Physiological changes in androgen plasma levels with elapsing of time from castration in adult male rats

In the present study we investigated in adult male rats the effects of castration on Dehydroepiandrosterone (DHEA), Androstenedione (delta 4), Testosterone (T) and Dihydrotestosterone (DHT) plasma levels: five days (group II), seven weeks (group III) and eleven weeks (group IV) after orchiectomy. The same hormone assays were performed in rats approximately 60 days of age which underwent a sham-operation for orchiectomy (group I). Our data show that five days following orchiectomy (group II) delta 4, T and DHT were decreased with respect to sham-operated rats. (Group I: delta 4: 83.3 +/- 14.9 (SEM) ng/dl (n = 12); T: 435.32 +/- 51.45 (n = 12); DHT: 51.47 +/- 6.54 (n = 12); Group II: delta 4: 44.81 +/- 6.09 (n = 12) P = 0.05; T: 25.54 +/- 2.88 (n = 12) P less than 0.01; DHT: 12.9 +/- 2.51 (n = 12) P less than 0.01). Seven weeks afterwards T and DHT remained significantly lower (group III: T: 54.37 +/- 12.21, n = 16) (P less than 0.01; DHT: 33.22 +/- 4.49 (n = 16) P less than 0.01) while eleven weeks after all steroids were significantly decreased with respect to the values observed in sham-operated rats. (Group IV) delta 4: 32.01 +/- 5.7 (n = 10) P less than 0.01: T: 27.29 +/- 7.05 (n = 10) P less than 0.01; DHT: 29.03: 5.34 (n = 10) P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

Elevated oestrogen and reduced testosterone levels in the serum of male septic shock patients

The variations in oestrogen levels which occur in men with septic shock were determined and analysed in terms of the changes seen in the levels of other steroid hormones of testicular and adrenal origin. The concentrations of the hormones, oestrone (E1), oestradiol (E2), testosterone (T), delta 4-androstenedione (delta 4), cortisol (F) and progesterone (P4) were determined by radioimmunoassay. The serum levels of cholesterol, triglycerides, phospholipids and non-esterified fatty acids (NEFAs) were also determined. Two groups of male septic shock patients were studied within the first 24 h following the admission to the Intensive Care Unit. Group I (n = 24) patients died. Group II (n = 22) patients recovered. Both groups were compared to a control group (n = 44) of healthy men. In group I patients, serum E1 levels were 3900 +/- 900 pmol/l, 12-fold higher than controls (296 +/- 22 pmol/l) [P less than 0.001], serum E2 levels were 880 +/- 170 pmol/l, 6-fold above control levels (158 +/- 30 pmol/l) [P less than 0.001] and serum T levels were 1.7 +/- 0.3 nmol/l, 11-fold lower than in controls (18.7 +/- 1.9 nmol/l) [P less than 0.001]. Serum P4 and F levels were slightly increased (P less than 0.05) and delta 4 androstenedione levels were unchanged. Groups II serum estrogen levels (814 +/- 350 pmol/l) [P less than 0.01] were higher than controls and serum T levels were 2-3 times less than control levels (5.5 +/- 2 nmol/l) [P less than 0.01]. The group II serum P4, F and delta 4 androstenedione levels did not differ from control levels. The levels of cholesterol, triglycerides, phospholipids and NEFAs were all decreased to similar, significant, degrees in both groups of shock patients. The dramatic increase in E1 levels associated with the decrease in T suggests an adrenal-testicular relationship with possible potentiation of aromatization of adrenal or testicular androgens in men in septic shock. The determination of serum E1 and T during septic shock in men could form the basis for prognostic estimations of septic shock severity and for a new therapeutic approach to shock.     

Cyclooxygenase pathway mediates lung injury induced by phorbol and platelets

The role of platelets in lung injury has not been well defined. In the present study of isolated perfused rat lungs, phorbol myristate acetate (PMA; 0.15 microgram/ml) or platelets (6.7 X 10(4)/ml) alone did not discernibly change the pulmonary arterial pressure (PAP) or lung weight (LW). However, the combination of platelets and PMA drastically increased the PAP and LW (delta PAP 26.2 +/- 1.0 mmHg, delta LW 2.7 +/- 0.4 g). delta PAP was positively correlated with the increase in thromboxane B2 produced by infusion of platelets and PMA (thromboxane B2 = 35.6 + 0.97 delta PAP, r = 0.67, P less than 0.01). The hypertension and edema formation induced by PMA and platelets were strongly attenuated by indomethacin, an inhibitor of platelet cyclooxygenase (delta PAP 5.6 +/- 2.0 mmHg, P less than 0.001; delta LW 0.0 +/- 0.1 g, P less than 0.001), and by imidazole, an inhibitor of thromboxane A2 synthase (PAP 8.0 +/- 2.5 mmHg, P less than 0.001; LW 0.0 +/- 0.3 g, P less than 0.01). Inactivation of platelet lipoxygenase with nordihydroguaiaretic acid mildly depressed pulmonary pressure but did not affect delta LW (delta PAP 18.9 +/- 1.6 mmHg, P less than 0.05; delta LW 3.1 +/- 0.3 g, P greater than 0.05). In vitro experiments showed that the capacity of platelets to release oxygen radicals was only 2.6% of that found for granulocytes. These results suggest that platelets may be activated by PMA to increase PAP and vascular permeability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the effect of hydration on the dynamics of various globular proteins by Rayleigh scattering of M?ssbauer radiation

Hydration relationships of the elastic scattering fraction of M?ssbauer radiation were studied for human serum albumin (HSA), pancreatic trypsin inhibitor and lysozyme within hydration degrees 0 less than or equal to h less than or equal to 0.75 g/g (at T = 295 degrees K) and temperatures 100K less than or equal to T less than or equal to 320 K (for HSA only at h = 0.03; 0.25; 0.41; 0.65). It is shown that the increase of both hydration degree above h greater than 0.1 and temperature above T greater than 200K leads to the appearance of intramolecular mobility in these proteins.     

Deficient protein kinase C-dependent Na+/H+ exchanger activity in T cells from bone marrow transplantation recipients

The early Na+/H+ exchanger-mediated alkalinization of intracellular pH (pHi) was analyzed in peripheral blood T cells from 23 bone marrow transplantation (BMT) recipients (17 allogeneic and 6 autologous) and a group of 13 healthy controls, in response to stimulation of protein kinase C (PKC) with a phorbol ester. In parallel we evaluated the proliferative response of peripheral blood T cells to an anti-CD3 mAb in the presence of either IL-2 or PMA. The pHi increase (delta pHi) observed in control samples ranged from 0.14 to 0.23 pH units (X +/- SD = 0.17 +/- 0.03). In 10 allogeneic and four autologous BMT recipients the delta pHi was under the lower limit of the control range (range: 0.01 to 0.09, X +/- SD = 0.05 +/- 0.02), whereas the remaining nine cases responded similarly to control samples (range: 0.14 to 0.24, X +/- SD = 0.17 +/- 0.04). The response of the Na+/H+ antiporter to a PKC-independent osmotic stimulation appeared to be normal, thus indicating that the intrinsic Na+/H+ exchanger activity was unaltered. The anti-CD3 induced proliferative response of the group of samples displaying a suboptimal delta pHi, was significantly lower (p less than 0.01) than that detected in control samples. T cell proliferation in samples from BMT recipients displaying a normal delta pHi was undistinguishable from the control group (p greater than 0.05). Our results provide the first evidence for a defective early metabolic event, closely related to PKC activity, in T cells from BMT recipients displaying a low proliferative response to T cell mitogens.     

Oxygen binding and acid base status of the blood from the freshwater turtle, Phrynops hilarii

Oxygenation studies with the whole blood of Phrynops hilarii show a P50 of 38 torr at extracellular pH (pHe) of 7.4 which corresponds to an intracellular pH (pHi) of 7.05 at 25 degrees C. The blood CO2 Bohr effect was -0.56 when related to pHi. pHi is related to pHe by the following equation: pHi = 0.75.pHe + 1.54 (r = 0.99); pHi = 0.72. pHe + 1.72 (r = 0.96) at 10 and 25 degrees C respectively. Blood pHe, for 25 degrees C, was 7.519 +/- 0.254 (n = 6). Blood gas partial pressures were: pCO2 = 25.8 +/- 3.8 torr (n = 6); pO2 = 61.7 +/- 21.2 torr (n = 6). The major red cell phosphates, in mmole/l erythrocytes, n = 6, were: ATP (3.66 +/- 0.86); GTP (0.53 +/- 0.28); 2.3-DPG (0.32 +/- 0.12) and inorganic phosphates (2.00 +/- 0.35). The plasma inorganic ion composition, n = 6, was, in mEq/l: K+ (3.04 +/- 0.40); Na+ (148.4 +/- 12.6); Ca2+ (4.75 +/- 1.32); Cl- (106.6 +/- 5.0). Additional blood parameters of interest (n = 6) were: lactate (2.07 +/- 1.72 mM in plasma); erythrocytes/mm3 (416 X 10(3) +/- 4.6 X 10(3)); leucocytes/mm3 (44636 +/- 2618); haematocrit (%) (14.5 +/- 3.6); haemoglobin, g/dl (3.2 +/- 0.5); plasma protein g/dl (4.4 +/- 0.4); osmolarity (293 +/- 10 mOsm/l). The non-bicarbonate buffer value was -22.6 mmol/kg H2O/pH. For a constant CO2 content, delta pHe/delta t = 0.0141 +/- 0.002 (n = 18) and delta pHi/delta t = 0.0157 +/- 0.003 (n = 18).     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

Non-standard O(2) consumption-temperature curves during rest and isometric exercise in human skeletal muscle

The present work was aimed at measuring intramuscular oxygen consumption (O(2)) as a function of temperature (T), in human forearm, during rest and aerobic isometric exercise (4% of the maximal voluntary contraction, MVC). Based upon results from in vitro experiments performed on isolated mitochondria of animal species, it was hypothesised that, during isometric exercise, the O(2)-T curve should display a maximum for some 'optimal' T. Intramuscular T and measurements were performed using a combined deep body temperature/near infrared probe during muscle cooling. At rest, O(2) increased non-linearly and monotonically as a function of T (n=8). O(2) increased approximately 2 times when going from 26 to 36 degrees C. A log(O(2))-T plot or a log(O(2))-1/T did not linearise the data. During isometric contraction, O(2) values at 26.8+/-0.6, 28.6+/-0.9, 31.9+/-0.9 and 35.9+/-0.9 degrees C were 3.04+/-1.26, 7.60+/-1.64, 4.43+/-1.95, and 6.64+/-1.37 micromol 100 g(-1) min(-1), respectively (n=6). The O(2) value at 28.6 degrees C was significantly higher (P<0.05) than that at 26.8 and 31.9 degrees C. The 'sudden' O(2) change at 28.6 degrees C is compatible with the phenomenon observed at the mitochondrial level.     

Static contraction increases arachidonic acid levels in gastrocnemius muscles of cats

Static contraction of hind-limb muscles is well known to increase reflexly cardiovascular function. Recently, blockade of cyclooxygenase activity has been reported to attenuate the reflex pressor response to contraction, a finding which suggests that working skeletal muscle releases arachidonic acid metabolites. Therefore, we measured the effects of static contraction and ischemia on arachidonic acid levels in the gastrocnemius muscles of barbiturate-anesthetized cats treated with indomethacin. Unesterified arachidonic acid levels were measured by high-pressure liquid chromatography. We found that static contraction of freely perfused gastrocnemius muscles increased arachidonic acid levels from 4.4 +/- 1.0 to 10.3 +/- 2.2 nmol/g wet wt (n = 12; P less than 0.005). Likewise, static contraction of gastrocnemius muscles made ischemic for 2 min before the onset of the contraction period increased arachidonic acid levels from 12.6 +/- 2.3 to 21.0 +/- 2.0 nmol/g wet wt (n = 12; P less than 0.01). Lastly, 2 min of ischemia with the gastrocnemius muscles at rest increased arachidonic acid levels from 5.9 +/- 1.1 to 10.5 +/- 3.0 nmol/g wet wt (n = 18; P less than 0.02). We conclude that both static contraction and ischemia increase arachidonic acid levels in working hindlimb muscle.     

NMR study of the alkaline isomerization of ferricytochrome c

The pH-induced isomerization of horse heart cytochrome c has been studied by 1H NMR. We find that the transition occurring in D2O with a pKa measured as 9.5 +/- 0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0 +/- 0.6 s-1 at 27 degrees C and is independent of pH; the reverse rate constant is pH-dependent. The activation parameters are delta H not equal to = 12.8 +/- 0.8 kcal.mol1, delta S not equal to = -12.9 +/- 2.0 e.u. for the forward reaction and delta H not equal to = 6.0 +/- 0.3 kcal.mol-1, delta S not equal to = -35.1 +/- 1.3 e.u. for the reverse reaction (pH* = 9.28). delta H degree and delta S degree for the isomerization are 6.7 +/- 0.6 kcal.mol-1 and 21.9 +/- 1.0 e.u., respectively.     

An investigation of the equilibria between aqueous ribose, ribulose, and arabinose

The thermodynamics of the equilibria between aqueous ribose, ribulose, and arabinose were investigated using high-pressure liquid chromatography and microcalorimetry. The reactions were carried out in aqueous phosphate buffer over the pH range 6.8-7.4 and over the temperature range 313.15-343.75 K using solubilized glucose isomerase with either Mg(NO3)2 or MgSO4 as cofactors. The equilibrium constants (K) and the standard state Gibbs energy (delta G degrees) and enthalpy (delta H degrees) changes at 298.15 K for the three equilibria investigated were found to be: ribose(aq) = ribulose(aq) K = 0.317, delta G degrees = 2.85 +/- 0.14 kJ mol-1, delta H degrees = 11.0 +/- 1.5 kJ mol-1; ribose(aq) = arabinose(aq) K = 4.00, delta G degrees = -3.44 +/- 0.30 kJ mol-1, delta H degrees = -9.8 +/- 3.0 kJ mol-1; ribulose(aq) = arabinose(aq) K = 12.6, delta G degrees = -6.29 +/- 0.34 kJ mol-1, delta H degrees = -20.75 +/- 3.4 kJ mol-1. Information on rates of the above reactions was also obtained. The temperature dependencies of the equilibrium constants are conveniently expressed as R in K = -delta G degrees 298.15/298.15 + delta H degrees 298.15[(1/298.15)-(1/T)] where R is the gas constant (8.31441 J mol-1 K-1) and T the thermodynamic temperature.     

Energetics of DNA twisting. II. Topoisomer analysis

A gel electrophoresis method has been developed for resolving small (approximately equal to 250 bp DNA topoisomers. In this size range only one major topoisomer band is observed, except for ligase closure conditions in which the probabilities are nearly equal for circularization by untwisting and overtwisting the corresponding linear DNA. The two probabilities are nearly equal when delta Tw is close to 0.5, if the mean helical twist of the linear DNA is n + delta Tw, where n is an integer and delta Tw is the fractional twist. We determine delta Tw of the linear DNA in standard conditions (20 degrees C, no ethidium) by titration experiments in which delta Tw is varied at the time of ligase closure, either by changing temperature or ethidium concentration. The endpoint (delta Tw = 0.5) is found when the two topoisomers formed by untwisting and overtwisting are present at equal concentrations. This analysis assumes that the net writhe is zero and the DNA helix is isotropically bendable. The results confirm the analysis of cyclization probabilities given in the preceding paper: delta Tw = 0 at the two maxima in the curve of j-factor versus DNA length and delta Tw = 0.5 at the minimum. Consequently, we can determine the DNA lengths at which Tw takes on integral values and use them to measure precisely the average helix repeat. From the difference between the delta Tw values of DNAs with 237 and 247 bp, we obtain an approximate value for the helix repeat of h = 10.4 +/- 0.1 bp/turn, in good agreement with earlier values found by the band-shift and nuclease-cutting methods. The twist is integral at 250.8 +/- 0.4 bp and from h = 10.4 +/- 0.1 we find n = 24; then 250.8/24 gives h = 10.45 +/- 0.02 bp/turn. The mean linking number (Lk) changes in a stepwise manner as delta Tw is varied for 250 bp DNAs. This result is expected when the free energy of twisting half a turn becomes large compared to thermal fluctuations. In these experiments, it is possible to obtain the mean Tw value from the mean Lk value only when delta Tw = 0.5, and consequently the mean Lk value is not simply related to DNA length for 250 bp DNAs except when delta Tw = 0.5.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of secretion into human and feline airways by Pseudomonas aeruginosa proteases

We have investigated the effect of elastase and alkaline protease from Pseudomonas aeruginosa on airway secretion into the trachea of anesthetized cats and from human bronchial mucosa in vitro. Secretory macromolecules were radiolabeled biosynthetically with two precursors in the cat, [3H]glucose and [35S]sulfate, and with [35S]-sulfate only in human tissue. Both enzymes (2.6 x 10(-9) to 1.3 x 10(-6)M elastase and 8 x 10(-9) to 2.4 x 10(-6)M alkaline protease) released radiolabeled macromolecules in a concentration-dependent manner from the two preparations. Purified elastase, 1.3 x 10(-6)M, released radiolabeled macromolecules (delta 3H = +397 +/- 72%, delta 35S 225 +/- 40% over control, P less than 0.001) and periodic acid-Schiff- (PAS) reactive glycoconjugates (delta PAS = +4.1 +/- 0.96 micrograms/min or +102 +/- 20%; P less than 0.01) from cat trachea, as did alkaline protease, 2.4 x 10(-6)M (delta 3H = +356 +/- 57%, delta 35S = +176 +/- 25%, delta PAS = +7.5 +/- 1.3 micrograms/min or 194 +/- 36%, P less than 0.001). Increases in 3H exceeded those of 35S, suggesting surface epithelium as the main source of secretion. Inhibition of enzyme activity abolished secretory effects. Both enzymes also stimulated secretion from human bronchus (e.g., with elastase, 1.3 x 10(-6)M: delta 35S = +331 +/- 67%, delta PAS = +4.3 +/- 0.92 micrograms/min or +131 +/- 24%, P less than 0.001; with alkaline protease, 2.4 x 10(-6)M: delta 35S = +220 +/- 67%, delta PAS = +12.7 +/- 3.2 micrograms/min or +575 +/- 245%, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sarcomere shortening in pressure overload hypertrophy

Sarcomere shortening during contraction was measured by using laser diffraction, in thin, rabbit right ventricular (RV) trabeculae from normal hearts (N) (n = 5) and from hearts subjected to RV pressure overload by pulmonary banding (H) (n = 5). Banding resulted in substantial RV hypertrophy after 2 wk. Hypertrophied preparations had the same resting muscle length (H = 3.15 +/- 0.29 mm) and resting sarcomere lengths (H = 2.16 +/- 0.005 micron) as the normal preparations (3.10 +/- 0.37 mm, 2.16 +/- 0.008 micron, respectively). Total tension at the peak of isometric twitches was the same as normal in the hypertrophied muscles (N = 8.06 +/- 1.20, H = 8.51 +/- 1.95 g/mm2). However, the amount of auxotonic sarcomere shortening was much less than normal in the hypertrophied preparations (N = 0.39 +/- 0.028, H = 0.19 +/- 0.034 micron; P less than 0.001). In isotonic contractions in which the ratio of muscle shortening to resting muscle length was the same in both the normal and hypertrophied muscles (ratio of 0.05 in both groups), the extent of sarcomere shortening relative to resting sarcomere length was less in the hypertrophied muscles than in the normal preparations (N = 0.14 +/- 0.01), H = 0.07 +/- 0.01; P less than 0.01). Series elasticity was the same as normal in the hypertrophied muscle P less than 0.05). Less auxotonic sarcomere shortening for a given level of isometric tension development and less isotonic sarcomere shortening per unit muscle shortening indicate that there is less than normal work per sarcomere during contraction in hypertrophied myocardium. These findings may have important implications for intracellular compensatory adaptation in pressure overload cardiac hypertrophy.     

Calorimetric study of tryptophan synthase alpha-subunit and two mutant proteins

Heat-denaturation of tryptophan synthase alpha-subunit from E. coli and two mutant proteins (Glu 49 leads to Gln or Ser; called Gln 49 or Ser 49, respectively) has been studied by the scanning microcalorimetric method at various pH, in an attempt to elucidate the role of individual amino acid residues in the conformational stability of a protein. The partial specific heat capacity in the native state at 20 degrees, Cp20, has been found to be (0.43 +/- 0.02) cal . k-1 . g-1, the unfolding heat capacity change, delta dCp, (0.10 +/- 0.01) cal . K-1 . g-1, and the unfolding enthalpy value extrapolated to 110 degrees, delta dh110, (9.3 +/- 0.5) cal . g-1 for the three proteins. The value of Cp20 was larger than those found for "fully compact protein" and that of delta dh110 was smaller. Unfolding Gibbs energy, delta dG at 25 degrees for Wild-type, Gln 49, and Ser 49 were 5.8, 8.4, and 7.1 kcal . mol-1 at pH 9.3, respectively. Unfolding enthalpy, delta dH, of the three proteins seemed to be the same and equal to (23.2 +/- 1.2) kcal . mol-1 at 25 degrees. As a consequence of the same value of delta dH and the different value in delta dG, substantial differences in unfolding entropy, delta dS, were found for the three proteins. The values of delta dG for the three proteins at 25 degrees coincided with those from equilibrium methods of denaturation by guanidine hydrochloride.     

The influence of the sampling point on testicular steroid concentrations in spermatic venous blood: a physiological approach to evaluate testicular secretion

In the present study, we have evaluated the influence of the location of the blood sampling in the spermatic vein on the steroid concentrations observed. Simultaneous blood sampling at two different points of the spermatic vein (iliac level and pampiniform plexus) was perfomed in the same patients during a surgical protocol for varicocelectomy. In order to further evaluate which of the two sampling points is more useful to investigate testicular secretion, we have performed both forms of sampling in 4 volunteers given an HCG stimulation 24 h before the surgical procedure. It was found that levels of testosterone (T) and 17 alpha-hydroxyprogesterone (17-OHP) were higher in the pampiniform plexus (scrotal) than at the iliac sampling point (T scrotal 1,168.343 +/- 142.65 nmol/l, iliac 850.63 +/- 143.411 nmol/l, n = 21, p less than 0.01; 17-OHP scrotal 260.130 +/- 43.14 nmol/l, iliac 164.46 +/- 31.02 nmol/l, n = 17, p less than 0.01). This indicates that spermatic blood collected at the scrotal sampling point has received more blood coming from the testis than the blood collected at the iliac point. We did not observe significant differences in progesterone and delta 4-androstenedione (delta 4) levels between the two samplings. The T/delta 4 ratio was significantly lower in the iliac than in the scrotal sampling (T/delta 4 scrotal 31.420 +/- 6.69; iliac 15.41 +/- 3.84; p less than 0.05). After HCG stimulation, testosterone concentrations were higher in the pampiniform plexus than in the iliac sample. This suggests that the first sampling point is more proper for studying testicular secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand-affected shift of Na+/H+ exchange pHi set point in human blood platelets, rapidly revealed by a novel approach.

The Na+/H+ exchange time-course of BCECF-loaded human platelets, suspended in isotonic media containing NaCl and sodium propionate and activated by intracellular acidification, was measured spectrofluorimetrically. Sequential alkalinization rates decline exponentially as a function of the changing intracellular pH (pHi) and its linear expression (log rate vs. pHi) extrapolates reproducibly to the pHi set point for the Na+/H+ exchange activation. The set point of control platelets (7.28 +/- 0.01) is shifted rapidly (discernibly less than or equal to 30 s) and markedly to alkaline pHi (7.62 +/- 0.03) by PMA, that activates protein kinase C and is shifted to acidic pHi (7.05 +/- 0.01) by staurosporine, which inhibits protein kinases. The addition of 5-N-(3-aminophenyl)amiloride reveals that the alkalinization measured is predominantly Na+/H+ exchange with only a minute contribution (delta pHi = 0.012 +/- 0.002 in 1 min) of an acid loading component, at pHi greater than 7.2. The results support recent studies concluding that the set point indeed reflects the phosphorylation state of the Na+/H+ exchanger.     

Effects of hypothermia on rat brain pHi and phosphate metabolite regulation by 31P-NMR

The effects of arterial alphastat regulation on brain intracellular pH (pHi) and several phosphate metabolites were assessed in anesthetized rats during hypothermia (28.6 +/- 0.2 degrees C) and normothermia (36.2 +/- 0.2 degrees C) by using 31P high-field (8.5 T) nuclear magnetic resonance (NMR). There were significant differences in pHi and metabolite ratios at the two temperatures under conditions of equal minute ventilation. During hypothermia, the brain pHi was 0.09 U higher, the phosphocreatine-to-inorganic phosphate (PCR/Pi) ratio 49% larger, and Pi-to-ATP 20% lower than at normothermia. These changes were fully reversible on warming the animal. The change in brain pHi/temperature was -0.011U/degrees C (95% confidence interval -0.007 to -0.016). The brain's ability to regulate its pHi and phosphate metabolism during hypercapnic acid-base stress was studied by using 10% CO2 ventilation. Hypothermic rats showed a larger fall in brain pHi (0.145 +/- 0.01 U, 7.15-7.01) with 10% CO2 than normothermic rats (0.10 +/- 0.02 U, 7.06-6.96). Similarly ventilated rats had a larger fall in arterial pH with 10% CO2 at hypothermia (0.36 +/- 0.04 U) than normothermia (0.24 +/- 0.01 U), so the delta brain pH/delta arterial pH was the same at both temperatures. The brain PCr-to-Pi ratio decreased approximately 20% during 10% CO2 breathing in both hypothermic and normothermic animals. Brain pHi and metabolite ratios returned to base line 30-50 min after CO2 washout in both groups. In summary, lowering body temperature while maintaining constant ventilation leads to changes in brain pHi and metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational properties of streptokinase--differential scanning calorimetric investigations.

The two-domain structure of streptokinase (Sk) was demonstrated by scanning calorimetric investigations at neutral pH and low ionic strength. The melting pattern of the protein is composed of two two-state transitions at TtrS1 = 45.9 +/- 0.4 degrees C with delta H1 = 431 +/- 18 kJ/mol, and TtrS2 = 60.1 +/- 1.3 degrees C with delta H2 = 306 +/- 16 kJ/mol. The partial specific heat capacity of native Sk was determined to be Cp = 1.42 +/- 0.17 J/K/g and the denaturational heat capacity change associated with the two transitions, delta Cp1 = 0.21 J/K/g and delta Cp2 = 0.38 J/K/g, respectively. The overall melting pattern of Sk remains almost unchanged at a variety of tested solvent compositions, except at pH 4 (and below) and in the presence of denaturants. The two domains show different susceptibility to urea. It is proposed that the less thermostable domain is located within the N-terminal part (residues 1-230), and the more thermostable one, within the C-terminal region.