findings suggest that CUS may be a useful model of human conditioned anxiety that develops subsequent to chronic stress exposure.

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system for studying the regulation of gene activity and chromatin structure. We demonstrated previously that the six known serpin genes in this region were organized into two subclusters of three genes each that occupied approximately 370 kb of DNA. To more fully understand the genomic organization of this region, we annotated a 1-Mb sequence contig from data from the Genoscope sequencing consortium (http://www.genoscope.cns.fr/ ). We report that 11 different serpin genes reside within the 14q32.1 cluster, including two novel alpha1-antiproteinase-like gene sequences, a kallistatin-like sequence, and two recently identified serpins that had not been mapped previously to 14q32.1. The genomic regions proximal and distal to the serpin cluster contain a variety of unrelated gene sequences of diverse function. To gain insight into the chromatin organization of the region, sequences with putative nuclear matrix-binding potential were identified by using the MAR-Wiz algorithm, and these MAR-Wiz candidate sequences were tested for nuclear matrix-binding activity in vitro. Several differences between the MAR-Wiz predictions and the results of biochemical tests were observed. The genomic organization of the serpin gene cluster is discussed.

This essay responds to D. Davis and W. C. Follette (2002), who question the value of motive evidence in murder cases. They argue that the evidence that a husband had extramarital affairs, that he heavily insured his wife's life, or that he battered his wife is ordinarily of infinitesimal probative value. We disagree. To be sure, it would be foolish to predict solely on the basis of such evidence that a husband will murder his wife. However, when this kind of evidence is combined with other evidence in a realistic murder case, the evidence can be quite probative. We analyze cases in which it is virtually certain that the victim was murdered but unclear who murdered her, and in which it is uncertain whether the husband murdered the wife or she died by accident. We show that in each case motive evidence, such as a history of battering or of infidelity, can substantially increase the odds of the husband's guilt. We also consider the actual case on which Davis and Follette base their paper. We argue that testimony of Davis on the basis of the analysis presented in their paper was properly excluded, for it would have been misleading and unhelpful.

Serotonin (5-HT) is generally inhibitory to masculine sexual behavior. It has been suggested that 5-HT released after ejaculation may promote the sexual quiescence of the postejaculatory interval (PEI). The following experiments were conducted to test (1) whether extracellular 5-HT increases in either the anterior lateral hypothalamic area (LHAA) or the medial preoptic area (MPOA) of male rats after ejaculation; (2) whether increasing 5-HT in these sites, by microinjecting the selective serotonin reuptake inhibitor alaproclate, could inhibit copulatory abilities; and (3) whether copulation deficits produced by alaproclate were attributable to locomotor impairments. The effects of local application of alaproclate on extracellular 5-HT levels in the LHAA and the MPOA were also tested. Extracellular serotonin was measured in all experiments using in vivo microdialysis. Ejaculation was correlated with enhanced 5-HT release from the LHAA; no 5-HT increases were observed before ejaculation, and levels were decreased toward basal values during a subsequent copulatory series. Elevating 5-HT in the LHAA by microinjecting alaproclate inhibited copulation by increasing the latency to mount, intromit, and ejaculate. This inhibition did not result from nonspecific locomotor impairments. In the MPOA, 5-HT release remained stable throughout copulation, and microinjecting alaproclate into this site did not significantly alter sexual behavior. These data support the large body of evidence suggesting that 5-HT is inhibitory to masculine sexual behavior. Furthermore, the LHAA, but not the MPOA, may be one site responsible for serotonergic inhibition of copulation during the PEI.

We investigated the in vitro interaction with and antitumor effect on several murine and human leukemic cell lines of diaziquone (AZQ). L1210 cells accumulated AZQ from Roswell Park Memorial Institute Medium 1640 with or without newborn calf serum by a temperature-dependent and sodium azide-resistant process. AZQ inhibited, in a dose-dependent fashion, [3H]thymidine incorporation into L1210 cells, but this inhibition was slow to develop, requiring approximately 6 hr to become apparent. The minimal inhibitory concentration of AZQ for this process was 0.05 to 0.25 nmol/ml. AZQ was a much less effective inhibitor of L1210 cell [3H]uridine and [14C]valine incorporation. In suspension cultures, AZQ inhibited growth of L1210 and HL-60 cells at minimal inhibitory concentrations of 0.5 to 1 nmol/ml. In soft agar cultures, AZQ inhibited HL-60 cell cloning at minimal inhibitory concentrations of 0.1 to 0.3 nmol/ml. AZQ provoked a dose-dependent increase in oxygen consumption when added to intact L1210, HL-60, and K562 cells and was converted to an AZQ anion free radical by these cells. When the aziridine rings of AZQ were opened by acid treatment, the resulting molecule was not accumulated by L1210 cells, did not provoke O2 consumption, did not form free radicals when added to L1210 cells, and was a much less effective inhibitor of [3H]thymidine incorporation by L1210 cells than was AZQ.

Nuclei isolated from rat liver, heart, and kidney catalyze oxygen consumption in the presence of reduced pyridine nucleotide (NADPH) and quinone or quinone-imine antibiotics such as Adriamycin, daunorubicin, actinomycin D, mitomycin C, and streptonigrin. The Km and Vmax values for NADPH were 2.4 x 10(-3) M and 3 x 10(-8) mol O2 per min per mg protein and Km values for the antibiotics ranged from 1.4 x 10(-4) M to 5.9 x 10(-6) M. Metabolism of the anthracycline antibiotics, i.e., reductive glycosidase reaction, occurs in reaction mixtures after all oxygen is consumed. During the reaction, free-radical species of Adriamycin and daunorubicin are detectable by electron paramagnetic reasonance spectrometry. These observations indicate that some cytotoxic antibiotics can be activated to a free-radical state at the site where damage to nuclear DNA may result.

The aziridinyl quinone 3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone is an antitumor agent that enhances electron flow from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen in rat liver microsomes, rat liver nuclei, and purified NADPH-cytochrome c reductase. The process is enzymatic and has an optimum pH of 7.5; NADPH is the preferred cofactor. Electron spin resonance studies show the presence of a free radical semiquinone characterized by five hyperfine lines at g = 2.0046.

A polymorphic acidic protein (Pa) has been isolated from human parotid saliva by the use of ion-exchange and gel filtration chromatography. Following these purification procedures, analytical anionic polyacrylamide disc gel electrophoresis revealed a single stainable band. Amino acid analysis demonstrated a protein particularly rich in proline, glutamic acid, and glycine, but with reduced amounts of threonine and no tyrosine. Only a very small percentage of carbohydrate was detected. Isoelectric focusing at pH 3-10 verified the acidic character of this protein with an isoelectric point in the range pH 3.9-4.5. Other salivary proteins called Pa-II, possibly related physiologically and genetically to the Pr system, were also partially purified and studied. Differences were noted between Pa and Pa-II proteins in molecular size and amino acid composition.

A sample consisting of 92 black subjects was examined in this study. According to results of preliminary statistical tests there is a significant relationship between certain genetically determined salivary factors and individual susceptibility to dental disease in the racial group studied. These findings are been validated by the examination of data from several additional studies involving large samples.

A brief description of the systemic and oral manifestations and the inheritance pattern of a family with myotonic dystrophy (MD) is presented. Relevant intraoral findings in this case included constricted maxillary and mandibular dental arches with adequate intercuspation and functional occlusion. A possible explanation for these atypical findings is discussed. Considerations for providing complete dental care and treatment for patients with MD are emphasized.

The acidic proline-rich proteins (Pr) showing genetic polymorphism were purified from human parotid salivas by gel filtration and ion exchange chromatography. Molecular weight determinations, amino acid composition analyses, and polypeptide mapping experiments indicate that the Pr 3 protein is a fragment of the Pr 1 protein. Studies of a parotid saliva factor capable of converting Pr 1 to Pr 3 and Pr 2 to Pr 4 indicate that Pr 3 and Pr 4 are generated from Pr 1 and Pr 2, respectively. Evidence suggests that the converting factor is a protease capable of posttranslationally cleaving Pr 1 and Pr 2, the primary or derived products of alleles Pr1 and Pr2.

Isoelectric focusing was performed on parotid salivas selected for their electrophoretic phenotypes of proline-rich acidic salivary proteins. Fractions encompassing narrow pH regions were pooled and examined by polyacrylamide gel electrophoresis. Isoelectric focusing yielded partial purification of major and minor acidic proline-rich proteins which were subsequently compared by immunoelectrophoresis and double immunodiffusion against goat anti-human parotid saliva. Cross-reactivity without spurring between all fractions containing major Pr proteins in both immunoelectrophoresis and double immunodiffusion suggests that these proteins are immunologically very similar or identical.

Mice treated with lethal doses of adriamycin (A1) (IP) are rescued with a single IP dose of 3,5,5-trimethyl-2-morpholinon-3-yl radical dimer (TM3). The in vivo rescue is assumed to be analogous to the in vitro reaction of TM3 with A1 that produces the non-toxic 7-deoxy-adriamycinone (7dAone). TM3 prevents death if given within 60 min following A1 administration. Control A1-treated mice died by 8 days (median survival time) whereas TM3 rescued A1-treated mice had a median survival time of greater than 60 days.

Previous studies have reported antitumor activity and reduced cardiotoxicity for a putative 3:1 complex of iron:Adriamycin (ADR). We have studied the tissue distribution and metabolism of a wide variety of freshly prepared and lyophilized iron:ADR preparations after administration to BALB/c mice (ADR, 16 mg/kg i.v.). Tissue concentrations of ADR given without iron were initially highest in kidney and liver, and ADR fluorescence was lost from all tissues except the spleen with a t1/2 of 15 to 18 hr. ADR remained the major fluorescent species in liver and kidney from 0.5 to 72 hr after treatment. Freshly prepared iron:ADR (1:1) behaved similarly to ADR except for a slightly longer tissue t1/2 in heart, liver, and kidney. The tissue distribution of freshly prepared 2:1 and 3:1 iron:ADR was very different from that of ADR without iron; lung containing the highest concentrations of ADR fluorescence. Administration of freshly prepared 1:1, 2:1, and 3:1 iron:ADR resulted in some increase in adriamycinol in the liver, but ADR was always the major fluorescent species present. The tissue distribution of 1:1 iron:ADR that had been aged for 48 or 96 hr was similar to that of fresh 2:1 and 3:1 iron:ADR rather than ADR or fresh 1:1 iron:ADR. When lyophilized iron:ADR preparations were reconstituted and administered, the 0.5-hr tissue distribution of 0.1:1, 0.2:1, 0.25:1, and 0.33:1 iron:ADR was the same as ADR alone, but 0.5:1, 1:1, 2:1, and 3:1 iron:ADR were all accumulated primarily in the lung. Physicochemical studies confirm the production of microaggregated iron:ADR complexes and light microscopy allows visualization and sizing of these aggregates. We feel that trapping of these iron:ADR aggregates in the pulmonary vascular bed accounts for the observed dramatic alteration in tissue distribution. Light and electron microscopic studies confirm the intravascular sequestration of iron in pulmonary capillaries.

Urinary bladders from Fischer rats organ cultured in a chemically defined medium, Ham's F12, underwent transitional cell hyperplasia which persisted for the duration of the culture period (10 days). The hyperplastic response was initiated at 2 days of culture in basal epithelial cells as evidenced by [3H]thymidine autoradiography. After 2 days, cells classified as intermediate cells were observed replicating DNA in increasing numbers, whereas the frequency of basal cells replicating DNA decreased. The peak periods of basal and intermediate cell DNA replication were at 2 and 5 days, respectively. The total increase in the number of cells in the epithelium during a 10-day culture period was approximately 2.6-fold. The appearance of DNA-replicating cells before the appearance of mitotic figures indicated that the cells of the transitional epithelium are primarily G1 cells, The hyperplastic response in the transitional epithelium was significantly inhibited by hydrocortisone. Epithelia cultured in the presence of hydrocortisone also displayed less atypia than those epithelia cultured in its absence. Hydrocortisone concentrations of 2.1 and 21 micronM inhibited hyperplasia by 75 and 84%, respectively, Cells replicating DNA at 2 days of culture were considerably less sensitive to the hydrocortisone inhibition of [3H]thymidine incorporation into DNA than cells replicating DNA at 4 days of culture. The possibility is discussed that basal and intermediate cells may have different sensitivities to hydrocortisone.

The phenotypic expression of a dominantly inherited human salivary acidic protein (Pa) has been described in acid-urea starch and in Tris-borate acrylamide gel systems. Estimates of the Pa+ allelic frequencies in American Caucasians, American blacks, and Orientals are .21, .14, and .42, respectively. The genetic and biochemical similarities to another series of proline-rich salivary proteins, Pr, and to a pair of similarly staining salivary proteins, Db (double band), are evaluated. It is concluded that either one locus or two (or three) tightly linked loci are viable explanations for this polymorphic system(s). It is suggested that the three factors, Pa, Pr, and Db, be treated as separate loci to allow clarification of their genetic relationships.

Thirty-eight urinary bladders from Fischer rats were organ cultured for 7 days in Ham's Medium F12 with varying concentrations of hydrocortisone added. Measurements of cell numbers and relative areas of nuclei and cytoplasm (N/C ratios) were made with an automated image analyzer on 1.5-micronm-thick plastic sections of the tissues. Epithelia cultured in the absence of hydrocortisone became hyperplastic and dysplastic and contained approximately 2.5 times as many cells per unit length as uncultured controls. N/C ratios were increased by over 50%, caused mainly by a decrease in the cytoplasmic area. Concentrations of hydrocortisone below 10(-7) M had no significant effect on the hyperplasia or N/C ratios. Increasing concentrations above 10(-7) M resulted in progressive decreases of both the number of cells per unit length and the N/C ratios. Epithelia cultured in 2 X 10(-5) M hydrocortisone were not significantly different from uncultured controls. N/C ratios from 85 fields with a wide range of values were quanititated both with the image analyzer and a manual point-counting technique. Linear regression analysis revealed a close linear relationship and a correlation coefficient of 0.922. The analyzer provided an efficient, accurate tool for quantitating morphological observations.

In view of the correlation shown in vivo between vitamin A deficiency and increased transitional cell proliferation, the ability of retinoic acid to block hyperplasia in organ-cultured bladder epithelium was investigated. Retinoic acid, at concentrations shown to block hyperplasia in other in vitro systems, was ineffective in blocking hyperplasia in bladder epithelium. The data suggest the possibility that, in vivo, vitamin A may not act directly on the transitional epithelium to control cell proliferation. The cytoplasmic volume, which normally decreases during the period of hyperplasia in vitro, was increased by retinoic acid. The increase in cytoplasmic volume stimulated by 0.3 micrometer retinoic acid was sufficient to maintain normal nuclear:cytoplasmic ratios during the hyperplastic response. The increase in cytoplasmic volume could not be correlated with increased protein, mucin, or glycogen synthesis. The data suggest that retinoic acid may have increased the permeability of the luminal membrane to water, possibly by interfering with the synthesis of the keratin component of the membrane.

A system for organ culture of rat bladder in defined medium, without serum, is described. Transitional epithelial morphology of normal bladder was well maintained in Waymouth's Medium MB 752/1, while Ham's Medium F12 caused marked epithelial hyperplasia. Hyperplastic epithelial states induced by in vivo administration of the carcinogen, N-methyl-N-nitrosourea, were well maintained in vitro during organ culture in Medium MB 752/1. The combination of hydrocortisone and insulin markedly inhibited the proliferative response of normal bladder epithelium grown in Medium F12.

Ferric ions and adriamycin in solution interact to form complexes that can yield colloidal and flocculant mixtures. At high concentrations (Fe3+ greater than or equal to 10(-4) M, adriamycin greater than or equal to 10(-5) M) an absorption appears at 600 nm, indicating colloid formation, which is directly responsive to concentrations of the reactants. Evidence from dilution experiments by spectral analysis, ultracentrifugation, titration, and filtration indicate that phase transition that is sensitive to pH and time occurs with iron-adriamycin complexes to yield flocculated drug. We conclude that patients and animals treated with the iron-adriamycin preparations known as 'quelamycin' received flocculated iron-adriamycin, which accounts for the toxic and pharmacologic effects reported. It may be useful to utilize colloidal preparations of reactive or irritating drugs to avert acute toxic effects and to produce slower release of active drug.

The effects of the bladder carcinogens butyl(4-hydroxybutyl)nitrosamine (BBN) and butyl(3-carboxypropyl)-nitrosamine (BCPN) on proliferating transitional rat epithelium in organ culture were studied. At low to intermediate concentration ranges (0.5--2.9 mM), both compounds appeared to stimulate hyperplasia in some regions of epithelia. The major effect of both carcinogens, however, was to suppress hyperplasia in other regions of epithelia and, at higher concentrations (5--6 mM), to cause necrosis. For comparable concentrations, BBN was more effective in suppressing proliferation and causing necrosis than was BCPN.