The activity of the RGA5 sensor NLR from rice requires binding of its integrated HMA domain to effectors but not HMA domain self-interaction

The rice nucleotide-binding (NB) and leucine-rich repeat (LRR) domain immune receptors (NLRs) RGA4 and RGA5 form a helper NLR/sensor NLR (hNLR/sNLR) pair that specifically recognizes the effectors AVR-Pia and AVR1-CO39 from the blast fungus Magnaporthe oryzae. While RGA4 contains only canonical NLR domains, RGA5 has an additional unconventional heavy metal-associated (HMA) domain integrated after its LRR domain. This RGA5_HMA domain binds the effectors and is crucial for their recognition. Investigation of the three-dimensional structure of the AVR1-CO39/RGA5_HMA complex by X-ray crystallography identified a candidate surface for effector binding in the HMA domain and showed that the HMA domain self-interacts in the absence of effector through the same surface. Here, we investigated the relevance of this HMA homodimerization for RGA5 function and the role of the RGA5_HMA effector-binding and self-interaction surface in effector recognition. By analysing structure-informed point mutations in the RGA5_HMA-binding surface in protein interaction studies and in Nicotiana benthamiana cell death assays, we found that HMA self-interaction does not contribute to RGA5 function. However, the effector-binding surface of RGA5_HMA identified by X-ray crystallography is crucial for both in vitro and in vivo effector binding as well as effector recognition. These results support the current hypothesis that noncanonical integrated domains of NLRs act primarily as effector traps and deepen our understanding of the sNLRs' function within NLR pairs.     

Comparisons of the release of vasodilator substances from left and right cardiac chambers of the isolated perfused rabbit heart: Implications for intraventricular thrombus formation

Prostacyclin (PgI₂) and endothelium-derived nitric oxide (EDNO) are produced by the arterial and venous endothelium. In addition to their vasodilator action on vascular smooth muscle, both act together to inhibit platelet aggregation and promote platelet disaggregation. EDNO also inhibits platelet adhesion to the endothelium. EDNO and PgI₂ have been shown to be released from the cultured endocardial cells. In this study, we examined the release of vasoactive substances from the intact endocardium by using isolated rabbit hearts perfused with physiological salt solution (95% O₂/5% CO₂, T = 37 °C). The right and left cardiac chambers were perfused through separate constant-flow perfusion loops (physiological salt solution, 8 ml min⁻¹). Effluent from left and right cardiac, separately, was bioassayed on canine coronary artery smooth muscle, which had been contracted with prostaglandin F_{2α_}(2 × 10⁻⁶ M) and no change in tension was exhibit. However, addition of calcium ionophore A23187 (10⁻⁶ M) to the cardiac chambers’ perfusion line induced vasodilation of the bioassay coronary ring, 61.4 ± 7.4% versus 70.49 ± 6.1% of initial prostaglandin F_2α contraction for the left and right cardiac chambers perfusate, respectively (mean ± SEM, n = 10, p > 0.05). Production of vasodilator was blocked totally in the left heart but, only partially blocked in the right heart by adding indomethacin (10⁻⁵ M) to the perfusate, respectively, 95.2 ± 2.2% versus 41.5 ± 4.8% (mean ± SEM, n = 10, p < 0.05). 6-Keto prostaglandin F_1α, measured in the endocardial superfusion effluent was also higher for the left cardiac chambers than for the right at the time of stimulation with the A23187, respectively, 25385.88 ± 5495 pg/ml (n = 8) versus 13,132.45 ± 1839.82 pg/ml (n = 8), (p < 0.05). These results showed that cyclooxygenase pathway plays major role in generating vasoactive substances for the left cardiac chamber endocardium; while it is not the main pathway for the right ventricular endocardium at which EDNO and PgI₂ could act together and potentiate their antithrombogenic activities in isolated perfused rabbit heart. This may be an explanation for the intraventricular thrombus mostly seen in left ventricle rather than in right ventricle as a complication of myocardial infarction.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

TPA Increases Conductance but Decreases Permeability in Neonatal Rat Cardiomyocyte Gap Junction Channels

Short-term (10 min) effects of 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), the protein kinase C (PKC) activator, on cardiac macroscopic (g_j) and single channel (γ_j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell (WC) or perforated patch (PP) voltageclamp, g_j increased by 15.5 ± 7.2% (mean ± SD, n = 9) and by 46.3 ± 17.0% (n = 5), respectively. The latter difference is not related to intracellular calcium concentration, because raising the Ca²⁺ concentration in the electrode solution did not change the TPA-induced increase in g_j observed under WC conditions. The inactive phorbol ester, 4α-phorbol 12,13-didecanoate (αPDD), did not affect g_j. Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two γ_j sizes of 20 and 40-45 pS. Under control conditions, the larger events were most frequently observed. Whereas αPDD did not change this distribution, TPA shifted the γ_j distribution to the lower sizes. Diffusion of Lucifer Yellow (LY) and 6-carboxyfluorescein (6-CF), gap junction permeant tracers, was studied on small clusters of cardiomyocytes. Under control conditions, LY labeled 19.4 ± 7.2 cells (mean ± SD, n = 18) and 6-CF labeled 8.4 ± 2.2 cells (n = 20). Whereas αPDD did not change the extent of dye transfer, TPA restricted the diffusion of LY to 2.8 ± 1.3 cells (n = 11) and of 6-CF to 2.4 ± 1.4 cells (n = 20). This suggests that permeability and single channel conductance of connexin 43 channels are parallely related. Altogether, these results point to the opposite modulation of electrical and metabolic coupling of cardiac cells evoked by TPA.     

Comparisons Between the Kinetic Behaviour of the Muscle Carnitine Palmitoyltransferase I of a Higher and Lower Vertebrate

The V_max of rat muscle mitochondrial CPT I toward the coenzyme A derivatives of 16:0, 16:1n-7, 18:1n-9, and 22:6n-3 were far lower than those recorded previously for this enzyme in rat liver at the same temperature (37°C). However, the V_max of 7.0 nmol · min⁻¹ · mg mitochondrial protein⁻¹ for linoleoyl CoA (18:2n-6), which was the greatest recorded for the five acyl CoAs examined in muscle, was similar to that in liver. These comparisons presumably reflect a difference in the essential fatty acid requirements of these two rat tissues. Although the V_max values for CPT I in the musculature of a lower vertebrate (larval lamprey) at 20°C were similar to those exhibited toward the coenzyme A derivatives of 16:0, 16:1n-7, 18:1n-9, and 22:6n-3 by the CPT I of rat musculature at 37°C, the corresponding V_max toward 18:2n-6 (3.2 nmol · min⁻¹ · mg mitochondrial protein⁻¹) was lower. The latter relatively low activity may spare from oxidation this essential fatty acid, which is in low abundance in the diet of larval lampreys. Although the V_max values toward the four nonessential fatty acids in larval lamprey muscle were similar to those in rat muscle, the corresponding K_0.5 values were lower, thus indicating that the musculature of larval lampreys has a high capacity for energy generation through β-oxidation.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

Determination of oocyte membrane permeability coefficients and their application to cryopreservation in a rabbit model

Having an effective means to cryopreserve human oocytes would offer more flexibility in healthcare services for infertility patients, and obviate cryopreservation of preimplantation embryos. It is essential to establish good animal models for human oocyte cryopreservation and the rabbit is a good candidate. Attempts to improve oocyte cryopreservation are often empirical, with results often being irreproducible. Cryopreservation protocols may be optimized by modeling the changes in oocyte volume and the associated damages incurred during the addition and dilution of cryoprotective agents (CPA). The objectives of the current study were to determine cryobiological properties of rabbit oocytes, including the isotonic volume, osmotically inactive cell fraction (V_b), hydraulic conductivity (L_p), permeability (P_s) to dimethylsulfoxide (Me₂SO), ethylene glycol (EG), and glycerol (GLY) and to examine the correlation between cell volume excursions and viability. This has led to the development of the accumulative osmotic damage (AOD) model associated with the processes of CPA addition/dilution. Mature rabbit oocytes were perfused with 15% (V/V) CPA medium (dissolved in 1× PBS). The osmotic responses of the oocytes were videotaped. A two-parameter model was fit to the experimental data to determine the values of L_p and P_s. Oocyte volumes reached upon equilibration with 285, 600, 900, and 1200 mOsm (milliosmolal) solutions of non-permeating compounds were plotted in a Boyle van’t Hoff plot. The average radius of rabbit oocytes in an isotonic solution was determined to be 55.7 ± 1.2 μm (n = 16). The rabbit oocyte exhibited an “ideal” osmotic response in the range from iso-osmolity to 1200 mOsm. The V_b was determined to be 20% of the isotonic value with r² = 0.97. The values of L_p were determined to be 0.79 ± 0.26, 0.82 ± 0.22, and 0.64 ± 0.16 μm min⁻¹ atm⁻¹ and the P_s values were determined to be 2.9 ± 1.3, 2.7 ± 1.3, and 0.27 ± 0.18 × 10⁻³ cm min⁻¹ for Me₂SO, EG and GLY, respectively. There were no significant differences (p > 0.05) between values for L_p and P_S in the presence of the Me₂SO and EG. However, these values were significantly different from the values in presence of GLY. We calculated the AOD values of those oocytes that experienced the process of CPA additions/dilutions and found that these values were highly correlated to the development rates of these oocytes after parthenogenetic activation (r = −0.98).     

Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors

Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11β-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC₅₀ values of 1.6 ± 0.1 and 9.9 ± 0.9 nM (mean ± SEM, n = 3–6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K_i values of 0.8 ± 0.04 and 2.2 ± 0.2 nM (n = 3–4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.     

Volume regulation of human peripheral blood lymphocytes and stimulated proliferation of volume-adapted cells

Human peripheral blood lymphocytes exposed to hypotonic media (Ca/Mg-free, room temp.) first swell and then shrink. This shrinking response is characterized by a simple exponential with a half-time of 1.44±0.60 min (n=11) and its extent but not the half-time for a given hypotonicity is influenced by [K⁺]₀. Using K-selective electrodes, we observe a change in [K⁺]₀ when cells are diluted into hypotonic media. A half-time of 1.55±0.06 min (n=4) was obtained. A similar half-time was obtained by assay of total cell K using atomic absorption spectroscopy. At all osmolarities [K⁺]_i was decreased from control values and was constant as [K⁺]₀ was increased. Short-term incubation with ouabain (10⁻⁴ M) had no effect. Decreasing osmolarities progressively inhibited phytohemagglutinin-stimulated DNA synthesis, yet cell number and viability remained unaltered. Our evidence indicates that the volume response is mediated by a change in the passive permeability of the plasma membrane to K and/or to the accompanying anions, and that the consequently volume-adapted cells are growth-inhibited.     

High-salt intake primes the rat kidney to respond to a subthreshold uroguanylin dose during ex vivo renal perfusion

In a variety of animal models, uroguanylin causes diuresis, natriuresis and kaliuresis and is found in larger concentrations in the urine compared to controls after oral salt intake or in conditions of excess salt and fluid retention. It has been proposed that uroguanylin functions as an intestinal natriuretic hormone following intake of meals high in salt content. In the present work, we examined if 10 days of salt ingestion resulted in an enhanced response to uroguanylin in the isolated perfused rat kidney. Rats were given normal water, 1% NaCl (HS1%), or 2% NaCl (HS2%) for 10 days, at which time the right kidneys were surgically removed and perfused with a modified Krebs-Henseleit solution for 30 min. After a 30-min control period, the kidneys were perfused with a modified Krebs-Henseleit solution containing 0.06 μM uroguanylin for an additional 90 min. Compared to vehicle-matched time controls, 0.06 μM uroguanylin perfusion of kidneys from rats maintained on HS2% resulted in a significantly increased urine flow (UF; from 0.17 ± 0.01 to 0.23 ± 0.01, after 60 min, n = 6, P < 0.05), fractional Na⁺ excretion (%E_Na+; from 16.6 ± 0.7 to 30 ± 2, after 60 min, n = 6, P < 0.05), fractional K⁺ excretion (%E_K+; from 20.5 ± 0.58 to 37.4 ± 2.1, after 60 min, n = 6, P < 0.05), and fractional Cl⁻ excretion increased from 18.16 ± 0.52 to 35.2 ± 2.0 at 60 min, n = 6, P < 0.05. With the exception of a significant increase in the %E_K+, no other effect was observed in the kidneys from the rats maintained on HS1%, and no significant effects were seen in those that were maintained on normal water. The effect of a higher dose (0.6 µM) of uroguanylin on urinary flow, sodium or potassium excretion was also significantly increased by 2% NaCl (HS2%) treatment (P < 0.05). We also observed an expressive upregulation of the GC-C and a slight downregulation of the GC-A receptor in high-salt treated rats. These data demonstrate that prolonged salt ingestion primes the kidney to enhanced renal responses to uroguanylin.     

High-performance liquid chromatographic assay of human lymphocyte kynureninase activity levels

Human lymphocyte kynureninase activity was assessed in homogenized cells by determination of 3-hydroxyanthranilic acid production as a function of time after addition of the substrate, 3-hydroxykynurenine. The product, 3-hydroxyanthranilic acid, was determined by isocratic high-performance liquid chromatography and fluorescence detection. Mean (± S.D.) lymphocyte kynureninase activity in a group (n = 12) of vitamin B₆-deficient men was 5.04 ± 0.81 pmol 3-hydroxyanthranilic acid formed per mg protein per min, which was significantly (p=0.005) lower than the 6.69 ± 1.70 pmol 3-hydroxyanthranilic acid formed per mg protein per min in men with a normal vitamin B₆ status. This indicates that lymphocyte kynureninase activity is depressed during a vitamin B₆ deficiency.     

Informativeness of human (dC-dA)n · (dG-dT)n polymorphisms

Abundant human interspersed repetitive DNA sequences of the form (dC-dA)_n · (dG-dT)_n have been shown to exhibit length polymorphisms. Examination of over 100 human (dC-dA)_n · (dG-dT)_n sequences revealed that the sequences differed from each other both in numbers of repeats and in repeat sequence type. Using a set of precise classification rules, the sequences were divided into three categories: perfect repeat sequences without interruptions in the runs of CA or GT dinucleotides (64% of total), imperfect repeat sequences with one or more interruptions in the run of repeats (25%), and compound repeat sequences with adjacent tandem simple repeats of a different sequence (11%). Informativeness of (dC-dA)_n · (dG-dT)_n markers in the perfect sequence category was found to increase with increasing average numbers of repeats. PIC values ranged from 0 at about 10 or fewer repeats to above 0.8 for sequences with about 24 or more repeats. (dC-dA)_n · (dG-dT)_n polymorphisms in the imperfect sequence category showed lower informativeness than expected on the basis of the total numbers of repeats. The longest run of uninterrupted CA or GT repeats was found to be the best predictor of informativeness of (dC-dA)_n · (dG-dT)_n polymorphisms regardless of the repeat sequence category.     

Isolation,characterization and nucleotide sequence of the muscle isoforms of creatine kinase from the Antarctic teleost Chaenocephalus aceratus

Creatine kinase (CK) was isolated from the white muscle of the Antarctic icefish Chaenocephalus aceratus, which is deficient in glycolytic capacity. C. aceratus white myotomal creatine kinase (MMCK) displayed an apparent K_m at 0.5 °C of 0.06 mM for ADP and 17 mM for Phosphocreatine. These K_m values are similar to those reported for other vertebrate MMCKs at their physiologically relevant body temperatures. C. aceratus MMCK exhibited optimal activity at pH of 7.6–7.7 at 0.5 °C, in contrast to rabbit MMCK which had optimum activity at pH 6.2 at 30 °C. The apparent V_max of C. aceratus MMCK at 0.5 °C is 94±4 S.D. (n=9) μmol ATP/min/mg (i.e. U/mg), which is comparable to rabbit MMCK assayed at 20 °C and 8-fold greater than rabbit MMCK measured at 0.5 °C. DEAE chromatography of C. aceratus white muscle CK resolved two distinct activity peaks. Cloning and sequencing of C. aceratus CK cDNAs confirmed that two muscle-specific isoforms of CK were expressed that were distinct from the mitochondrial and brain isoforms. Icefish MMCK was sensitive to transient temperature elevation, and the DEAE-fractionated forms were highly unstable. These results indicate that C. aceratus MMCK displays significant activity at physiological temperature and intracellular pH of icefish muscle that could contribute to sustaining energy charge during burst-swimming.     

Plasma pro- and anti-inflammatory cytokine levels and outcome prediction in unselected critically ill patients

Purpose. To determine the inter-relationships between cytokine levels and physiological scores in predicting outcome in unselected, critically ill patients. Methods. To this end, 127 patients (96 men), having a mean ± SD age of 45 ± 20 years, with a wide range in admission diagnoses (medical, surgical, and multiple trauma patients) were prospectively investigated. Severity of critical illness and organ dysfunction were graded by acute physiology and chronic health evaluation (APACHE II) and sequential organ failure assessment (SOFA) scores, respectively. Blood samples were drawn on admission in the ICU to determine pro- and anti-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, and IL-10. The main outcome measure was 28-day mortality. Results. Overall, 88 patients survived and 39 patients died. Univariate logistic regression analysis showed that SOFA, APACHE II, IL-8, IL-6, and IL-10 on admission in the ICU were related to mortality. Multiple logistic regression analysis in the entire cohort of critically ill patients revealed that SOFA (OR = 1.341, p < 0.001) and IL-6 (OR = 1.075, p = 0.01) constituted independent outcome predictors. receiver operator characteristics curve analysis showed that SOFA, APACHE II, and IL-6 had the highest area under the curve values. IL-6 correlated with APACHE II (r_s = 0.44, p < 0.0001) and SOFA (r_s = 0.40, p < 0.0001) scores. Conclusions. In mixed ICU patients cytokine concentrations on admission in the ICU represent independent outcome predictors in the presence of disease severity scores.     

α-Factor inhibition of the rate of cell passage through the “start” step of cell division in Saccharomyces cerevisiae yeast: Estimation of the division delay per α-factor·receptor complex

A highly sensitive, kinetically unambiguous assay for α-factor-induced delay of cell passage through the “start” step of cell division in yeast is presented. The assay employs perfusion with periodic microscopy to monitor the bud emergence kinetics on the 20% of cells within an exponentially growing population which exist prior to the α-factor execution point of start. The t_1/2 for cell passage through start by this population of cells is 31 min in the absence of α-factor. The inhibition constant, K_I, represents the α-factor concentration which produces a 50% inhibition of this rate and is equal to 2×10⁻¹⁰M. A second assay for maximal cell division arrest by α-factor on whole populations of cells is presented. This assay shows a maximum cell division arrest time of 125±5 h at saturating α-factor, and a K₅₀ (that is, an α-factor concentration which produces a half-maximal response) of 2.5×10⁻⁸M. Both assays were performed in the effective absence of α-factor inactivation. Values of the dissociation constant K_D and total number of receptors per cell which specifically mediate cell division arrest or delay were estimated to be 2.5×10⁻⁸M and 10⁴, respectively. These estimates, along with the quantitative dose-response data for division arrest which are presented here, are consistent with each receptor·α-factor complex which is present on the cell at equilibrium producing a 43±10 s delay of cell passage through start. Surprisingly, this number is constant within twofold over the entire range of cellular division arrest responses to α-factor, that is, from a 1.9-fold inhibition of the rate of cell passage through start at 0.17 nM α-factor to a 125±5 h maximum arrest at saturating α-factor concentrations of >170 nM. The possible significance of this observation toward the mechanism of α-factor-induced cell division arrest is discussed.     

Effects of indomethacin on cardiac output distribution in normal and asphyxiated piglets

We determined the effect of breathing 9% CO₂/10% O₂/81% N₂ (asphyxia) on cardiac output distribution (microspheres) in 4–5 day old unanesthetized, chronically instrumented piglets prior to and following intravenous indomethacin administration. Thirty minutes of asphyxia caused Pa_CO2 to increase from 35 ± 2 mmHg to 66 ± 2 mmHg, Pa_O2 to decrease form 73 ± 4 mmHg to 41 ± 1 mmHg, and pH to decrease from 7.52 ± 0.05 to 7.21 ± 0.07. Arterial pressure was increased slightly but cardiac output was not changed significantly. Asphyxia caused blood flow to the brain, diaphragm, liver, heart, and adrenal glands to increase while causing decreases in blood flow to the skin, small intestine, and colon. Blood flows to the stomach and kidneys tended to decrease, but the changes were not significant. Treatment with indomethacin during asphyxia did not alter arterial pressure or cardiac output but decreased cerebral blood flow to the preasphyxiated level and decreased adrenal blood flow about 20%. Indomethacin did not alter blood flow to any other systemic organ. At this time the piglet was allowed to breathe air for 2.5 hr undisturbed. Two and a half hours after indomethacin administration, blood flows to all organs returned to the preasphyxia control levels with the exception of cerebral blood flow which was reduced (93 ± 13 to 65 ± 5 ml/100 g·min. Three hours after indomethacin administration, the cerebral hyperemia caused by asphyxia was less (134 ± 17b ml/100 g·min) than prior to indomethacin (221 ± 15 ml/100 g·min. Indomethacin did not alter the asphyxia-induced changes to any other systemic organ. We conclude that in newborn pigs, systemic treatment with indomethacin decreases cerebral blood flow and cerebral hyperemia in response to asphyxia, without affecting blood flow to any other systemic organ.     

Inhibitory effect of aluminium on calcium absorption in small intestine of rats with different thyroid hormone status

To analyse the influence of thyroid status on the effect of aluminium (Al) upon intestinal calcium (Ca) absorption, adult male Wistar rats with experimentally altered thyroid hormones circulating levels, were orally treated (o.g.) with 0 (control), or 50 mg elemental Al (as chloride)/kg body weight (b.w.) per day, for a 14 d period. Hyper- and hypo-thyroid conditions were respectively achieved by means of administration of either sodium levothyroxine (50 μg/kg b.w. per day, o.g.) or methimazole, a thyroxine synthesis inhibitor (1 mg/kg b.w. per day, o.g.). In duodenum–jejunum segments, in vitro mucosa-to-serosa ⁴⁵Ca flux (JCa_ms) and kinetics of ⁴⁵Ca uptake in isolated enterocytes, were determined. In serum, concentrations of thyroxine (T4) and triiodothyronine (T3) were measured by chemiluminescent enzyme immunoassay. Unlike non-Al-treated rats, JCa_ms of Al-exposed rats decreased as serum levels of T4 and T3 increased, showing a significant inverse correlation in both cases (T4: r² = 0.414, P = 0.024; T3: r² = 0.443, P = 0.018). Enterocytes isolated from rats treated with Al plus thyroxine showed a reduction of both maximum Ca uptake (4.86 ± 0.44 vs. 6.85 ± 1.04 nmol Ca/mg protein, P < 0.05) and K_m (0.84 ± 0.18 vs. 1.05 ± 0.36 mM, P < 0.05) when compared to control. The observed variability in the Al effect on Ca transport with thyroid status of rats could be reflecting a negative interaction of Al with thyroid hormone action mechanisms on intestinal Ca absorption, which would take place mainly at Ca entry into enterocyte from lumen.     

Cryopreservation of composite tissues and transplantation: preliminary studies

Despite advances in cryobiology, the reliable cryopreservation of complex tissues has not yet been achieved. This study evaluates the viability of cryopreserved composite flaps and demonstrates the feasibility of their transplantation. Epigastric flaps were harvested from male Lewis rats. 1.5 M dimethyl sulfoxide (Me₂SO) was used as the initial cryoprotectant agent (CPA). Samples were frozen at controlled rate to −140 °C and transferred to liquid nitrogen for at least two weeks. Hematoxylin and eosin (H/E) staining, MTT tetrazolium salt assay, and factor VIII immunostaining were used to evaluate the overall histology, epithelial viability, and vascular endothelial integrity, respectively, of cryopreserved flaps. For the in vivo phase, flaps were isotransplanted to 35 recipient animals, divided into three groups: fresh (n = 10), perfused (n = 8), and cryopreserved (n = 17). Blood vessel patency was assessed via Doppler at 1, 7, and 60 days post-transplantation. For in vitro studies, cryopreserved samples (10/10) retained normal cell architecture and vascular endothelial integrity upon H/E and factor VIII staining. The viability index of cryopreserved composite flap skin (n = 10) was 11.17 ± 2.01, which was not significantly different from fresh controls (n = 10, 12.15 ± 1.32). All transplanted flaps in the fresh and perfusion groups survived with healthy color and hair growth at 60 days after operation. Survival in the cryopreserved group ranged from 2 to 60 days, with a mean of 12 days. These results demonstrate that the long term survival of cryopreserved composite tissue transplants is possible. Further studies are needed to refine protocols for the reliable cryopreservation of composite parts.     

Metabolic rate, maximum metabolism, and advantages of torpor in the fat mouse Steatomys pratensis natalensis Roberts, 1921 (Dendeomurinae)

1. The fat mouse Steatomys pratensis natalensis (mean body mass 37.4±0.43 (se)) has a low euthermic body temperature T_b=30.1–33.8 °C and a low basal metabolic rate (BMR)=0.50 ml O₂ g⁻¹ h⁻¹.

2. Below an ambient temperature (T_a)=15 °C, the mice were hypothermic.

3. The lowest survivable T_a=10 °C.

4. Torpor is efficient in conserving energy between T_a=15–30 °C, below T_a=15 °C, the mice arouse.

5. Euthermic and torpid mice were hyperthermic at T_a=35 °C.

6. Thermal conductance was 0.159 ml O₂ g⁻¹ h⁻¹ °C⁻¹, 98.8% of the expected value.

7. Non-shivering thermogenesis (NST) was 2.196 ml O² g⁻¹ h⁻¹ (3.69×BMR).

8. Maximal oxygen consumption, however, was 3.83 ml O₂ g⁻¹ h⁻¹ (6.44×BMR), indicating that other methods of heat production are additive.

9. Because fat mice conserve energy by torpor only between T_a=15–30 °C, we suggest that torpor may be a more important mechanism for surviving food shortages than for surviving cold weather.

Delayed treatment with GnRH agonist, hCG and progesterone and reduced embryonic mortality in buffaloes

The present study examined the effect of delayed treatment with tropic hormones and progesterone (P₄) on embryonic mortality in buffaloes. Buffaloes with a conceptus on Day 25 after AI were assigned to the following treatments: Control (n = 41), i.m. physiological saline; GnRH agonist (n = 36), i.m. 12 μg buserelin acetate; hCG (n = 33), i.m. 1500 IU hCG; P₄ (n = 38), i.m. 341 mg P₄ every 4 days on three occasions. Control buffaloes had an embryonic mortality of 41.4% (17/41) between Days 25 and 45, and this was reduced (P < 0.01) by treatment with GnRH agonist (11.1%, 4/36), hCG (9.0%, 3/33) and P₄ (13.1%, 5/38). On Day 45, buffaloes treated with hCG and which ovulated had greater (P < 0.05) concentrations of P₄ in whey (453 ± 41 pg/ml) than buffaloes in the same treatment that did not ovulate (297 ± 32 pg/ml). A similar but non-significant trend was observed for buffaloes treated with GnRH agonist. It was concluded from the findings that the treatment of buffaloes on Day 25 after AI with tropic hormones or P₄ is beneficial to processes associated with embryonic implantation.