Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver

The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. delta-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.58 +/- 0.09, 0.91 +/- 0.19 and 0.61 +/- 0.12 nmol delta-aminolevulinic acid/mg per h). Copro- and protoporphyrinogen oxidase activities were increased (20 and 60% above controls) in iron-deficient animals. In contrast, coproporphyrinogen oxidase was decreased by 20%, while protoporphyrinogen oxidase remained unchanged in iron-overloaded rats. These variations of activities were not due to changes in the affinity of these enzymes toward their substrates, as coporphyrinogen had the same Km in each case (0.62 +/- 0.05 M) as did protoporphyrinogen (0.22 +/- 0.035 M). Thus, the Km did not vary with the treatment received by the animals. Ferrochelatase activity was measured by both the pyridine hemochromogen method and by measurement of zinc protoporphyrin with endogenous zinc as substrate. In all cases, ferrochelatase was found to be able to synthesize zinc protoporphyrin with endogenous zinc as substrate. However, the apparent Km of zinc chelatase for protoporphyrin was significantly different in the three groups of animals with Km,appProto, app = 2.4 +/- 0.1 10(-7), 4 +/- 0.3 10(-7) and 9.10 +/- 0.05 10(-7) M in iron-overloaded, control and iron-deficient animals, respectively. When ferrochelatase activity was measured by pyridine hemochromogen, identical results were observed in iron-deficient and control animals but decreased by 45% in iron-overloaded animals. The mitochondrial heme content was also decreased by 40% in iron-overloaded rats but unchanged in either iron-deficient or control rats.     

Development in ontogeny of the effect of ACTH fragment 4-7 on rat learning

Studies have been made of the effect of ACTH fragment 4-7 on learning in rats in early postnatal ontogenesis, as well as of the possibility of preservation of early learning during administration of this peptide to adult animals. It was shown that conditioned reaction of active avoidance practically cannot be formed in normal 13-15-day animals; however, administration of ACTH 4-7 increases the number of animals exhibiting adequate reactions. Weak effect of ACTH 4-7 in 12-15-day animals, in older ones (20-24 days) is changed by a significant stimulating effect.     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondria of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 +/- 0.0043, 4.11 +/- 0.58 and 22.2 +/- 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4-5 weeks, respectively. The energy coupling of the mitochondria was greatly diminished after 4-5 weeks of treatment, and the ferrochelatase activity was inhibited 80-90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36 +/- 0.15, 3.97 +/- 0.40 and 3.59 +/- 0.23 nmol/mg protein for control animals, 3 days- and 4-5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tanger?s, A. (1985) Biochim. Biophys. Acta 843, 199-207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.     

Inhibition of in-stent restenosis by oral copper chelation in porcine coronary arteries

Stress-induced release of IL-1alpha and fibroblast growth factor-1 is dependent on intracellular copper and is a major driver of neointimal hyperplasia. Therefore, we assessed the effect of tetrathiomolybdate (TTM), a clinically proven copper chelator, on in-stent restenosis. Nine pigs were treated with TTM (5 mg/kg po) twice daily for 2 wk before stent implantation and for 4 wk thereafter, and nine pigs served as controls. In-stent restenosis was assessed by quantitative coronary angiography (QCA), intravascular ultrasound (IVUS), and histomorphometry. Serum ceruloplasmin activity was used as a surrogate marker of copper bioavailability. In TTM-treated animals, ceruloplasmin dropped 70 +/- 10% below baseline levels. Baseline characteristics were comparable in TTM-treated and control animals. At 4-wk follow-up, all parameters relevant to in-stent restenosis were significantly reduced in TTM-treated animals: minimal lumen diameter by QCA was 2.03 +/- 0.57 and 1.47 +/- 0.45 mm in TTM-treated and control animals, respectively (P < 0.05), percent stenosis diameter was 39% less in TTM-treated animals (27.1 +/- 16.6% vs. 44.5 +/- 16.1%, P < 0.05), minimal lumen area by IVUS was 60% larger in TTM-treated animals (4.27 +/- 1.56 vs. 2.67 +/- 1.19 mm(2), P < 0.05), and neointimal volume by histomorphometry was 37% less in TTM-treated animals (34.9 +/- 11.5 vs. 55.2 +/- 19.6 mm(3), P < 0.05). We conclude that systemic copper chelation with a clinically approved chelator significantly inhibits in-stent restenosis.     

Teratogenic, biochemical, and histological studies with mice prenatally exposed to 2.45-GHz microwave radiation

Pregnant CD-1 mice were exposed to 2.45-GHz continuous wave microwave radiation at an incident power density of 30 mW/cm2. The local specific absorption rate near the uterine area (deep colonic location), as determined from time-temperature profiles measured with a Vitek thermistor probe, was 40.2 mW/g. Groups of mice were exposed 8 hr per day through Days 1-6 or 6-15 of pregnancy. Other groups of animals were exposed to an elevated ambient temperature of 31 degrees C which increased the colonic temperature 2.3 degrees C, the same as that produced by the microwaves. Sham-irradiated groups of animals were treated exactly the same as the microwave-exposed animals. For the two conditions, temperature exposed and sham exposed, two groups of animals were used. One group was handled in the same manner as the microwave-irradiated group and the other group was not handled so as to evaluate the effects of stressing the animals by handling. Eleven groups of animals were used in the complete study: five groups for gestational Days 1-6, five groups for gestational Days 6-15, and one group of cage control animals. On Day 18 of gestation the dams of all experimental groups were sacrificed and their reproductive status was determined. The fetuses were examined for visceral and skeletal alterations. Brain cholinesterase activity and histology were evaluated in the groups exposed on Days 6-15. The results show that microwave radiation increases embryo lethality at the early stages of gestation (exposure Days 1-6). Fetal toxicity and teratogenicity were not significantly increased by exposure to microwaves on either Days 1-6 or 6-15 of gestation. Cholinesterase activity and histology of the brain of 18-day-old fetuses were not adversely affected.     

Susceptibility and sensitivity of the Siberian lemming and Middendorff's vole to leptospirae of the grippotyphosa serogroup]

Siberian lemmings and Middendorf's voles were found to be susceptible to infection caused by Leptospira of the Grippotyphosa serogroup after the intraperitoneal injection of Leptospira culture, the application of the culture or infected urine to the skin, as well as after Leptospira-carrying animals were placed together with the animals to be infected. The infectious sensitivity of these animals to Leptospira was not high: leptospiruria was observed for 1-3 weeks; in some of the voles leptospiruria was slightly pronounced, whereas other voles had a great number of Leptospira in urine. Antibodies appeared in the blood on day 5 after infection, and their titers increased till days 61-63. In no case could Leptospira be isolated from the kidneys of the animals killed on days 61-83 of the experiment.     

Fish oil decreases hepatic cholesteryl ester secretion but not apoB secretion in African green monkeys

Two groups of African green monkeys were fed diets containing 40% of calories as fat with half of the fat calories as either fish oil or lard. The fish oil-fed animals had lower cholesterol concentrations in blood plasma (33%) and low density lipoproteins (LDL) (34%) than did animals fed lard. Size and cholesteryl ester (CE) content of LDL, strong predictors of coronary artery atherosclerosis in monkeys, were significantly less for the fish oil-fed animals although the apoB and LDL particle concentrations in plasma were similar for both diet groups. We hypothesized that decreased hepatic CE secretion led to the smaller size and reduced CE content of LDL in the fish oil-fed animals. Hepatic CE secretion was studied using recirculating perfusion of monkey livers that were infused during perfusion with fatty acids (85% 18:1 and 15% n-3) at a rate of 0.1 mumol/min per g liver. The rate of cholesterol secretion was less (P = 0.055) for the livers of fish oil versus lard-fed animals (3.3 +/- 0.5 vs. 6.0 +/- 1.2 mg/h per 100 g, mean +/- SEM) but the rate of apoB secretion was similar for both groups (0.92 +/- 0.15 vs. 1.01 +/- 0.13 mg/h per 100 g, respectively). The hepatic triglyceride secretion rate was also less (P less than 0.05) for the fish oil-fed animals (8.3 +/- 2.5 vs. 18.3 +/- 4.4 mg/h per 100 g). Liver CE content was lower (P less than 0.006) in fish oil-fed animals (4.1 +/- 0.8 vs. 7.4 +/- 0.7 mg/g) and this was reflected in a lower (P less than 0.04) esterified to total cholesterol ratio of perfusate VLDL (0.21 +/- 0.045 vs. 0.41 +/- 0.06). The hepatic VLDL of animals fed fish oil had 40-50% lower ratios of triglyceride to protein and total cholesterol to protein. From these data we conclude that livers from monkeys fed fish oil secreted similar numbers of VLDL particles as those of lard-fed animals although the hepatic VLDL of fish oil-fed animals were smaller in size and relatively enriched in surface material and depleted of core constituents. Positive correlations between plasma LDL size and both hepatic CE content (r = 0.87) and hepatic VLDL cholesterol secretion rate (r = 0.84) were also found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Utilization of glucose during exercise in relation of meal-timing

The effect of glucose administration was studied on its utilization during exercise carried out in the hours 500-700, 1100-1300, 1700-1900, 2300-100. The control group comprised animals at rest which had one or two glucose loads. Circadian variability of blood glucose level was observed in response to glycaemic stimulation in control animals. In the animals during exercise the circadian changes of glucose level depended on the time after glucose administration and the duration of exercise. Glucose utilization during exercise was not identical at various times of the 24-hour period. The greatest fall of blood glucose was observed at 1800 after one as well as after two glucose loads. Glucose administration after one hour of exercise prevented hypoglycaemia development.     

Cx37 deletion enhances vascular growth and facilitates ischemic limb recovery

The unique contributions of connexin (Cx)37 and Cx40, gap junction-forming proteins that are coexpressed in vascular endothelium, to the recovery of tissues from ischemic injury are unknown. We recently reported that Cx37-deficient (Cx37(-/-)) animals recovered ischemic hindlimb function more quickly and to a greater extent than wild-type (WT) or Cx40(-/-) animals, suggesting that Cx37 limits recovery in the WT animal. Here, we tested the hypothesis that enhanced angiogenesis, arteriogenesis, and vasculogenesis contribute to improved postischemic hindlimb recovery in Cx37(-/-) animals. Ischemia was induced unilaterally in the hindlimbs of WT or Cx37(-/-) mice (isoflurane anesthesia). Postsurgical limb appearance, use, and perfusion were documented during recovery, and the number (and size) of large and small vessels was determined. Native collateral number, predominantly established during embryonic development (vasculogenesis), was also determined in the pial circulation. Both microvascular density in the gastrocnemius of the ischemic limb (an angiogenic field) and the number and tortuosity of larger vessels in the gracilis vasculature (an arteriogenic field) were increased in Cx37(-/-) animals compared with WT animals. Cx37(-/-) mice also had an increased (vs. WT) number of collateral vessels in the pial circulation. These findings suggest that in Cx37(-/-) animals, improved recovery of the ischemic hindlimb involves enhanced vasculogenesis, resulting in increased numbers of collaterals in the hindlimb (and pial circulations) and more extensive collateral remodeling and angiogenesis. These results are consistent with Cx37 exerting a growth-suppressive effect in the vasculature that limits embryonic vasculogenesis as well as arteriogenic and angiogenic responses to ischemic injury in the adult animal.     

Desensitisation of beta-adrenergic responsiveness in vivo. Decreased coupling between receptors and adenylate cyclase in isolated brown-fat cells

Chronic catecholamine stimulation in vivo of brown adipose tissue during acclimation of hamsters to cold does not result in any alteration of beta-adrenergic receptor number or affinity when determined in isolated adipocytes by (-)-[3H]dihydroalprenolol binding. Norepinephrine displacement of (-)-[3H]dihydroalprenolol showed the same Ki for both groups. However, the slope of the displacement curve was shallower for cells from cold-acclimated animals than for controls. Cyclic AMP accumulation was stimulated by norepinephrine in cells from both groups of animals, although the dose-response curve for cells from cold-acclimated animals was shifted to the right and the maximum value obtained was less than half that found in cells from control animals. The slope of the curve was again lower. Other catecholamines stimulated cAMP accumulation with an order of potency in agreement with a response mediated through beta 1-adrenergic receptors. The dose-response curve for norepinephrine-stimulated oxygen consumption was also shifted to the right for cells from cold-acclimated animals, although the maximal respiration was only slightly reduced. The slope factor was again decreased. The results are interpreted in terms of a reduced coupling between the beta-receptor and the metabolic response in isolated brown adipocytes from cold-acclimated animals as a result of chronic catecholamine stimulation in vivo.     

Effects of progesterone antagonist, lilopristone (ZK 98.734), on induction of menstruation, inhibition of nidation, and termination of pregnancy in bonnet monkeys

The effects of a progesterone antagonist, lilopristone (ZK 98.734), on induction of menstruation, inhibition of implantation or pregnancy, and termination of early and mid-pregnancy were studied in bonnet monkeys. In the regularly menstruating animals, administration of lilopristone (25 mg/day, s.c.) during the mid-luteal phase (Days 20-22 of the menstrual cycle) induced menstruation within 2-4 days after the initiation of treatment. A premature drop in circulating progesterone levels was also observed. The luteolytic effect of lilopristone was prevented by exogenous treatment with hCG; however, the animals showed premature menstruation, in spite of high progesterone levels (above 4 ng/ml). Treatment around the time of implantation (between Days 8 and 12 after the mid-cycle peak in estradiol levels) in mated animals provided 100% pregnancy protection. Treatment of pregnant animals on Days 30-32 of the menstrual cycle, i.e. about Day 20 after the estradiol peak, induced abortion in 8 of 10 animals. A significant (p less than 0.05) decrease in serum progesterone levels was observed on Day 3 after the initiation of treatment. However, the decrease was slower (slope: -0.36, r: 0.96) compared to that observed in nonpregnant animals (slope: -0.72, r: 0.95). In the other two animals, pregnancy was not affected. However, when the treatment was delayed until about Day 50 after the estradiol peak, all four animals aborted. This study suggests that lilopristone is a progesterone antagonist with a potential to induce menstruation, inhibit nidation, and terminate pregnancy. The antifertility effects are mediated through blocking progesterone action at the endometrium as well as decreasing progesterone bioavailability, which appears to be due to its effects on gonadotropin release.     

Expression of CD80 promoter in transgenic mice

CD80 is a very potent co-stimulatory factor which is required for complete T-cell activation. Here, we use transgenic mice as a tool to map the promoter of the CD80 gene. We engineered three different CD80 promoter driven luciferase transgenes: -3084, -1073 and -215. With these transgenes, we have generated three groups of transgenic mice. Our results showed that the -3084 CD80 promoter/luciferase transgene was sufficient to confer tissue-specific expression of the CD80 gene. When the promoter sequence was deleted to -1073, the normal tissue-specific expression was lost. A brain-specific element was mapped between -1073 nt and -215 nt. This element caused up to ninefold higher expression of the CD80 promoter/luciferase in brain tissue of -1073 CD80 promoter/luciferase transgenic animals as compared to -3084 CD80 promoter/luciferase transgenic animals. In contrast to results with a cell culture system, little luciferase activity was detected in -215 CD80 promoter/luciferase transgenic animals.     

Post-partum release of prostaglandin F(2alpha) and uterine involution in the cow

Peripheral plasma levels of the main blood plasma metabolite of PGF(2alpha) (15-keto-13,14-dihydro-PGF(2alpha)) and progesterone were investigated during the immediate, post-partum period in 59 normally calving cows. Uterine involution was monitored by weekly rectal palpations. The levels of the prostaglandin metabolite were high at parturition and remained thereafter elevated for periods varying up to 7-23 days. Uterine involution was completed during periods ranging from 16-53 days. According to the clinical findings, the animals were divided into three groups. Group A comprises 46 animals which had an uncomplicated, puerperal period. A significant (p<0.001) correlation between the duration of elevated prostaglandin levels and the time for completed uterine involution (Y=29.6 - 1.3 (X - 13.5)) was found for these animals. Group B animals (n=8) had periods of varying length with uterine discharge during the first 30 days post-partum. When compared to group A animals, the animals in group B had comparatively longer periods of prostaglandin release and also longer periods for completion of uterine involution. Group C animals (n=5) at times had palpable, thin-walled, cystlike structures in the ovaries during the first 30 days post-partum. In this group of animals, the periods of high prostaglandin levels, as well as for the completion of uterine involution, were similar to those for the animals in group A. Progesterone levels remained low during the immediate post-partum period and in no case were elevated levels found until the prostaglandin release had ceased.     

Effect of hematocrit on cerebral blood flow with induced polycythemia

Cerebral blood flow (CBF) is lowered during polycythemia. Whether this fall is due to an increase in red blood cell concentration (Hct) or to an increase in arterial O2 content (Cao2) is controversial. We examined the independent effects of Hct and Cao2 on CBF as Hct was raised from 30 to 55% in anesthetized 1- to 7-day-old sheep. CBF was measured by the radiolabeled microsphere technique before and after isovolemic exchange transfusion with either oxyhemoglobin-containing erythrocytes (in 5 control animals) or with methemoglobin-containing erythrocytes (in 9 experimental animals). Following exchange transfusion in the control animals, Hct rose (30 +/- 1 vs. 55 +/- 1%, mean +/- SE), Cao2 increased (15.1 +/- 0.8 vs. 26.7 +/- 0.9 vol%), and CBF fell (66 +/- 9 vs. 35 +/- 5 ml X min-1 X 100 g-1). Because the fall in CBF was proportionate to the rise in Cao2, cerebral O2 transport (CBF X Cao2) was unchanged. Following exchange transfusion in the experimental animals, Hct rose (32 +/- 1 vs. 55 +/- 1%) but Cao2 did not change. Nevertheless, CBF still fell (73 +/- 4 vs. 48 +/- 2 ml X min-1 X 100 g-1) and, as a result, cerebral O2 transport also fell. The latter cannot be attributed to a fall in cerebral O2 uptake, as cerebral O2 uptake was unaffected during each of these conditions. Comparison of the two groups of animals showed that approximately 60% of the fall in CBF may be attributed to the increase in red cell concentration alone. It is probable that this effect is due largely to changes in blood viscosity.     

Enhanced efficiency of lactate removal after endurance training

The effects of endurance training (running 1 h/day at 40 m/min, 10% grade) on net lactate removal at various lactate concentrations were assessed in resting rats by use of constant exogenous lactate infusion (0, 69.3, 123.6, and 175.0 mumol.kg-1.min-1). No consistent difference in resting lactate concentrations, 1.17 +/- 0.09 mM, was observed between control and trained animals with no exogenous infusion of lactate. With increasing lactate infusion rates, control animals demonstrated a twofold greater increase in blood lactate concentration (range 1.2-11.4 mM) compared with trained animals (range 1.0-5.5 mM). This response resulted from a more rapid rise in net lactate removal with changes in blood lactate concentration for trained animals. The estimated maximal reaction velocity for net lactate removal in trained animals was 19% lower than in control animals; however, the Michaelis-Menten constant was greater than 66% lower in trained animals (4 mM) compared with controls (12 mM). Control animals also demonstrated a twofold greater increase in lactate concentration as a function of the tracer-estimated lactate turnover. The ratio of 14CO2 yield to lactate specific activity as a function of total tracer removal was not significantly different between groups, suggesting that the relative contributions of oxidation and gluconeogenesis to lactate removal were similar for both groups. At blood concentrations greater than 1 mM, trained animals achieve higher rates of lactate removal for any given lactate concentration.     

Plasmodium berghei: gluconeogenesis in the infected mouse liver studied by 13C nuclear magnetic resonance

Using 13C nuclear magnetic resonance, we have compared the gluconeogenic activity of perfused livers isolated from normal starved mice and mice highly parasitized with Plasmodium berghei, using [2-13C]pyruvate as substrate. In both types of livers, 13C labeling of glucose carbons occurred in positions 1, 2, 5, and 6. The equal proportions of [1,6-13C]- and [2,5-13C]glucose in livers from malarial and normal mice suggests that pyruvate enters the gluconeogenic pathway directly and, to an equal extent, via the tricarboxylic acid cycle. The normalized signal heights indicated that at a given time after the addition of [2-13C]pyruvate the degree of 13C labeling in glucose carbons was reduced in livers from malarial animals, when compared to livers from normal animals. During the course of the perfusion experiment, the [2-13C]lactate resonance signal was always more intense from livers of malarial animals than from normal animals. A reduced activity of hepatic gluconeogenesis in malarial animals was further confirmed by a separate set of perfusion experiments which showed a 56% reduction of the measured rate of glucose production in livers from malarial animals, with respect to that of normal animals. A lowered NAD/NADH ratio in livers from malarial animals would explain the increased proportion of lactate observed in the spectra and be related to a decreased gluconeogenic rate. A more reduced oxidoreduction level in the hepatocytes of a malarial animal would result from a defect in the oxidative phosphorylation activity of mitochondria.     

Parathyroid hormone in sodium-dependent hypertension

Plasma parathyroid hormone (pPTH) levels have been assessed in three separate radioimmunoassay systems in samples from Wistar-Kyoto rats. The animals were subjected to one of three dietary regimens throughout the study period: Group 1 animals consumed normal rat chow and drank tap water; Group 2 animals consumed normal rat chow and tap water was replaced with 0.5% saline solution; Group 3 animals consumed normal rat chow to which 2.5% CaCO3 (by weight) had been added and also drank 0.5% saline solution. Animals had consumed these diets for approximately 7 months prior to sacrifice for blood collection. Blood pressure was measured by tail cuff plethysmography in these animals and, as previously reported, saline consuming animals showed a moderate hypertension (Gp 2) only when diets did not contain added calcium (Gp 3). In the week prior to sacrifice, mean blood pressures were: Gp 1: 128.0 +/- 3.46 mmHg; Gp 2: 140.2 +/- 3.15 mmHg; and Gp 3: 133.5 +/- 2.90 mmHg. Three assay systems were used to measure pPTH levels from trunk blood samples obtained by guillotine decapitation. One assay used an antiserum directed toward the vasoactive N terminal fragment 1-34 and produced pPTH measurements of 0.74 +/- 0.05 ng/ml in Gp 1 animals, 1.04 +/- 0.07 ng/ml in Gp 2 animals and 1.12 +/- 0.08 ng/ml in Gp 3 animals. This pattern was consistent with that obtained by another antiserum which had been raised against the intact 1-84 PTH molecule and produced values of 0.25 +/- 0.03 ng/ml in Gp 1 animals, 0.55 +/- 0.07 ng/ml in Gp 2 animals and 0.74 +/- 0.04 ng/ml in Gp 3 animals. Antiserum raised against the C-terminal did not show any difference in pPTH across groups. We conclude that saline consumption may increase some portions of circulating PTH. Such elevation of pPTH may not be a pathophysiological component in the sodium dependent elevation of blood pressure since animals concurrently consuming both saline and calcium supplemented diets retained elevated pPTH levels even though blood pressures did not differ from controls. Rather, elevation of circulating PTH levels may be a response to prolonged increases in sodium consumption.     

Age-Related Changes of the Anxiety Level of Male and Female Rats in Elevated Cross-Maze Test

Age-related peculiarities of formation behavior in the elevated cross-maze was studied in male and female Wistar rats of 6 age groups: the 17-, 21-, 30-, and 36-day-old rat pups as well as adult animals. Recorded were duration of animal stay in the open arms of the maze and the number of hanging-down reactions, parameters of the level of anxiety. In the 17-day-old rat pups the anxiety level was higher than the older animals, the 17-day-old pups being characterized by frequent manifestation of freezing reaction. Duration of stay in the open arms of the maze increased in the 26-day-old rat pups. In the 21-day-old animals the number of hanging reactions rose as compared with the 17-day-old ones, but did not differ from that in the 26-day-old animals. The anxiety level in the 30-day-old rat pups was much lower than in animals of younger age groups. In the rats aged 36 days (the age directly preceding sexual maturation) there was a pronounced enhancement of anxiety, recorded using both parameters. In the 42-day-old animals an increased duration of stay in the open maze arms was again observed, which indicates a decrease of the anxiety level. The obtained data demonstrate complex non-linear development of this form of emotional behavior in ontogenesis and allow suggesting an important role of the hormonal-humoral system in its formation.     

Neutral endopeptidase terminates substance P-induced inflammation in allergic contact dermatitis

Sensory nerve-derived neuropeptides such as substance P demonstrate a number of proinflammatory bioactivities, but less is known about their role in inflammatory skin disease. The cell surface metalloprotease neutral endopeptidase (NEP) is the principal proteolytic substance P-degrading enzyme. This study tests the hypothesis that the absence of NEP results in dysregulated inflammatory skin responses. The effector phase of allergic contact dermatitis (ACD) responses was examined in NEP(-/-) knockout and NEP(+/+) wild-type mice and compared with the irritant contact dermatitis response in these animals. NEP was found to be normally immunolocalized in epidermal keratinocytes and dermal blood vessels. The ACD ear swelling response was 2.5-fold higher in animals lacking NEP and was accompanied by a significant increase in plasma extravasation and infiltration of inflammatory leukocytes. The augmented ACD response in NEP(-/-) animals was abrogated by either administration of a neurokinin receptor 1 antagonist or by repeated pretreatment with topical capsaicin. Similar to NEP(-/-) mice, the acute inhibition of NEP in NEP(+/+) animals resulted in an augmented ACD response. In contrast to the ACD responses, little differences were observed in the irritant contact dermatitis response of NEP(-/-) compared with NEP(+/+) animals after epicutaneous application of the skin irritants croton oil or SDS. Thus, these results indicate that NEP and cutaneous neuropeptides have a significant role in the pathogenesis of ACD.     

Targeted disruption of Ras-Grf2 shows its dispensability for mouse growth and development

The mammalian Grf1 and Grf2 proteins are Ras guanine nucleotide exchange factors (GEFs) sharing a high degree of structural homology, as well as an elevated expression level in central nervous system tissues. Such similarities raise questions concerning the specificity and/or redundancy at the functional level between the two Grf proteins. grf1-null mutant mice have been recently described which showed phenotypic growth reduction and long-term memory loss. To gain insight into the in vivo function of Grf2, we disrupted its catalytic CDC25-H domain by means of gene targeting. Breeding among grf2(+/-) animals gave rise to viable grf2(-/-) adult animals with a normal Mendelian pattern, suggesting that Grf2 is not essential for embryonic and adult mouse development. In contrast to Grf1-null mice, analysis of grf2(-/-) litters showed similar size and weight as their heterozygous or wild-type grf2 counterparts. Furthermore, adult grf2(-/-) animals reached sexual maturity at the same age as their wild-type littermates and showed similar fertility levels. No specific pathology was observed in adult Grf2-null animals, and histopathological studies showed no observable differences between null mutant and wild-type Grf2 mice. These results indicate that grf2 is dispensable for mouse growth, development, and fertility. Furthermore, analysis of double grf1/grf2 null animals did not show any observable phenotypic difference with single grf1(-/-) animals, further indicating a lack of functional overlapping between the two otherwise highly homologous Grf1 and Grf2 proteins.