X-ray-induced mutations affecting the level of the enzyme alcohol dehydrogenase in Drosophila melanogaster: frequency and genetic analysis of null-enzyme mutants.

The frequency of X-ray-induced (null-enzyme) mutations at the alcohol dehydrogenase locus in Drosophila melanogaster was measured. The rate of recovery of chromosomes that fail to direct the synthesis of a functional Adh protein is 3 x 10(-8) per R for chromosomes that do not include large chromosome rearrangements. However, this analysis excludes a larger number of chromosomes that are "null-enzyme mutations" because thye are deleted for the region of the Adh locus. The dose of X-rays required to induce a frequency of non-deletion null-enzyme mutants equal to the spontaneous frequency is about 73 rad calculated from the data reported in this communication.     

Uptake of the yolk protein, lipovitellin, by developing crustacean oocytes

A variety of cytochemical techniques were used to demonstrate how crustacean lipovitellin accumulates within the egg. It was found that a protein serologically identical to the lipovitellin of yolk spheres was present in the hemolymph of vitellogenic crustaceans, but was absent from the hemolymph of males and immature females.In the three crustacean species studied (Uca pugilator, Cambarus clarkii, and Libinia emarginata), pinocytosis of fluorescein-conjugated lipovitellin and trypan blue occurred only during those periods when oocytes were accumulating yolk.It may be concluded from the present studies that yolk spheres develop in crustacean eggs primarily through micropinocytotic uptake of lipovitellin from the hemolymph, although other oocyte proteins appear to be made in the oocyte.     

Prostaglandin E inhibition of mitogen stimulation in patients with multiple sclerosis

Kirbey et al have reported that leukocyte function from patients with multiple sclerosis is not suppressed by PGE₂, as are normal leukocytes. We examined the ability of PGE₂ (0.01–0.5 μg/ml) to suppress Phytohemagglutinin induced ³H-thymidine incorporation in peripheral blood lymphocytes from multiple sclerosis patients and normals. There was no difference in sensitivity between the two groups. There was also no difference in activity of the prostaglandin producing suppressor cell between the multiple sclerosis patients and controls.     

Magnetic resonance studies of the mitochondrial divalent cation carrier

Measurements of water proton spin relaxation enhancements (ε) can be used to discriminate high-affinity binding of Mn²⁺ or Gd³⁺ to biological membranes, from low-affinity binding. In rat liver mitochondria, ε_b values of approx. 11 are observed upon binding of Mn²⁺ to the inner membrane, while internal or low-affinity binding remains invisible to this technique. Energy-driven Mn²⁺ uptake by liver mitochondria results in the subsequent decay of ε¹.Comparison of ε¹ with the initial velocity of Mn²⁺ uptake in rat liver mitochondria reveals a linear correlation, which holds at all temperatures between 0 °C and 40 °C, regardless of the mitochondrial protein concentration. Consequently, enhancement appears to reflect the binding of Mn²⁺ to the divalent cation pump.Binding of Mn²⁺ to blowfly flight muscle also results in substantial ε¹, which is associated with the glycerol-1-phosphate dehydrogenase instead of divalent cation transport. Consequently, no decay in ε¹ due to uptake occurs after Mn²⁺ is bound.Lanthanide ions are also bound and transported by mitochondria. Addition of Gd³⁺ to pigeon heart or rat liver mitochondria results in ε_b ≈ 5–6, which decays with similar kinetics in both systems. The uptake velocity of Gd³⁺ in rat liver mitochondria is about $\text{1}\text{6}$ the rate with which Mn²⁺ is transported. Lanthanides also diminish ε¹ due to the addition of Mn²⁺, and greatly retard the Mn²⁺ uptake kinetics. The presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone depresses ε¹ upon addition of Mn²⁺ or Gd³⁺ and also uncouples energy-driven uptake. On the other hand, prolonged anaerobic incubation in the presence of antimycin and rotenone exhausts the mitochondria of their energy stores, blocks the uptake of Mn²⁺, but does not affect ε¹ significantly. Evidently, the uncoupler-induced disappearance of divalent cation binding sites is not the result of “de-energization”.Measurements of ε¹ at several NMR frequencies indicate a correlation time (τ_b) for carrier-bound Mn²⁺ in rat liver mitochondria between 20 ns and 4 ns as one varies the temperature between 10 °C and 30 °C. The 13 Kcal/mole activation energy for τ_b suggests that the 11 ns time constant at room temperature represents the movement of the Mn^II-carrier complex. On the other hand, τ_b is probably approx. 100 times too short to represent the rotational motion of a carrier protein. Apparently, Mn²⁺ binds to a small arm of the carrier which moves independently of the main body of any protein.In addition to Mn(H₂O)₆²⁺, other complexes of Mn²⁺ may also be bound and transported by rat liver mitochondria. Only a small increase in ε¹ occurs upon addition of MnHPO₄, yet this species is accumulated by the mitochondria. Consequently, the carrier does not recognize divalent metal ions on the basis of charge.     

The effects of desiccation and rehydration on the lone star tick

This study describes the effects of desiccation and rehydration on the water content, haemolymph volume (per cent), osmolarity, and concentrations of Na, K, Mg, and Ca in the haemolymph of the lone star tick, Amblyomma americanum.The water content percentages of ‘severely desiccated’, ‘moderately’ and ‘fully hydrated’ ticks were 46·0, 52·8, and 60·3 per cent respectively. The lowest and highest of these were near the minimum and maximum possible.The haemolymph volume (per cent) of ‘severely desiccated’ ticks was regulated near the level of ‘moderately hydrated’ ticks despite significant decreases in total body water content and increases in osmolarity and concentration of sodium. Conversely, the change from ‘severely desiccated’ to ‘moderately hydrated’ ticks can be viewed as causing an increase in total body water, decrease in blood osmolarity and sodium, but little change in haemolymph volume (per cent).Most of the water taken up by ‘moderately hydrated’ ticks (while becoming ‘fully hydrated’) was added to the haemolymph. At the same time, there was little change in the blood osmolarity or haemolymph concentration of sodium. Conversely, the change of ‘fully’ to ‘moderately hydrated’ ticks was marked by a substantial loss of haemolymph volume (per cent) but little change in osmolarity and concentration of sodium.The concentration of potassium was regulated over the full range of desiccating and hydrating conditions. The lone star tick appeared less able to regulate its haemolymph concentrations of Ca and Mg; both fluctuated at the same rate, but inversely as the haemolymph volume (per cent).It appears that a carefully controlled movement of solutes (Na the predominant cation) between haemolymph and non-haemolymph tissue is intimately linked with haemolymph volume regulation and movement of water into the haemolymph during hydration.     

Effects of indomethacin on moloney sarcoma virus-induced tumors

Chronic administration of indomethacin to weanling (20 day old) BALB/c mice inoculated with Moloney sarcoma virus (MSV) delayed the onset of the tumor and suppressed the tumor growth. As expected, the prostaglandin (PG) levels associated with the MSV-injected leg muscle of the indomethacin treated mice were greatly depressed when compared to the elevated PG content associated with the tumors of MSV injected control mice. There was no effect of indomethacin on the cyclic AMP or cyclic GMP levels.The elevated levels of PG and cyclic AMP associated with the tumor were found to parallel the tumor growth.Administration of antilymphocyte serum (ALS) to the mice at the time of MSV inoculation resulted in accelerated and enhanced tumor growth. Indomethacin treatment of these mice similarly suppressed the tumor growth, but less dramatically.     

A rapid technique to evaluate two high-affinity binding proteins in the same preparation

A modified DEAE-cellulose filtration assay was developed to estimate the capacities of two types of high-affinity progesterone-binding sites. The method was tested using a mixture containing rat corticosteroid-binding globulin and progesterone-binding protein from pregnant guinea pig plasma and by simulation. The assay has proved useful in studying the progesterone-binding proteins in uterine cytosol of the rat and other species and in following the separation of the two types of binding sites in rat uterine cytosol by ammonium sulfate fractionation or differential hydroxylapatite adsorption.     

Activation of protein kinase in the guinea pig fundic gastric mucosa by histamine.

The effect of histamine, 1,4-methylhistamine and ethanol on cyclic AMP levels and protein kinase activation was measured in tissue strips from the fundic region of guinea pig gastric mucosa. Histamine induced a significant elevation of tissue cyclic AMP levels and also $\text{in situ}$ activation of the protein kinase. 1,4-methylhistamine, an inactive analog of histamine, and ethanol had no effect on these two parameters. Results suggest that protein kinase activation is involved in the cyclic AMP-mediated action of histamine on the gastric fundic mucosa.     

Properties of the genome in experimental hepatomas: variations in the composition of chromatin

The chromosomal proteins of two rapidly growing and poorly differentiated Morris hepatomas were compared with those of liver from normal and tumor bearing animals. While the total quantity of histone associated with DNA in all tumor and liver chromatin preparations studied were similar, tumor chromatin contained an increased quantity of nonhistone chromosomal proteins. Variations in specific classes of histones and nonhistone chromosomal proteins associated with the genome of the two tumors, host liver and liver of tumor bearing animals were observed.     

Albumin synthesis during induced and spontaneous metamorphosis in the bullfrog Rana catesbeiana

Treatment of premetamorphic tadpoles with triiodothyronine (T₃) alters the in vivo distribution of radioactive amino acids among serum protein fractions. The effects on the albumin fraction have been interpreted as reflections of the relative rate of synthesis. About 12 hr after intraperitoneal injection of 2.5 × 10^?10 mole of T₃ per gram, there is an increase in the relative rate of albumin synthesis. The effect peaks on day 3 at 5 × the untreated level and returns to near the untreated level by day 6. Continuous immersion in 1 × 10^?7M T₃ results in a similar stimulation of albumin synthesis, but with no decline after day 3. The timing of the response is independent of dose or route of T₃ administration. The effect of T₃ on the relative rate of albumin synthesis is also observed in froglets. There is a 6-fold increase in the relative rate of albumin synthesis during spontaneous metamorphosis peaking at stage XXI and returning to the premetamorphic level by stage XXV. The following was concluded: (1) The increase in the relative rate of albumin synthesis during metamorphosis results from increased endogenous thyroid levels. (2) Following a peak, the decline in albumin synthesis observed in induced and spontaneously metamorphosing animals is a result of decreasing thyroid hormone levels. (3) The effect of T₃ on albumin synthesis may be the summation of two effects, a direct effect of T₃ and a stimulation by amino acids from the resorbing tail. (4) A decreased relative rate of albumin degradation or a sparing of albumin is probably responsible for the elevated relative concentration of albumin in the serum of postmetamorphic animals.     

Hog thyroid peroxidase: physical, chemical, and catalytic properties of the highly purified enzyme.

Studies are reported on the purity and on the physical, chemical, and catalytic properties of a highly purified, stable, thyroid peroxidase (TPO). The enzyme was solubilized by treatment with deoxycholate and trypsin, and it was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. The final product, designated TPO VII, had a value for A₄₁₀/A₂₈₀ of 0.54, and its specific activity based on the guaiacol assay (794 μmol of guaiacol oxidized/min/mg) was considerably greater than that of any previously described TPO. Specific activity values based on other peroxidase-catalyzed reactions were also higher for TPO VII than for previous TPO preparations. Purity estimates for TPO VII, based on polyacrylamide disc gel electrophoresis and on isoelectric focusing in polyacrylamide gels, ranged from 80 to 95%. The molecular weight, determined by sedimentation equilibrium, was 93,000. Results of sodium dodecyl sulfate-gel electrophoresis also indicated a molecular weight of approximately 90,000. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions indicated that TPO VII is composed of two peptide chains of unequal size, with the larger about 2.5-fold the size of the smaller. Carbohydrate analysis revealed that TPO is a glycoprotein containing about 10% by weight of carbohydrate. The predominant sugars were mannose and N-acetyl glucosamine. A significant amount of glucose was also found, along with small amounts of galactose, fucose, and xylose. The amino acid composition of TPO VII showed a high proline content, a predominance of arginine over lysine, and a ratio of [Asp] plus [Glu] to [Lys] plus [Arg] of over 2. Isoelectric focusing in polyacrylamide gels indicated an isoelectric pH of 5.75. In agreement with observations made on earlier preparations of TPO, heme spectral data showed significant differences between the pyridine hemochromogens of TPO VII and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. Circular dichroism measurements indicated that approximately 40% of TPO VII involves α helix or β structure.     

Tetrahydrocannabinol, sesame oil and biogenic amines: a preliminary report

Sesame oil, a vehicle we used to solubilize tetrahydrocannabinol for chronic oral administration to mice, unexpectedly reduced norepinephrine in the brain, heart and spleen. This indicates that some solubilizing agents (at least sesame oil) may not be pharmacologically inert. Tetrahydrocannabinol significantly increased the concentration of brain serotonin without a concomitant change in 5-hydroxyindoleacetic acid, suggesting that inhibition of monoamine oxidase had not occurred.