Inhibition of colloid cell swelling in rabbit kidney cortex by disodium glycerophosphate.

Solution based on disodium glycerophosphate have been assessed for ability to inhibit cellular swelling during hypothermic storage of slices of rabbit renal cortex. It was found that slices stored in glycerophosphate media for 3 days gained about 50% of their control weight, whereas control slices stored in chloride based media gained 50% of their control weight after only 2 hr of storage and gained about 128% of their initial weight after 3 days. Electrolyte content of slices stored at 4 °C reflected the change in hydration. When slices were warmed after various storage times, it was found that viability was much better preserved in glycerophosphate saline than in chloride based saline, although the slices stored in glycerophosphate media contained more sodium prior to warming than slices stored in conventional media. Glycerophosphate solutions appear to be suitable for routine use in experiments with renal cortical slices in the vicinity of 0 °C when cell volumes are to be held constant for prolonged periods.     

Cadmium(II)-113 NMR studies of the mechanism of metal ion activation of yeast enolase

Yeast enolase binds one mole of 113Cd2+ per subunit at a site that consists of all oxyligands in a distorted octahedral environment. This "conformational" metal ion's environment undergoes further distortion on addition of substrate/product or analogs. At pH's below the optimum value the shifted resonance tends to break up into several, suggesting the existence of several slowly exchanging intermediate forms. At acid pH's, on addition of one additional mole/subunit of 113Cd2+, which greatly increases catalysis, "conformational" resonance(s) further broadens, suggesting that the second, "catalytic" metal ion increases the rates of interconversion between "conformational" species. At more alkaline pH's, near the optimum pH, the "conformational" peak is sharpened, which suggests that very fast interconversion is occurring. The position of the "catalytic" metal ion resonance also suggests all oxyligands in a distorted octahedral geometry. The "catalytic" resonance is often broadened to the point where it cannot be seen, suggesting rapid changes in its geometry due to interconversion of substrate and product.     

Stimulation of minor fetal hemoglobin synthesis in cord blood reticulocytes by butyrate

As it had been shown that sodium butyrate promoted hyperacetylation of nuclear histones the effect of 5, 10, and 50 mM sodium butyrate on the $\text{in}\text{vitro}$ synthesis of Hb F_Ic (an acetylated component of Hb F) and Hb F in cord blood reticulocytes was studied. The presence of 5 mM butyrate showed a significant increase in the production of Hb F_Ic, whereas Hb F synthesis was unaffected. Higher concentrations of butyrate, however, had no stimulatory effect on Hb F_Ic synthesis. Results of pulse-chase studies indicated that sodium butyrate inhibited the turnover of preformed Hb F_Ic, probably by inhibiting deacetylase.     

Antitumor activity and toxicity of peroxo heteroligand vanadates(V) in relation to biochemistry of vanadium

A selected set of 14 V(V) complexes was tested for toxicity and antitumor activity against L1210 murine leukemia, to examine the biological properties of peroxoheteroligand vanadates(V) of the formula (NH4)4[O(VO(O2)2)2], M3I[VO(O2)2C2O4], and MI[VO(O2)L], L = malate, citrate, iminodiacetate, nitrilotriacetate, and EDTA. The x-ray structure is known for five of these compounds. A relationship has been found between the chemical composition and the biological activity (antitumor activity-toxicity) of these complexes. Activity in the L1210 system is defined as greater than or equal to 25% increase in life span, and this was seen with (NH4)4[O(VO(O2)2)2]; M3[VO(O2)2(C2O4)]2H2O, M = K, NH4; and NH4[VO(O2)Malato]H2O. These observations are important for the biochemistry of vanadium. The special nature of electron transfer within the V(V)-peroxo moiety is proposed to be responsible for this phenomenon. Peroxo heteroligand vanadates(V) therefore represent a model system for studying some biochemical interactions of vanadium in living matter.     

Group mating among Norway rats II. The social dynamics of copulation: Competition,cooperation, and mate choice

The social interactions within groups of Norway rats (Rattus norvegicus) had a strong impact on the individual pattern of copulation which, in turn, affects sperm precedence and the probability of implantation in this species. Males alternated uninterrupted ejaculatory series, augmenting each others' copulatory investment. Females took turns mating after receiving an intromission, collectively potentiating the males' copulatory behaviour; increasing the number of oestrous females increased the number of intromissions and ejaculations achieved by each male but did not affect the amount of copulation experienced by each female. These turn-taking patterns within each sex provided the opportunity to change partners and permitted the emergence of different sex-typical patterns of copulation. Furthermore, the dominant male contributed more intromissions and tended to give each female more ejaculations than the subordinates did. Dominant males were also more likely to inhibit the subordinates' sperm transport. Females competed among themselves for the opportunity to mate with a male as he approached ejaculation and were likely to protect more of the dominant male's sperm transport than the subordinate male's.     

Dissolution half-times of nickel compounds in water,rat serum,and renal cytosol

Seventeen nickel compounds were incubated in water, rat serum, and rat renal cytosol for 72 hr at 37°C. Concentrations of dissolved nickel were analyzed by electrothermal atomic absorption spectrophotometry, and dissolution half-times (T50) were computed by the Weibull equation. Eleven of the nickel compounds (Ni, βNiS, amorphous NiS, αNi₃S₂, NiSe, Ni₃Se₂, NiTe, NiAs, Ni₁₁As₈, Ni₅As₂, and NiFeS₄) dissolved more rapidly in serum or cytosol than in water. Four of the compounds (NiO, NiSb, NiFe alloy, and NiTiO₃) had no detectable dissolution in any of the media (i.e., T50 > 11 yr). One compound (NiAsS) had approximately equal T50 values in the three media; the T50 value of one compound (NiS₂) could not be determined in serum or cytosol owing to precipitation. T50 values of 34 and 21 days for dissolution of αNi₃S₂ in serum and cytosol, respectively, agree closely with the excretion half-time of 24 days derived from previously reported data for excretion of ⁶³Ni in urine and feces of rats after intramuscular injection of α⁶³Ni₃S₂. These findings suggest that in vitro dissolution half-times of nickel compounds may be used to predict their in vivo excretion half-times, since the dissolution process is rate-limiting to their metabolism and excretion.     

Group mating among Norway rats I. Sex differences in the pattern and neuroendocrine consequences of copulation

The copulatory pattern of groups of rats (Rattus norvegicus) was studied in the laboratory in a seminatural environment. In a given mating session, every oestrous female copulated with each male; likewise, every male copulated with each oestrous female. While individual males and females experienced similar amounts of copulation, there were dramatic sex differences in sequence and temporal pattern. Males mated in a multiple intromission pattern and had more ejaculatory series when several females were in oestrus. In contrast, females received intromissions and ejaculations in a random order, not in the sequence of a male ejaculatory series. Males copulated at shorter intervals than females did, a temporal sex difference that was determined by the pattern of female solicitations and male approaches. These sex differences are used to discuss the different units of analysis that are appropriate for male and female sexual behaviour in this species. Furthermore, the sex differences in the temporal pattern of copulation which emerged during group mating parallel the known sex differences in the temporal parameters of the neuroendocrine reflexes which mediate successful reproduction in the domestic strain.     

Some studies on the metabolism of labelled molybdenum compounds in cattle

An initial experiment showed that [99Mo]di- and trithiomolybdates could be detected in bovine plasma after the introduction of [99Mo]molybdate into the rumen. It was felt that this justified the use of [99Mo]trithiomolybdate for the subsequent studies made of plasma thiomolybdate metabolism in vivo in cattle. Rapid intravenous injection of [99Mo]trithiomolybdate into cattle showed that doses of 50 mg Mo were subject to extensive hydrolysis over the first few minutes postinjection, but at lower dose rates this was reduced so that tracer doses (less than 1.5 mg Mo) were relatively stable. The plasma metabolism was unaffected by copper status within the limits of the experiments (that is, liver copper levels down to 9 mg/kg d.m.) The disappearance of [99Mo] and [35S]trithiomolybdate (1 mg Mo) from plasma was delayed for up to 10 hr by the immediate preinjection of copper, although no chemical modification of the thiomolybdate appeared to occur.     

Activation of yeast enolase by Cd(II)

Activation of yeast enolase by Cd2+ exhibits properties similar to activation by the physiological cofactor Mg2+. The activity is weakly stimulated, then inhibited by increasing ionic strength. The activity increases, then falls with increasing Cd2+ concentration. The effect of pH on activity produced by Cd2+ is very similar to that produced by Mg2+, except that the Cd2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd2+-enzyme is about 10% of that of the Mg2+-enzyme. The apparent kinetic parameters of Cd2+ activation show little effect of pH except for inhibition by high concentrations of Cd2+: the apparent Ki increases sharply with pH. This is interpreted as the result of Cd2+ being a less effective "catalytic" metal ion, and Cd2+ being more effective in stabilizing the enzyme at alkaline pH's. The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme. We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc. The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd2+-enzyme complex. The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7. There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg2+ or Cd2+ as cofactor. The slowness of the Cd2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen. The apparent reaction rate constant increases linearly with Cd2+ concentration. Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly. The binding of Cd2+ to the catalytic sites is characterized by low association and dissociation rate constants.     

An easy and rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with 32Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.     

Protein intake regulation in the weanling rat: Effects of additions of lysine,arginine and ammonia on the selection of gluten and energy

The effects of adding lysine, arginine and ammonia to gluten on the self-selection of protein and energy by the weanling rat simultaneously offered a choice of two diets differing only in gluten concentration (15 and 55%) were tested. Previous studies have shown that while lysine (6 g/100 g) additions to gluten decreased the amount of gluten selected by the rat from 40 to 20 g per 100 g of food eaten, selection was not related to the nutritional quality of the gluten. When graded levels of arginine (1.8, 3.6 or 7.2 g/100 g) were added to the gluten with or without lysine (0 or 6 g/100 g) the dietary protein selection was unaffected. The addition of ammonia (1.4 g/100 g as NH₄Cl) to gluten had initially the same effect as lysine (6 g/100 g) but with time protein intake returned to control levels. This effect of ammonia was unaltered by arginine additions. It is concluded that the mechanisms which lead to decreases in gluten selection caused by lysine or ammonia are not similar, and that the effects of lysine on gluten selection are not caused by an increased arginine requirement for urea cycle activity.     

Neural crest origin of cardiac ganglion cells in the chick embryo: Identification and extirpation

Using interspecific grafting of neural crest between quail and chick embryos, it was determined that the cardiac ganglia originate from the cranial region (somites 1–2) of the vagal neural crest (somites 1–7). Neuronal uptake of [³H]choline was used as an index of neuronal development in the chick atrium. Normal uptake was found to be quite high between Days 8 and 14 of incubation. Following extirpation of neural crest over somites 1 to 3 at stages 8 to 10, neuronal uptake in 8-day chick atrium was decreased by 25–60% depending on the stage at which the lesion was performed. It is thought that the residual uptake represents preganglionic terminals from the dorsal motor nucleus of the vagus. Embryos with extirpations of neural crest over somites 1–3 performed at stage 9 showed the greatest decrease of neuronal choline uptake and did not live beyond 11 days of incubation. However, hearts from embryos with partial lesions (performed at stage 10) were treated on incubation Days 12 and 15 for demonstration of acetylcholinesterase in the subepicardial plexus. These hearts showed much less extensive neural plexus with sparse, small cardiac ganglia.     

Deep hypothermia and circulatory arrest: effect on cerebrospinal fluid electrolytes in newborn lambs.

Cerebrospinal fluid (CSF) Na, K, and acid-base changes were studied in 13 new-born lambs anesthetized with α-chloralose (60 mg/kg) or diethylether during 90 min of normothermic (37 °C) or hypothermic (20 °C) circulatory arrest. CSF K concentration increased linearly from 3.1 to 23.2 meq/liter during 90 min of normothermic circulatory arrest. During hypothermic circulatory arrest, animals anesthetized with α-chloralose exhibited an exponential increase in CSF K concentration from 3.1 to 13.6 meq/liter and animals anesthetized with diethylether had an exponential increase in CSF K concentration from 3.3 to 12.7 meq/liter. The rate of increase in CSF K concentration in hypothermic and normothermic animals between 60 and 90 min of circulatory arrest was the same. CSF Na concentration decreased slightly in both hypothermic and normothermic animals, with a greater decrease in the normothermic group.Although CSF pH and bicarbonate were significantly decreased during normothermia as well as hypothermia, both CSF pH and bicarbonate showed greater decreases during normothermia. Mean pH values after 90 min of circulatory arrest were 6.34, 6.87, and 6.77, respectively, in the normothermic, α-chloralose-hypothermic, and diethylether-hypothermic groups; corresponding values for bicarbonate were 7.7, 13.8, and 12.2 meq/liter.CSF pCO₂ increased linearly from 40.2 to 190.0 Torr during 90 min of normothermic circulatory arrest, from 28.6 to 92 Torr in the ether-hypothermic group, and from 28 to 81 Torr in the α-chloralose-hypothermic group.     

Activation of pyruvate dehydrogenase complex (PDC) of rat heart mitochondria by glyburide

The effects of the second generation sulfonylurea, glyburide, on the pyruvate dehydrogenase multienzyme complex (PDC) of rat myocardial tissue were examined using rat ventricular slices and isolated mitochondria. Therapeutic concentrations (10(-7) to 10(-6)M) of glyburide produced a 30% increase in the decarboxylation of [1(-14)C] pyruvate by the PDC of ventricular tissue. Addition of glyburide to intact rat heart mitochondria stimulated activity of the PDC in a time- and concentration-dependent manner. Half-maximal stimulation of the enzyme occurred with 6 X 10(-5)M glyburide and maximal activation of the enzyme was achieved with 1 X 10(-4)M glyburide. At the height of stimulation, PDC activities were 6-fold greater than those observed under control conditions with succinate alone. When mitochondria were disrupted by sonication or freeze-thawing, glyburide produced no stimulation of pyruvate decarboxylation. We conclude that glyburide directly stimulates the decarboxylation of pyruvate by the PDC of the myocardium. Furthermore, the presence of intact mitochondria is necessary for the stimulatory action of glyburide on the PDC.     

Preliminary studies on the inhibitory effect of novel Pd(II) complexes of vitamin B6 on cell divisions

Two novel complexes of Pd(II) involving vitamin B₆ compounds have been synthesized. They are compatible with the compositions Pd(P.H.)₂ C₂(P=pyridoxol) and Pd(PL.H)₂C₂ (PL = pyridoxal). The complexes inhibited the growth as well as the biosynthesis of RNA, DNA, and protein of E. coli B-766. Photoacoustic spectral (PAS) measurements showed that the complexes bound to DNA of the bacteria and were present only in the kidney of treated mice. The complexes inhibited the incorporation of ³H-thymidine as well as ¹⁴C-leucine in the DNA and protein, respectively, of liver cell cultures (BL8L). The inhibition of cell division of Walker-S-cells and human lymphocytes by the complexes was highly significant.     

Studies of the role of catalytic and conformational metals in producing enzymatic activity in yeast enolase

Spectrophotometric titrations of yeast apoenolase with magnesium, the metal that produces the highest level of activity, nickel, which produces a very low level, and calcium, which produces no activity, suggest strong binding of 2 mol (1 per subunit) of all three metals at the same sites, called “conformational” sites. About two-thirds of the possible absorbance change in the chromophoric competitive inhibitor 3-aminoenolpyruvate-2-phosphate (AEP) that occurs when it binds to the enzyme in the presence of saturating levels of magnesium is produced when just 2 mol (1 per subunit) of magnesium is added. Since additional “catalytic” metal won't bind unless the AEP does, and the AEP won't bind unless the “conformational” sites are filled with metal, much of the absorbance change in the AEP must be produced by conformational metal.Metals that do not produce enzymatic activity do not produce the absorbance change in AEP whereas metals that permit any level of enzymatic activity produce the same absorbance change that magnesium does-the reaction is “all or none.” Studies of the effect of calcium, nickel, and magnesium on the CD spectrum of apoenzyme-AEP solutions suggest that activating metals produce an asymmetric chromophore in the AEP. This is interprested as indicating the chromophore in AEP bound to enzyme in the presence of an activating metal is a twisted carbon-carbon double bond.Calorimetric studies show the competitive inhibitor 3-phosphoglycerate binds to the calcium- and magnesium-enzyme with about the same change in enthalpy. The substrate or AEP reduces the rate of the apparent reaction of the calcium- or magnesium-enzyme with excess EDTA, suggesting that both substrate and AEP bind to the calcium-enzyme. The interpretation of these data is that the conformational metal plays a crucial role in activating the substrate while the catalytic metal controls the reaction rate. This interpretation is supported by experiments in which an enzyme with one type of conformational metal is reacted in the stopped-flow with catalytic metal and substrate. If an activating metal is the conformational metal, the initial activity is greater.     

Regulation of peroxo-bridge formation between cobalt(II)-carnosine complexes by histidine: Possible involvement in polycythemia

The mechanisms by which histidine stabilizes the cobalt(II)-carnosine complex from oxidation to cobalt(III) in aqueous solution are investigated with ¹H-nmr, laser Raman, and Fourier transform-infrared spectroscopy. Histidine has at least three effects on the cobalt(II)-carnosine complex. First, over the concentration range of at least 5 to 250 mM, histidine stabilizes the cobalt(II)-carnosine complex from oxidation by excluding solvent molecules from the equatorial coordination positions. Second, at the upper end of this concentration range, histidine reduces the strained nonplanarity of the equatorial coordination positions around the cobalt(II) ion that results from tridentate chelation by carnosine. Bidentate ligation by histidine causes the carnosine to bind as a bidentate ligand also. Third, bidentate ligation of two carnosine molecules to the equatorial coordination positions of Co(II) ion places the β-alanyl residues inthe vicinity of the two axial coordination positions and thereby inhibits the binding of molecular oxygen. Substitution of a molecule of histidine for one of these two carnosine molecules makes an axial coordination position available for binding oxygen. The first two effects are expected to stabilize the cobalt(II) ion from rapid oxidation, whereas the third effect is expected to give long-term stability of the peroxo-bridged complex. Since bidentate ligation of histidine is favored over monodentate ligation only when the concentration of Co(II) ion is not limiting and is inhibited by high concentrations of carnosine in the same solution, the results presented provide a possible explanation for the observation that the stability of the Co(II) complexes toward oxidation and their ability to bind molecular oxygen depend on both the relative and absolute concentrations of Co(II) ion, carnosine, and histidine in solution. Furthermore, these results provide additional support to the suggestion that the high activity of carnosinase in kidney is involved in part in regulation of the oxygen sensor in this organ.     

Direct radioimmunoassay of specific urinary estrogen glucosiduronates in normal men and nonpregnant women.

Direct radioimmunoassay are described for the measurement of each of three specific estrogen glucosiduronates: estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate and estriol-16 alpha-glucosiduronate in urine. Each assay utilizes a specific antiserum prepared by complexing the carboxylic acid group of the appropriate glucosiduronate to the epsilon-amino group of lysine in bovine serum albumin or bovine thyroglobulin. The antisera showed little or no cross reactivity toward other estrogens that might be present in significant amounts in urine. These antisera were used for the direct assay of the conjugates in urine from normal men and nonpregnant women without prior extraction or chromatography. The values were similar to those obtained after extraction, chromatographic purification on DEAE-Sephadex and subsequent immunoassay; The following mean values +/- SE (microgram/g creatinine) were obtained: estrone glucosiduronate, male 10.1 +/- 0.6, follicular phase female 17.3+/- 1.6, luteal phase female 31.8 +/- 2.5; 17 beta-estradiol-17-glucosiduronate, male 1.7 +/- 0.3, follicular phase female 2.4 +/- 0.1, luteal phase female 4.2 +/- 0.4; estriol-16 alpha-glucosiduronate, male 1.8 +/- 0.2, follicular phase female 4.7 +/- 0.9, luteal phase female 10.0 +/- 1.6.     

Membrane associations between subsurface cisternae of endoplasmic reticulum and the plasma membrane of rat Sertoli cells

Close membrane associations between the endoplasmic reticulum and the plasma membrane (ER-PM) occur in specialized regions of the rat Sertoli cell cytoplasm. They are characterized, in freeze fracture replicas, as mesa-like modifications of E membrane fracture faces or as corresponding discoid depressions on P membrane fracture faces. When these structures lie along transitional regions in the membrane fracture plane, they are seen to be complementary, and the space between them to be greatly reduced. These specialized close membrane associations may represent adhesive sites between the endoplasmic reticulum and the plasma membrane. However, their resemblance to vascular endothelial fenestrae which are known to be sites of increased membrane permeability may suggest other functional roles.