Cilazapril reverses endothelium-dependent vasodilator response to acetylcholine in mesenteric artery from spontaneously hypertensive rats

This study was designed to evaluate the effect of chronic treatment with cilazapril on vascular reactivity of aorta and mesenteric artery from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Cilazapril (5 mg/kg), an angiotensin converting enzyme inhibitor, was injected intraperitoneally twice a day for 4 weeks. Results demonstrated that acetylcholine (ACh)-induced relaxation in aorta and mesenteric artery from SHR was significantly less than that from WKY, cilazapril-treated WKY, and SHR. The impairment of ACh-induced relaxation in SHR was significantly reversed after cilazapril treatment and there were no significant differences among WKY, cilazapril-treated WKY, and SHR. Meanwhile, both N omega-nitro-L-arginine (LNNA; 10(-4) mol/L) and methylene blue (MB; 10(-5) mol/L) completely blocked the vasodilator response to ACh in aorta but only partly inhibited in mesenteric artery from WKY, cilazapril-treated WKY, and SHR. These LNNA- and MB-resistant vasodilator responses to ACh in mesenteric artery were only slightly inhibited by TEA (10(-3) mol/L) but not by indomethacin (5 x 10(-6) mol/L). These findings suggest that there may be an unidentified endothelium-dependent relaxing factor(s) (EDRF), which exists in the endothelium and may participate in the modulation of blood pressure in SHR. Results further demonstrate that the antihypertensive effect of cilazapril may be partly mediated by the reversing function of endothelium to release EDRF and LNNA-resistant, unidentified relaxing factor(s).     

Facilitation of glutamate receptors reverses an age-associated memory impairment in rats

The aim of this study was to clarify the inheritance of the nuclear ribosomal DNA (rDNA) in the ectomycorrhizal basidiomycete Laccaria bicolor S238N in order to resolve inter- and within-strain relationships in forest ecosystems. PCR amplification of the intergenic spacer (IGS) was carried out in the dikaryotic mycelium and its haploid progeny. In the dikaryotic mycelium, multiple amplification products were produced for the 25s/5s (IGS1) and 5s/17s (IGS2) intergenic spacers. The 4.5- and 4.0-kb fragments of IGS2 (haplotypes alpha and beta, respectively) were observed to occur in a 1:1 ratio within the haploid progeny as a result of divergent IGS haplotypes in the two separate nuclei. Recombinant monokaryons having both types of IGS2 occurred at a low frequency (6.5%; 60 kb per centimorgan) during meiosis. Haplotypes alpha and beta of IGS1 cross-hybridized forming heteroduplexes during the PCR temperature cycle. The two IGS1 haplotypes differed only by the repeat number of a TA2C3 motif and co-segregated with the IGS2 haplotypes. Heteroduplex formation and IGS polymorphism provide information that is helpful in distinguishing between introduced exotic L. bicolor S238N and indigenous populations of Laccaria spp. in forest ecosystems.     

Reversal of innate aversions: Attempts to induce a preference for chili peppers in rats.

Although humans frequently develop preferences for innately unpalatable bitter or irritant substances, such preferences are extremely rare in animals. An attempt was made to understand the nature of this difference by 5 experiments with Charles River albino rats, using chili pepper as the unpalatable substance. In parallel with major aspects of the human experience with chili pepper, Ss were exposed to it as a flavoring in all their food for periods up to 11 mo from birth without significant preference enhancement. Gradual introduction of chili into the diet also had no effect, nor did poisoning and safety experiences designed to teach Ss that only chili-flavored foods were safe to eat. Seven pairings of chili-flavored diet with prompt recovery from thiamine deficiency did significantly attenuate the innate aversion and may have induced a chili preference in at least 1 case. Extensive experience with chili did not reliably make Ss much less sensitive to its oral effects. The only reliable way to eliminate chili aversion in rats is to destroy their chemical irritant sense, which was accomplished in 1 group. (29 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Comparison of the abilities of MHC-compatible bone marrow cells and lymph node cells to induce tolerance of skin allografts in rats

A comparison has been made of the abilities of bone marrow cells and lymph node cells to induce tolerance of skin when inoculated into neonatal rats known to differ only with regard to non-MHC incompatibilities, including putative skin-specific (Skn) antigens. Each recipient received 50 x 10(6) cells, and tolerance was confirmed by the permanent acceptance of donor-strain neonatal heart tissue transplanted to the pinna of the ear. In 5 of the 8 MHC-compatible strain combinations tested, BMC were significantly more effective than LNC in inducing tolerance of skin, whereas in one situation LNC proved more efficient. Although the results are in accord with the occurrence of Skn antigens in rats, it appears that at least some of the antigens involved must also be expressed by BMC or LNC, but not equally by both of these tissues. The results also provide evidence that in rats, as in mice, the MHC can play a major role in determining the response to, and/or the immunogenicity of, Skn antigens.     

Adjustment of adolescents to chronic disability.

Investigated the relationship between adolescents' adjustment to a chronic disability and 8 demographic and attitudinal variables. Adjustment was defined as the total positive score on the Tennessee Self-Concept Scale. Ss were 59 12–18 yr old females with idiopathic scoliosis and their mothers. Analysis identified the following 4 variables, in decreasing order, that correlated with the adjustment of adolescents to scoliosis: (a) adolescents' tendency toward the container-isolator end of B. A. Wright's (1960) theoretical continuum of coping responses to a physical disability as measured by an attitude toward disability test, (b) a dimension of maternal attitude toward disability as measured by a disability factor scale, (c) a dependency factor of maternal attitude toward females and persons with scoliosis as measured by a factor analyzed 70-item semantic differential scale, and (d) a maternal high self-concept factor from the semantic differential scale. Ss did not differ significantly from peers on the adjustment measure, and implications for treatment and rehabilitation are discussed. (19 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The association of yogurt starters with Lactobacillus casei DN 114.001 in fermented milk alters the composition and metabolism of intestinal microflora in germ-free rats and in human flora-associated rats

The aim of this study was to compare the effects of milk and of various fermented milks on the composition and metabolic activities of the intestinal microflora. Groups of eight rats were fed for 6 wk a diet containing 30% nonfermented milk (M), yogurt (Y), milk fermented with Lactobacillus casei (LcFM) or milk fermented with the association of L. casei DN 114.001 and yogurt starters (LcYFM). In the first study, the survival of the lactic acid bacteria from the fermented milks was assessed by bacterial enumeration in feces of germ-free rats (GF rats) fed milk or fermented milks. The metabolic activities of the lactic acid bacteria were studied in these rats by the measurement of glycolytic activities and products of bacterial fermentation, i.e., acetate and lactate (isoforms L and D). In a second study, the effects of fermented milks on the composition and metabolism [gas, glycolytic activities, short-chain fatty acids (SCFA), alcohol and ammonia] of human flora were studied using human flora-associated rats (HF rats). In GF rats, the survival of L. casei in the feces did not differ between those fed the LcFM and LcYFM diets. L. bulgaricus was detected in the feces of the rats fed Y, whereas Streptoccus thermophilus was found in the feces of the LcYFM group. In HF rats, fecal concentration of Bifidobacteria was greater in the LcFM group than in the others. beta-Glucuronidase (EC 3.2.1.31) activity was lower in rats fed LcFM and Y than in those fed M and LcYFM, whereas beta-galactosidase (3.2.1.23), alpha-glucosidase (EC 3.2.1 20) and beta-glucosidase (EC 3.2.1.21) activities were higher in the LcYFM group compared with the others. Methane excretion was higher in rats fed Y than in other groups. Cecal SCFA concentrations did not differ in LcFM, Y and M groups, but total SCFA, acetate, propionate and butyrate were significantly greater in the LcYFM group. These results suggest that milk fermented with the combination of L. casei and yogurt starters leads to specific effects that are different from the simple addition of the effects found with yogurt and milk fermented with L. casei. These specific effects are potentially beneficial to human health.     

Estradiol implants in the arcuate nucleus induce lactogenesis in virgin rats. Role of progesterone

Peroxisomal proliferators induce in rodents hepatic hyperplasia and hypertrophy; the significant increase in the peroxisomal population is accompanied by specific and reversible induction of some peroxisomal enzymes. In suckling rats born from clofibrate-treated mothers, a massive removal of proliferated organelles occurs within 3 days of recovery. In the present paper we examined the early stages of the recovery period in liver of male rats treated with clofibrate for 5 days. The lysosomal involvement in the removal of drug-induced peroxisomes was investigated under physiological conditions, i.e. in the absence of inhibitors of the autophagic process. Biochemical results indicate that peroxisomal beta-oxidation, but not catalase activity, returns to the control values within the examined period. Total acid phosphatase activity is not affected by clofibrate treatment, but following fractionation on a linear density gradient the lysosomal marker enzyme activity is shifted towards lower density values, particularly at day 1 and 2 of recovery. This class of organelles possibly represents lysosomes involved in active autophagic processes. Acid phosphatase cytochemistry shows an increase of lysosome number at day 1 of recovery. Combination of acid phosphatase cytochemistry either with catalase cytochemistry or with catalase immunogold labelling allows to reveal organelles containing both marker enzymes. These results strongly support the involvement of autophagic processes in the removal of proliferated peroxisomes.     

Retrograde injections of formaldehyde into the biliary tree induce alterations of biliary epithelial function in rats

Formaldehyde may induce severe lesions of intrahepatic and extrahepatic bile ducts. The purpose of this study was to examine in vivo the functional consequences of an alteration of the biliary epithelium induced by a retrograde intrabiliary injection of formaldehyde in rats. After basal bile collection, a 10% formaldehyde solution was injected into the biliary tree of anesthetized rats, and the cannula was occluded for 30 minutes. Choleresis was then reestablished, and bile flow, bile acid, and bicarbonate secretion were measured both spontaneously and during ursodeoxycholate infusions. Formaldehyde injections induced a significant increase in bile flow and a marked inhibition of ursodeoxycholate-induced increase in biliary bicarbonate concentration and secretion. Biliary glucose secretion, which is normally very low, was increased about 20-fold in animals injected with formaldehyde. Histological and ultrastructural examination of the liver showed alterations of biliary epithelial cells, whereas hepatocytes, bile canaliculi, and canalicular tight junctions remained normal. Hepatocytic excretory function, as assessed by biliary secretion of bile acids, was not affected. It was concluded that short-term formaldehyde intrabiliary injections cause an inhibition of ursodeoxycholate-induced hypersecretion of bicarbonate, an increase in biliary glucose secretion, and selective structural alterations of biliary epithelial cells. These results suggest that formaldehyde retrograde biliary injection may be a useful model to study alterations of biliary epithelial function in vivo.     

Naloxone and initial estrogen action to induce lordosis in ovariectomized rats: the effect of a cut between the septum and preoptic area

The objective of the study was to investigate the sociodemographic and psychological factors which are associated with choice or refusal of regional anesthesia for lower segment Caesarean section. A semi-structured questionnaire was administered preoperatively to consecutive women presenting for elective operation at two hospital sites in the same health authority. The questionnaires were administered to 39 pregnant women by two anesthetists, one at each site. Sociodemographic and past and present obstetric and anesthetic details were obtained. The Hospital Anxiety and Depression scale and the FEAR questionnaire were used with an additional visual analog scale to assess fearfulness associated with the proposed operation. A 'panic checklist' of ten items was also constructed to identify procedures associated with feelings of panic. Ten women requested general anesthesia. These women had a similar level of anxiety to the others, but were significantly more depressed, had had more pregnancies overall and more without live babies. There were also more aspects of the procedure at which they thought they might panic. Women who have suffered the loss of a conception with its associated grief and often obstetric intervention are more reluctant to be conscious during an operative delivery for a subsequent baby. Preventive measures should be considered at the time of fetal loss to avoid this. However, preoperatively a simple visual analog scale for fearfulness would identify these women so that any unresolved fears could receive full psychological assessment.     

Reframing the meaning of disability to families: the embrace of paradox

In our society, families turn to medical science and health practitioners for assistance when a family member becomes disabled. In the cases of chronic illnesses and developmental disabilities, health professionals may focus interventions on the disease processes, feeling unprepared to deal with the broader issues families struggle with in managing disability. In addition, collaboration between professionals and parents may be strained when family's discourse is viewed as unrealistic, overly emotional, exceedingly optimistic and confusing by professionals. Using in-depth case studies, this qualitative research examined the daily occupations and subjective well-being of six Mexican-origin mothers parenting children with disabilities. Emergent findings demonstrated that these mothers do express apparently contradictory emotions of grief and joy, hope and fear that influenced their subjective well-being. The meaning of the mothers' contrary statements was illuminated in a life metaphor, the embrace of paradox. The embrace of paradox was the management of the internal tension of opposing forces between loving the child as he or she was and wanting to erase the disability, between dealing with the incurability while pursuing solutions and between maintaining hopefulness for the child's future while being given negative information and battling their own fears. The tensions created by the paradox promoted an internal striving to maintain a tenuous hopefulness despite "crashes" in their maternal work, and promoted an extension of maternal skills working toward a hopeful life trajectory for the mother and her child. In the embrace of paradox mothers created a positive bias and regained a sense of control that fueled their optimism in maternal work.     

Ex vivo gastrointestinal biotransformation of chlorothalonil in the germ-free and conventional rat

1. The metabolism and absorption of chlorothalonil and corresponding diglutathione and dicysteine conjugates was studied using isolated everted gastrointestinal sacs of the conventional and germ-free rat. An HPLC method was used to analyse mucosal and serosal fluids. Thiol metabolites of chlorothalonil were determined by GC/MS. 2. Low absorption of the substrates was observed, with < 4% of the radioactivity being recovered from the serosal buffers and the digestive tissues. A major part of the radioactivity was recovered from the mucosal fluids and it corresponded to unchanged chlorothalonil. Traces of unchanged chlorothalonil and mono-, di- and trimethylthio metabolites were present in serosal fluids as well as unidentified polar peaks. An important transformation (> 75%) of the chlorothalonil conjugates was observed. The di- and trimethylthio metabolites of chlorothalonil were detected from both sides of the everted gut sac of rat incubated with the diglutathione and dicysteine conjugates. 3. Few differences were observed between the conventional and germ-free rat: absorption was higher in the duodenum of germ-free rat, but tissue retention was more significant in the duodenum of the conventional rat.     

A-ring reduced derivatives of two synthetic progestins induce anxiolytic effects in ovariectomized rats

This study was to determine the extent of alteration of enolase specific activities in chicken embryo retina primary cells in culture when exposed to an ELF (extremely low frequency) electric field at 60 Hz. Results showed no alteration of enolase activity and enolase mRNA levels. In this study, sham vs. control experiments were also conducted to neutralize ambient AC magnetic fields, stray magnetic fields and variations in field uniformity. Under similar conditions, the specific activity of enolase is decreased in neuroblastoma cell line (NG108). It is apparent from this study that primary cells either are not affected by these exposure conditions or the effect is transient and warrants no damage.     

Changing the relative amplitudes of wave packets to induce nonlocal effects

The short- and long-term effects of an elemental diet in children with acute Crohn's disease were compared with those of prednisolone in historical controls. Clinical remission was induced in 25 of 30 and in 18 of 28 episodes treated for six weeks with an elemental diet and prednisolone. Patients with proximal disease had longer remission after treatment with an elemental diet (p < 0.05) than did patients with colonic disease after treatment with prednisolone (p < 0.01). Disease activity index score improved in both groups compared with the pretreatment scores (p < 0.05). However, the improvement in the elemental diet group was significantly better than in the prednisolone group (p < 0.001). Changes in linear growth were better after treatment with an elemental diet compared with steroids (p < 0.001). Serum albumin and haematocrit concentrations all improved significantly in the children treated with an elemental diet (p < 0.001) but not in those treated with steroids. Thus an elemental diet was better than prednisolone in proximal disease and confirmed improved growth and nutritional status.     

Barriers to the provision of mental health services to individuals with severe physical disability.

Surveyed 86 consumer advocates (67 with physical disabilities), 157 rehabilitation counselors, 94 rehabilitation administrators, 323 mental health administrators and professionals, and 145 severely disabled Ss concerning Ss' perceptions of the nature and extent of mental health services, barriers to mental health service provision for individuals with severe physical disabilities, and personal data. Overall, there was a general consistency among the sample groups in their perceptions of the extent to which each of 10 variables is a barrier to service delivery. The lack of accessible public transportation, especially in nonmetropolitan areas, and the accompanying limitation on costly mental health outreach services were viewed as serious problems. The limited knowledge and skills of many mental health professional regarding severe physical disability was perceived to be a moderate to substantial barrier to service provision. Findings indicate that individuals with severe physical disability are an underserved group. Recommendations regarding policy and program development and training of professionals are offered. (34 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effect of sodium rhein on electrically-evoked and agonist-induced contractions of the guinea-pig isolated ileal circular muscle

1. This study examined the effects of sodium rhein (0.03-30 microM) on the contractions of the isolated circular muscle of guinea-pig ileum induced by acetylcholine (100 nM), substance P (3 nM) and electrical stimulation (10 Hz for 0.3 s, 100 mA, 0.5 ms pulse duration). The effect of sodium rhein was also evaluated on the ascending excitatory reflex using a partitioned bath (oral and anal compartments). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 20 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the guinea-pig intestinal circular muscle in the oral compartment was recorded. 2. Sodium rhein (0.3, 3 and 30 microM) significantly potentiated (52+/-11% at 30 microM) acetylcholine-induced contractions. In the presence of tetrodotoxin (0.6 microM) or omega-conotoxin GVIA (10 nM) sodium rhein (3 and 30 microM) did not enhance, but significantly reduced (49+/-10% and 44+/-8%, respectively, at 30 microM) acetylcholine-induced contractions. 3. Sodium rhein (0.3, 3 and 30 microM) significantly increased (65+/-11% at 30 microM) substance P-induced contractions. In the presence of tetrodotoxin (0.6 microM), omega-conotoxin GVIA (10 nM) or atropine (0.1 microM), sodium rhein (3 and 30 microM) significantly reduced (50+/-10%, 55+/-8% and 46+/-10%, respectively, at 30 microM) substance P-induced contractions. 4. NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) abolished the potentiating effect of sodium rhein on acetylcholine and substance P-induced contractions. At the highest concentration (30 microM), sodium rhein, in presence of L-NAME, reduced the acetylcholine (30+/-6%)- or substance P (36+/-6%)-induced contractions. 5. Sodium rhein (30 microM) significantly potentiated (29+/-9%) the electrically-evoked contractions. L-NAME (100 microM), but not phentolamine, enhanced the effect of sodium rhein. Sodium rhein (30 microM) significantly increased (32+/-9%) the ascending excitatory reflex when applied in the oral, but not in the anal compartment. 6. These results indicate that sodium rhein (i) activates excitatory cholinergic nerves on circular smooth muscle presumably through a facilitation of Ca2+ entry through the N-type Ca2+ channel, (ii) has a direct inhibitory effect on circular smooth muscle and (iii) does not affect enteric ascending neuroneural transmission. Nitric oxide could have a modulatory excitatory role on sodium rhein-induced changes of agonist-induced contractions and an inhibitory modulator role on sodium rhein-induced changes of electrically-induced contractions.     

Exposure of female rats to a 100-microT 50 Hz magnetic field does not induce consistent changes in nocturnal levels of melatonin

The hypothesis whereby alternating (50 or 60 Hz) magnetic fields such as those produced by electric power reduce the nocturnal production of melatonin in the pineal gland and thereby indirectly enhance development and growth of breast cancer has attracted a great deal of interest. In view of the potential importance of this hypothesis that there is a link between electric power and breast cancer, which is also known as the "melatonin hypothesis", we undertook various experiments in female Sprague-Dawley rats to evaluate whether 100-microT 50 Hz magnetic-field exposure, i.e. a flux density shown recently to exert a tumor (co)promoting effect in the 7,12-dimethylbenz[a]anthracene (DMBA) model of breast cancer in Sprague-Dawley rats, consistently reduces melatonin levels and, if not, which factors may be involved in the inconsistent effects of magnetic-field exposure on production of melatonin. Long-term exposure of female Sprague-Dawley rats to magnetic fields for 13 weeks did not alter the nocturnal levels of melatonin in the pineal gland or serum (determined 5 h after the onset of darkness) significantly, irrespective of whether rats were treated with DMBA or not. In one experiment, when blood was sampled 3, 5 and 6 h after the onset of darkness after 2 weeks of magnetic-field or sham exposure, a significant decrease in melatonin was seen in magnetic-field-exposed rats at 6 h. However, the results could not be reproduced in two subsequent experiments in other groups of rats. Shorter (1 day, 1 week) or longer (4, 8, 13 weeks) exposure periods also did not result in any significant effects of the magnetic field on melatonin levels when blood sampling was performed either 5 or 6 h after onset of the dark phase. Various potential sources of variation in melatonin levels or in magnetic-field effects on melatonin levels were evaluated, but the reason(s) for the inconsistent effect of magnetic-field exposure remains unclear. Thus the present study failed to demonstrate a consistent effect of 100-microT 50 Hz magnetic-field exposure on melatonin levels in Sprague-Dawley rats.     

Development of a scoring system to assess disability in an Italian elderly population. Evaluation of a disability questionnaire

Patients with idiopathic and symptomatic restless legs syndrome (RLS) suffer from "dyskinesia while awake" or "daytime myoclonus" when at rest preceded by sensory symptoms. In order to characterise the RLS either as reflex movement or as voluntary movement we measured movement-related cortical potentials in 5 idiopathic and 8 uraemic RLS patients. Movements from both legs were polygraphically recorded concomitantly with cortical activity 2000 msec before to 500 msec after onset of EMG activity. These data were compared with a voluntary simulation of each patient's movement pattern and with 5 age-matched controls performing dorsiflexion of the right, left and both feet. Cortical activity preceding daytime myoclonus was absent in RLS patients whereas self-initiated leg movements in patients elicited onset times (1180-1380 msec) and amplitudes of Bereitschaftspotential (readiness potential) not significantly different from readiness potentials in control subjects (P > 0.05). Lack of movement-related potentials in myoclonus and/or dyskinesias during daytime in RLS patients is compatible with an involuntary mechanism of induction and points towards a subcortical or spinal origin of RLS.     

Modification of aspartic acid residues to induce trypsin cleavage

1,2-Diaminoethane and diaminomethane were coupled to aspartic acid residues in small peptides by means of a water-soluble carbodiimide. The resulting modified side chains sufficiently resembled lysine for trypsin to cleave the peptides. Similar modification of glutamic acid residues in peptides gave little or no susceptibility to trypsin.     

Ras signals to the cell cycle machinery via multiple pathways to induce anchorage-independent growth

Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.