Predicting cadmium concentrations in wheat and barley grain using soil properties.

The entry of Cd into the food chain is of concern as it can cause chronic health problems. To investigate the relationship between soil properties and the concentration of Cd in wheat (Triticum aestivum L.) and harley (Hordeum vulgare L.) grain, we analyzed 162 wheat and 215 barley grain samples collected from paired soil and crop surveys in Britain, and wheat and barley samples from two long-term sewage sludge experiments. Cadmium concentrations were much lower in barley grain than in wheat grain under comparable soil conditions. Multiple regression analysis showed that soil total Cd and pH were the significant factors influencing grain Cd concentrations. Significant cultivar differences in Cd uptake were observed for both wheat and barley. Wheat grain Cd concentrations could be predicted reasonably well from soil total Cd and pH using the following model: log(grain Cd) = a + b log(soil Cd) - c(soil pH), with 53% of the variance being accounted for. The coefficients obtained from the data sets of the paired soil and crop surveys and from long-term sewage sludge experiments were similar, suggesting similar controlling factors of Cd bioavailability in sludge-amended or unamended soils. For barley, the model was less satisfactory for predicting grain Cd concentration (22% of variance accounted for). The model can be used to predict the likelihood of wheat grain Cd exceeding the new European Union (EU) foodstuff regulations on the maximum permissible concentration of Cd under different soil conditions, particularly in relation to the existing Directive and the proposed new Directive on land applications of sewage sludge.

Cadmium content of wheat grain from a long-term field experiment with sewage sludge.

Grain Cd concentrations were determined in the wheat (Triticum aestivum L.) cultivars Soissons, Brigadier, and Hereward grown in 1994,1996, and 1999, respectively, in soils of a long-term field experiment to which sewage sludges contaminated with Zn, Cu, Ni, or Cr had previously been added. Soil pore water soluble Cd and free Cd2+ increased linearly with increasing total soil Cd (R2=0.82 and 0.84, respectively; P<0.001). Similarly, soil pore water free Cd2+ increased linearly with increasing soil pore water soluble Cd (R2=0.98; P<0.001). There was no evidence of a plateau in soil pore water Cd concentrations with increasing soil Cd concentrations. Grain Cd concentrations were significantly correlated with total soil Cd (P<0.001), soil pore water Cd (P<0.001), and free Cd2+ (P<0.001). A slight curvilinear relationship between grain Cd and soil Cd was apparent, but there was no plateau, even at the maximum soil Cd concentration of about 2.7 mg kg(-1). The relationship between soil pore water Cd and grain Cd was linear for all three cultivars. The slopes were in the order 1994 > 1996 > 1999, with more Cd being taken up into the grain by Soissons grown in 1994, and least by Hereward grown in 1999. For Soissons, Cd concentration in the grain greater than the EU limit (0.24 mg kg(-1) dry wt.) occurred at soil Cd less than the current UK limit of 3 mg kg(-1) for soils receiving sewage sludge. In contrast, for Brigadier and Hereward, grain Cd concentrations were near to and less than the EU limit, respectively, at soil Cd concentrations of 3 mg kg(-1).

Enhanced acetaminophen hepatotoxicity in transgenic mice overexpressing BCL-2.

Mitochondria play an important role in the cell death induced by many drugs, including hepatotoxicity from overdose of the popular analgesic, acetaminophen (APAP). To investigate mitochondrial alterations associated with APAP-induced hepatotoxicity, the subcellular distribution of proapoptotic BAX was determined. Based on the antiapoptotic characteristics of BCL-2, we further hypothesized that if a BAX component was evident then BCL-2 overexpression may be hepatoprotective. Mice, either with a human bcl-2 transgene (-/+) or wild-type mice (WT; -/-), were dosed with 500 or 600 mg/kg (i.p.) APAP or a nonhepatotoxic isomer, N-acetyl-m-aminophenol (AMAP). Immunoblot analyses indicated increased mitochondrial BAX-beta content very early after APAP or AMAP treatment. This was paralleled by disappearance of BAX-alpha from the cytosol of APAP treated animals and, to a lesser extent, with AMAP treatment. Early pathological evidence of APAP-induced zone 3 necrosis was seen in bcl-2 (-/+) mice, which progressed to massive panlobular necrosis with hemorrhage by 24 h. In contrast, WT mice dosed with APAP showed a more typical, and less severe, centrilobular necrosis. AMAP-treated bcl-2 (-/+) mice displayed only early microvesicular steatosis without progression to extensive necrosis. Decreased complex III activity, evident as early as 6 h after treatment, correlated well with plasma enzyme activities at 24 h (AST r(2) = 0.89, ALT r(2) = 0.87) thereby confirming a role for mitochondria in APAP-mediated hepatotoxicity. In conclusion, these data suggest for the first time that BAX may be an early determinant of APAP-mediated hepatotoxicity and that BCL-2 overexpression unexpectedly enhances APAP hepatotoxicity.

African-American women's perceptions of mammography screening.

While mammography has been shown to decrease breast cancer mortality, many African-American women are not receiving annual screenings. African-American women's reasons for not having mammograms are not well understood. This study therefore surveyed 164 African-American women concerning barriers to mammography screening. Outreach coordinators in two urban and one rural site in Texas asked African-American women to complete a checklist about the barriers to mammography screening. The 23-item Mammography Barriers Checklist, which was developed based upon one of the author's clinical experience and the research literature, included both internal and external barriers to screening. Women in all three geographic areas identified fear of finding cancer and mammography cost as the most important reasons for not having mammograms. These results suggest that outreach strategies that address fears related to mammography screening and help women find low-cost mammography resources may be more effective than those focusing strictly on providing information.

Experimental carpal reverse-flow pedicle vascularized bone grafts. Part I: the anatomical basis of vascularized pedicle bone grafts based on the canine distal radius and ulna.

We investigated the detailed extraosseous and intraosseous vascular anatomy of the distal radius and ulna in 55 pairs of canine forelimbs. Ten specimens were used for compartment identification. One hundred specimens were investigated after intra-arterial injection with red latex or Batson's compound. The extraosseous vascular network, including the nutrient artery location, was studied by delicate dissection and soft tissue digestion. Two superficial and 4 deep arteries were found on the dorsal aspect, with constant relationships to the retinaculum. On the palmar aspect, bony nutrient vessels rose from the branches of the radial artery (descriptive anatomy). The vascular supply of the canine distal forelimb was very consistent and enabled the design of 5 vascularized pedicle bone grafts similar to those described in humans (applied anatomy). These reverse-flow pedicle grafts may be placed in the carpus, allowing the canine forelimb to serve as an experimental model for the study of vascularized carpal bone grafting. (J Hand Surg 2000; 25A:34-45.

Experimental carpal reverse-flow pedicle vascularized bone grafts. Part II: bone blood flow measurement by radioactive-labeled microspheres in a canine model.

Reverse-flow pedicle vascularized bone grafts (VBGs) from the radius, commonly used for carpal nonunion and avascular necrosis, provide superior clinical results. In this experimental study bone blood flow in canine distal radius VBGs was measured to determine the status of the bone circulation immediately after elevation (group 1) and 2 weeks later (group 2). Immediate VBG flow (group 1) was substantial, but significantly less than the contralateral undisturbed distal radius (8.42 mL/min/100 g and 16.53 mL/min/100 g, respectively). At 2 weeks after surgery (group 2) VBG flow was compared with nonvascularized control grafts. Vascularized bone graft flow was significantly higher than group 1 (mean, 33.72 mL/min/100 g). Minimal flow was seen in the conventional graft control (0.62 mL/min/100 g). This study demonstrates that reverse-flow pedicle radius VBG maintain enhanced bone circulation long-term. Given the similarity of human and canine distal radius vascular anatomy, these data support the clinical use of reverse-flow VBG for carpal pathology. (J Hand Surg 2000; 25A:46-54.

Physician-patient interactions regarding diet, exercise, and smoking.

OBJECTIVES: The objectives were to determine the rate of physician/patient discussions regarding diet, exercise, and smoking and to assess the effect of such discussions on behavior change. METHODS: In a telephone survey of Connecticut adults, respondents who had a routine checkup in the past year (n = 433) were asked whether their physicians had asked them about their dietary habits, exercise, or smoking, and about any efforts to modify these behaviors during the preceding year. RESULTS: Diet was addressed with 50% of the subjects, exercise with 56%, and smoking status with 77%. Respondents who were asked about their diet were more likely to have changed their fat or fiber intake in the past year than those not asked (64 vs. 48%, P = 0.002) and were somewhat more likely to have lost weight (46 vs. 37%; P = 0.061); the differences were even greater among 94 overweight subjects (64 vs. 47%; P = 0.099). No behavior change was associated with discussions of exercise or smoking. CONCLUSIONS: Physicians have the potential to impact health behaviors, especially those related to diet, through simple discussions during routine checkups, but only about half are using this opportunity.

Nitric oxide mediates mecamylamine- and naloxone-precipitated nicotine withdrawal.

A nitric oxide synthase inhibitor blocked nicotine abstinence signs and increased weight loss in male, nicotine-dependent rats during withdrawal precipitated by the nicotinic receptor antagonist mecamylamine or the opioid receptor antagonist naloxone. These results indicate that nitric oxide systems mediate important aspects of the expression of nicotine physical dependence and suggest the potential use of nitric oxide synthase inhibitors as aids in tobacco smoking cessation.

Is phototherapy safe for HIV-infected individuals?

Patients infected with human immunodeficiency virus (HIV) have a high prevalence of UV radiation-responsive skin diseases including psoriasis, pruritus, eosinophilic folliculitis and eczemas. On the other hand, UV has been shown to suppress T cell-mediated immune responses and to induce activation and replication of HIV. These developments have prompted clinicians and investigators to question whether phototherapy is safe for HIV-infected individuals. We have reviewed these issues and hereby provide a summary and critique of relevant laboratory and clinical evidence.

Effect of human adrenomedullin on vascular resistance of the canine tibia.

An ex vivo model of a perfused canine tibia was used to investigate the effect of human adrenomedullin, a novel peptide with known vasodilator properties, on the vascular resistance of bone. Human adrenomedullin has a potent and long-lasting vasodilator effect in the canine tibia following precontraction of vascular smooth muscle by infusion of prostaglandin F2 alpha. A 0.1 ml bolus injection of 10(-5) M human adrenomedullin suppressed the pressor response of the canine tibia preparation to an infusion of norepinephrine by 43-52% for a duration of 100 minutes. An injection of 10(-6) adrenomedullin suppressed the pressor response to an infusion of norepinephrine by 22-23% for a duration of 40 minutes. These data suggest that human adrenomedullin may be a potent and long-acting vascular smooth-muscle relaxant in bone.

The public health impact and economic cost of smoking in Connecticut--1989.

Estimates of the smoking-attributable morbidity, mortality, and economic costs for Connecticut for 1989 were made using software distributed by the Centers for Disease Control and Prevention. The software calculations are based on relative risks for smoking-related diseases from major prospective studies. Using smoking prevalence, mortality, population, and health-care expenditure data for the state, 19.3% of all deaths in Connecticut in 1989 were estimated to be related to smoking. Cardiovascular disease and cancer accounted for the largest number of these estimated deaths. Men who died from smoking-related deaths lost an estimated 11.6 years of life while women died an estimated 12.8 years prematurely from smoking-related causes. The total cost, including direct and indirect smoking-related costs, was estimated to be $944 million, or $287 for each man, woman, and child in the state.

Outcome of carpal tunnel surgery in Washington State workers' compensation.

All cases of occupational carpal tunnel syndrome (OCTS) who received surgery for this condition in the Washington State workers' compensation system were identified using claim and physician billing databases. One hundred ninety-one incident surgical cases were identified between July 1, 1987 and December 31, 1987, and were followed up a mean of 3 years postoperatively for clinical, disability, and return to work outcomes. Medical record and claim file review was required for clinical and employment information. The mean age of all patients was 36.6 years, 48% were female, and 40% received bilateral surgery. The mean time from claim filing to surgery was 187 days. Ninety-eight percent of cases met the National Institute for Occupational Safety and Health (NIOSH) case definition for OCTS. Relief of pain was complete or modest in 86% (124/145) and only 14% of cases reported no improvement in symptoms. Mean duration of disability (time loss) postoperatively was nearly 4 months, and 8% of cases exceeded 1 year of time loss. The majority of cases returned to their same job (67%) or to a different job (15%). Workers in high risk occupations were less likely to return to the same job after CTS surgery compared to those in lower risk occupations (61% vs. 75%, p = 0.08). In this population, no association was seen between any outcome and age, gender, marital status, or baseline wage. Duration of disability was not significantly related to preoperative severity of OCTS or to more specific case criteria for this condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Preservation of bone graft vascularity with the University of Wisconsin cold storage solution.

The preservation of the microcirculation of bone has been evaluated with use of an in vitro canine tibia perfusion model. The production of relaxing factors by the osseous vascular endothelium was used as a metabolic marker for viability. This endothelial eccrine function was preserved for 5 days (120 h) by cold storage without continuous perfusion after a washout with the University of Wisconsin (UW) solution. This synthetic perfusate was superior to Krebs Ringer solution (p < 0.05), but storage without perfusion failed to prevent a significant rise in vascular resistance. Two techniques were effective for the preservation of bone vascularity for 24 h: washout with UW solution followed by nonperfusion cold (4 degrees C) storage, and vascular washout with mannitol solution followed by continuous hypothermic (5 degrees C) microperfusion (0.03 ml/min) with UW solution. The most consistent, and lowest, vascular resistance was produced by the microperfusion technique. However, UW solution does not consistently prevent an increase in vascular resistance with hypothermic ischemia. This technique may prove useful for the preservation of vascularized bone grafts, but it needs to be evaluated in a transplantation model.

In vivo evidence for a direct effect of naloxone on testicular steroidogenesis in the male rat.

It has been suggested that endogenous opioid peptides (EOP) exert paracrine or autocrine effects in the testes. To assess this hypothesis, we examined whether naloxone, by blocking the effects of EOP, influenced serum testosterone levels, apart from its effects on LHRH/LH, in the intact male rat. We found that naloxone increased serum LH and testosterone levels over essentially the same time course and produced dose-dependent increases in serum testosterone levels even though LH levels were maximally elevated at all doses. These data are not consistent with the view that naloxone exerts its effects on testosterone exclusively by altering LHRH/LH release. Additional, perhaps more definitive, evidence of a direct effect of naloxone on testosterone's biosynthesis was provided by our observations that 1) naloxone generated increases in serum testosterone levels in male rats in which the naloxone-induced surge in LH was blocked by nembutal; and 2) intratesticular injections of naloxone increased serum testosterone levels without increasing LH. Although these data suggest that naloxone influences steroidogenesis independently of its effects on LH, we found that the antagonist failed to increase serum testosterone levels in hypophysectomized animals or when serum LH levels were allowed to reach undetectable levels in nembutal-blocked animals. Consequently, our data are consistent with the hypothesis that naloxone facilitates the effects of LH on testosterone's biosynthesis rather than exerting an independent effect of its own. Whether the effects observed in these studies represent a negative autocrine effect of EOP on Leydig cells or a paracrine effect on Sertoli cells remains to be determined. Nevertheless, our results provide, to our knowledge, the first in vivo evidence that EOP modulate testicular steroidogenesis in the intact animal.

The effects of inhibition of heme synthesis on the intracellular localization of iron in rat reticulocytes.

These studies assessed the fate and localization of incoming iron in 6-8-day rat reticulocytes during inhibition of heme synthesis by succinylacetone. Succinylacetone inhibition of heme synthesis increased iron uptake by increasing the rate of receptor recycling without affecting receptor KD for transferrin, transferrin uptake, or total receptor number. Its net effect was to amplify the number of surface transferrin receptors by recruitment of receptors from an intracellular pool. Despite increased iron influx in inhibited cells, only 2-4% of total incoming iron was diverted into ferritin. The majority of incoming iron (65-80%) in succinylacetone-inhibited cells was recovered in the stroma, where ultrastructural and enzymic analyses revealed it to be accumulated mainly in mitochondria. Intramitochondrial iron (70-75%) was localized mainly in the inner membrane fraction. Removal of succinylacetone restored heme synthesis, utilizing iron accumulated within mitochondria for its support. Thus, inhibition of heme synthesis in rat reticulocytes results in accumulation of incoming iron in a functional mobile intramitochondrial precursor iron pool used directly for heme synthesis. Under normal conditions, there is no significant intracellular or intramitochondrial iron pool in reticulocytes, which are therefore dependent upon continuous delivery of transferrin-bound iron to maintain heme synthesis. Ferritin plays an insignificant role in iron metabolism of reticulocytes.

Age-related differences in the sensitivity to opiate-induced perturbations in reproductive endocrinology in the developing and adult male rat.

The effects of a single morphine pellet (75 mg) implanted in developing male rats at 27 days of age on reproductive endocrine parameters were compared to those found in adult (65-day-old) animals after the same treatment. The pellets were left in place to provide the release of morphine during critical phases of puberty and sexual maturation and to prevent an abrupt withdrawal syndrome upon pellet removal which would confound our results. Developing rats were sacrificed at representative intervals after pellet insertion to assess the development of key indices of reproductive endocrinology; adult rats were sacrificed at the same time intervals to permit an evaluation of age-related differences in the sensitivity to opiate-induced endocrine disturbances. Our results showed that morphine markedly influenced a number of endocrine parameters associated with the maturation of the hypothalamic-pituitary-gonadal axis in developing rats for prolonged periods of time, whereas the effects of the opiate in the adult rat were relatively modest and transient. In the developing rat, serum luteinizing hormone (LH), testosterone, the wet tissue weights of the seminal vesicles and testes and hypothalamic LH-releasing hormone (LHRH) levels were substantially depressed immediately after pellet implantation and these effects persisted for up to 4 weeks when compared to placebo-implanted, age-matched controls. In contrast to these results, adult rats showed only transient effects (less than 1 week) of morphine on certain reproductive endocrine parameters (e.g., serum LH, testosterone and the weights of the seminal vesicles) and no effects on others (e.g., testes weights and hypothalamic LHRH).(ABSTRACT TRUNCATED AT 250 WORDS)

Time-resolved solution X-ray scattering of tobacco mosaic virus coat protein: kinetics and structure of intermediates.

The kinetics of assembly and disassembly of tobacco mosaic virus coat protein (TMVP) following temperature jumps have been studied by small-angle X-ray scattering and turbidimetry. The structures of the principal aggregates of TMVP oligomers (A protein), intermediate size (helix I) and large size helical rods (helix II), have been characterized by their average radii of gyration of thickness, cross section, and shape obtained from the corresponding regimes of the small-angle scattering pattern. This structural information was obtained within seconds after the temperature-induced initiation of either polymerization or depolymerization and allowed us to detect transient intermediates. This methodology made it possible to observe and characterize the structure of a principal intermediate. Taken together with other kinetic information, these data suggest that polymerization of TMVP under virus self-assembly conditions may proceed via a single-layered helical nucleus that contains about 20 subunits. Previous studies have shown that overshoot polymerization of TMVP can occur and results in metastable long helical viruslike rods which subsequently depolymerize and then form short helical rods, depending on the conditions of the final equilibrium state. The longer rods (helix II) are overshoot polymers which form within seconds and contain 17 1/3 subunits per turn (helix IIB), in contrast to the subunit packing arrangement of 16 1/3 subunits per turn found in the shorter helical rods (helix IA). The latter packing arrangement is the one found in TMV. An overall polymerization scheme is proposed for the formation of these two helical forms of TMVP.

Tobacco mosaic virus protein aggregates in solution: structural comparison of 20S aggregates with those near conditions for disk crystallization.

Previous X-ray studies (2.8-A resolution) on the crystals of tobacco mosaic virus protein (TMVP) grown from solutions containing high salt have characterized the structure of the protein aggregate as a bilayered cylindrical disk formed by 34 identical subunits [Bloomer, A.C., Champness, J.N., Bricogne, G., Staden, R., & Klug, A. (1978) Nature (London) 276, 362-368]. Under low-salt conditions, 20S aggregates are in equilibrium with 4S species and involved in the efficient nucleation of TMV assembly in vitro [Butler, P.J.G. (1984) J. Gen. Virol. 65, 253-279]. We have investigated by sedimentation velocity and near-UV circular dichroism (CD) measurements the structure of 20S aggregates in low salt (I = 0.1 potassium phosphate at pH 7.0 and 20 degrees C) and the aggregates in high salt [0.2 M (NH4)2SO4 in I = 0.1 tris(hydroxymethyl)aminomethane hydrochloride at pH 8.0 and 20 degrees C, close to the conditions under which TMVP crystallizes as disk aggregates]. At high salt, we observe structures (presumably stacks of disks) having s20,w values around 40, 45, and 50 S, but not the 20S species present in low-salt buffers. The near-UV CD spectrum of 20S aggregates has been obtained for the first time, using computer techniques, from the spectra of the 4S-20S equilibrium mixture and the 4S species. This spectrum of 20S aggregates differs dramatically from that of the stacks of disks examined at both high and low salt (into which the stacks can be returned by dialysis), indicating that the difference is not a solvent effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism of assembly of tobacco mosaic virus.

Sedimentation and proton binding studies on the endothermic self-association of tobacco mosaic virus (TMV) protein indicate that the so-called "20S" sedimenting protein is an interaction system involving at least the 34-subunit two-turn yield cylindrical disk aggregate and the 49-subunit three-turn helical rod. The pH dependence of this overall equilibrium suggests that disk formation is proton-linked through the binding of protons to the two-turn helix which is not present as significant concentrations near pH 7. There is a temperature-induced intramolecular conformation change in the protein leading to a difference spectrum which is complete in 5 x 10(-6) s at pH 7 and 20 degrees C and is dominated at 300 nm by tryptophan residues. Kinetics measurements of protein polymerization, from 10(-6) to 10(3) s, reveal three relaxation processes at pH 7.0, 20 degrees C, 0.10 M ionic strength K (H) PO4. The fastest relaxation time is a few milliseconds and represents reactions within the 4S protein distribution. The second fastest relaxation is 50-100 x 10(-3) s and represents elementary polymerization steps involved in the formation of the approximately 20 S protein. Analysis of the slowest relaxation, approximately 5 x 10(4) s, suggests that this very slow formation of approximately 20 S protein may be dominated by some first order process in the overall dissociation of approximately 20S protein. Sedimentation measurements of the rate of TMV reconstitution, under the same conditions, show by direct measurements of 4S and approximately 20S incorporation at various 4S to approximately 20S weight ratios that the relative rate of approximately 20S incorporation decreases almost linearly, from 0 to 50% 4S. There appears to be one or more regions of TMV-RNA, approximately 1-1.5 kilobases long, which incorporates approximately 20S protein exclusively. Solutions of approximately 95-100% approximately 20S protein have been prepared for the first time and used for reconstitution with RNA. Such protein solutions yield full size TMV, but at a slower rate than if 4S protein is added. Thus the elongation reaction in TMV assembly, following nucleation with approximately 20S protein, is not exclusively dependent upon the presence of either 4S or approximately 20S protein aggregates. The initial, maximum, rate of reconstitution increases about threefold when the protein composition is changed from 5% to 30% 4S protein, at constant total protein concentration at pH 7.0, 20 degrees C in 0.10 M ionic strength K (H)PO4. The probable binding frame at the internal assembly nucleation site of TMV-RNA has been determined by measuring the association constants for the binding of various trinucleoside diphosphates to helical TMV protein rods. The -CAG-AAG-AAG-sequence at the nucleation site is capable of providing at least 10-14 kcal/mol of sites of binding free energy for the nucleation event in TMV self-assembly.

Kinetics and mechanism of tobacco mosaic virus assembly: direct measurement of relative rates of incorporation of 4S and 20S protein.

The mechanism of assembly of tobacco mosaic virus has been investigated under conditions in which the rates of incorporation of the 4S and 20S proteins can each be directly measured by analytical centfrifugation. Under these conditions, pH 6.5, 6.5 degrees C, 0.10 M ionic strength potassium orthophosphate, the protein can be made to exist as a metastable 20S aggregate that is necessary for efficient reconstitution. The overall assembly process consists of an initiation (nucleation) reaction that requires two to three 20S disk aggregates per RNA molecule and is followed by an elongation (growth) reaction. In the elongation phase of assembly the 4S protein is incorporated 50 to 70 times faster than the 20S disk, calculated on the basis of a steady-state kinetic analysis. Therefore, under these conditions, in which the rate of assembly is about 0.06 of that at pH 7, 20 degrees C, 0.10 M ionic strength orthophosphate, the 4S protein preferentially participates in the elongation phase. At this slow reconstitution rate intermediate assembly states (about 70-168 S) can be observed. The kinetics of both protein incorporation and nucleoprotein formation suggest that the elongation process is composed of at least two different, possibly sequential, rate-limiting reactions.