STUDIES ON THE METABOLISM OF THE AUTOTROPHIC BACTERIA : III. THE NATURE OF THE ENERGY STORAGE MATERIAL ACTIVE IN THE CHEMOSYNTHETIC PROCESS

In the autotrophic bacterium, Thiobacillus thiooxidans, the oxidation of sulfur is coupled to transfers of phosphate from the medium to the cells. CO₂ fixation is coupled to transfers of inorganic phosphate from the cells to the medium and is dependent, in the absence of concomitant sulfur oxidation, upon the amount of phosphate previously taken up during sulfur oxidation. The energy reservoir, which is formed by sulfur oxidation in the absence of CO₂ and which can be released for the fixation of CO₂ under conditions which do not permit sulfur oxidation, is a phosphorylated compound and the data suggest that the energy is stored in the cell as phosphate bond energy. It is possible to oxidize sulfur at a constant rate for hours in the absence of CO₂. The phosphate energy formed during this process is probably released by cell phosphotases. It is possible to inhibit these phosphotases by means of inorganic phosphate and thus to inhibit sulfur oxidation in the absence of CO₂. In the presence of CO₂, where alternative uses for the phosphate energy are available, the inhibition is relieved. Sulfur oxidation (energy input) is coupled, not to CO₂ fixation, but to phosphate esterification. CO₂ fixation (energy utilization) is coupled with phosphate release.     

THE PENETRATION OF THE MEMBRANE OF BRAIN MITOCHONDRIA BY ANIONS

The permeability of the membrane of rat brain non-synaptosomal mitochondria, towards inorganic and substrate anions, was assessed by measuring the rate of swelling that occurred when mitochondria were suspended in an iso-osmotic solution of a permeant anion, in the presence of a permeant cation such as NH⁺₄ or K⁺ in the presence or absence of valinomycin. In NH⁺₄-phosphate swelling was higher than it was in KCI or K⁺-phosphate, which showed the prevalence of the mechanism of phosphate transport previously demonstrated in liver mitochondria. The entry of succinate and L-malate seemed to require the presence in the inner mitochondrial membrane of specific carriers. as previously postulated for liver mitochondria, but the rate of swelling of brain mitochondria was lower than that of liver organelles. In K⁺-succinate, in the presence of antimycin, added ATP induced swelling and this was attributable to the simultaneous permeation both of the anion and the cation. Fumarate did not penetrate into brain mitochondria. Practically no swelling was recorded in NH⁺₄ or K⁺-citrate, which indicated that this anion penetrated poorly into the isolated brain mitochondria even in the presence of malate. Swelling occurred in NH⁺₄-L-glutamate in the presence of rotenone, and the entry of this anion seemed to follow a gradient of concentration although the presence of a specific translocator in the inner mitochondrial membrane might be concerned. The entry of glutamate was independent of that of phosphate and N-ethylmaleimide appeared to be a specific inhibitor of this entry. Swelling in K⁺-L-glutamate, in the presence of rotenone, was enhanced by the addition of valinomycin or ATP but in the latter case when osmotic equilibrium was reached swelling was not reversed by oligomycin. In conclusion, the lesser extent of swelling of isolated brain mitochondria compared with liver mitochondria could be attributed to the heterogeneity of the populations of these organelles, each population possessing its own characteristics of membrane permeability. Observations of electron micrographs of brain mitochondria incubated in iso-osmotic substrate anions confirmed the heterogeneous rate of swelling of these particles.     

THE ACTIVATION OF THIAMINE DIPHOSPHATASE BY ATP IN RAT BRAIN

• 1 Thiamine diphosphatase (TDPase) of brain was activated by a low concentration of ATP.
• 2 In thiamine-deficient rats TDPase activity in the brain increased significantly relative to that in pair-fed animals, while in liver it decreased by the same amount as in the pair-fed controls.
• 3 Liver TDPase was localized almost entirely in the soluble fraction, but in the brain it was bound to insoluble protein. On treatment with Triton X-100 brain TDPase activity increased.
• 4 The ATP content of the brain of deficient rats, but not the ADP or AMP content, was significantly higher than in the control group. The level of inorganic phosphate in the brain and spinal cord of deficient animals was elevated markedly, while that of P-creatine was unchanged.
• 5 The possible roles of brain TDPase in relation to nerve conduction and the blood-brain barrier are discussed.

     

The Absorption and Utilization of Phosphate by Young Barley Plants: IV. THE INITIAL STAGES OF PHOSPHATE METABOLISM IN ROOTS

Organic phosphorus compounds have been extracted from the rootsof intact plants which have absorbed radioactive phosphate.The distribution of phosphorus between different organic fractionsof the root during a 24-hour absorption period is markedly influencedby the concentration of phosphorus supplied. Less than 1 minute after entry a significant proportion of theabsorbed phosphorus is found to be in organic compounds. Incorporationinto nucleotides is particularly rapid, whereas incorporationinto hexose phosphates occurs more slowly. The pattern of esterificationis influenced by the phosphate status of the plants. 2: 4-dinitrophenol (10^–4M.) reduces the uptake of phosphorusand also the extent of esterification, the latter effect beingdue solely to reduced incorporation into the nucleotide fraction. Although extensive esterification of phosphate occurs in roots,it appears to be transported to the shoot as inorganic phosphateaccompanied by only a small amount of a single organic compound.     

BIOCHEMICAL CHARACTERIZATION OF β-N-OXALYL-l-α,β-DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN AS AN EXCITANT AMINO ACID

Abstract— β- N -Oxalyl- l -α,β-diaminopropionic acid (ODAP), the toxin isolated from the seeds of Luthyrus sativus produces head retraction, tremors and convulsions when injected into a variety of experimental animals. In 12-day-old rats, it has been found that the convulsive behaviour is accompanied by profound biochemical changes in the brain. The brain homogenates prepared from ODAP injected animals show a higher rate of respiration. There is a decrease in the brain glucose, glycogen, ATP, phosphocreatine and acetylcholine levels of the convulsing animals. The inorganic phosphate, lactic acid and acetylcholineesterase levels increase. These results establish that ODAP is a typical convulsant.     

THE DISTRIBUTION OF THE WATER-SOLUBLE INORGANIC ORTHOPHOSPHATE IONS WITHIN THE CELL: ACCUMULATION IN THE NUCLEUS : Electron Probe Microanalysis

Lead acetate treatment of unfixed cells immobilizes the intracellular water-soluble, inorganic orthophosphate ions as microcrystalline lead hydroxyapatite precipitates (see reference 1). These precipitates have been analyzed with the electron microprobe. A much higher concentration of phosphorus has been found in the nucleoli of maize root tip cells fixed in lead acetate-glutaraldehyde (organic phosphorus plus inorganic orthophosphate), as compared to the nucleoli of roots fixed in glutaraldehyde alone (organic phosphorus). The concentration of the inorganic orthophosphate pool in these nucleoli is three to five times as high as the concentration of the macromolecular organic phosphate. Since nearly all of the latter is in RNA, the concentration of inorganic phosphate in the nucleolus is calculated to be roughly 0.5–0.8 M. About 30%—and up to 50%—of the total cellular inorganic phosphate is accumulated in the nucleolus since the mean concentration per cell is about 10^-2 M. In the extranucleolar part of the nucleus the mean concentration was estimated by densitometry to be roughly six times less than in the nucleolus (⩽ 0.1 M), and appears more concentrated in the nucleoplasm than in the condensed chromatin. While there is no direct evidence for the concentration in the cytoplasm, it certainly must be much lower than the mean cellular level (i.e., < 10^-2 M) since the nucleus is about 10% of the total cell volume. The implications of this compartmentation in the intact cell are discussed in connection with (A) the availability of orthophosphate ions for the cytoplasm in those processes in which these ions affect the rate of enzymatic reactions, and (B) protein nucleic acid interactions within the nucleus and nucleolus.     

ACTIVATORS and INHIBITORS OF BRAIN GLUTAMINASE

(1) The glutaminase activity of a guinea pig brain dispersion (a 1500g supernatant solution) was tested at pH 7.5 in the presence of a series of organic acids at 20 mm with or without the further addition of 7.5 mm -phosphate. (2) In the absence of phosphate, glutaminase activity was strongly enhanced by tricarboxylic acids, less strongly by dicarboxylic acids, and slightly, if at all, by monocarboxylic acids. Acidic amino acids were intermediate between mono- and dicarboxylic acids. In the presence of 7.5 mm -phosphate, the addition of 20 mm organic acids resulted in strong potentiation of the activating effect in many cases. The activating effect of even the most active of the organic acids tested, citrate, was only about half of the effect of an equimolar amount of phosphate. (3) At phosphate concentrations approaching the saturation level for activation, the further addition of citrate was without effect. (4) Glutaminase was strongly activated by ITP which was about three times as active as inorganic phosphate. IMP was less active than inorganic phosphate and creatine phosphate had only slight activity which seemed to be accounted for by its content of inorganic phosphate. (5) Glutaminase was activated by fluoride, in the presence as well as in the absence of added phosphate. Chloride, bromide, and iodide, at 100 mm , produced increasing inhibition of the phosphate-activated reaction. The inhibiting effect of iodide was qualitatively competitive with phosphate. (6) The effects of various other potential inhibitors and activators, including SH-reagents, d -glutamine, several amino acids, and amino acid derivatives were studied. (7) The results have been discussed with particular reference to their significance in elucidating the natural function of brain glutaminase. It has been suggested that glutaminase is an allosteric enzyme and that the secondary active site requires a reaction with three anionic groups for full activation.     

THE MODE OF ACTION OF VITAMIN D

1. The purpose of this review article is to re-evaluate and integrate many of the observations related to the physiological effects of vitamin D, using as a working hypothesis the concept that the vitamin may be acting analogously to a steroid hormone in terms of its ability to interact with genetic information and ultimately elicit a physiological response. Prior to this time the problem of the mechanism of action of vitamin D has primarily been approached from the point of view that the vitamin was acting as a cofactor for some specific enzymic reaction. 2. The physiological activities of vitamin D are integrated with those of parathyroid hormone to provide a homeostatic control for the regulation of primarily calcium and secondarily phosphate metabolism. It is proposed that the role of vitamin D in this homeostatic control mechanism is older and more fundamental than that of parathyroid hormone. The interaction of vitamin D on skeletal calcium metabolism may have evolved before the effects of the vitamin on intestinal calcium absorption. 3. There are several physiological defects of calcium metabolism—rickets, osteo-malacia, vitamin D-resistant rickets and idiopathic hypercalcaemia—all of which may be a consequence of an aberration in one or another of the interlocking steps of the vitamin D-dependent and calcium-dependent homeostatic control mechanism. 4. The most thoroughly established action of vitamin D in vivo is to promote or facilitate the intestinal absorption of calcium. Although the exact biochemical details of this process are not available, this may involve vitamin D-mediated synthesis of the appropriate enzyme systems or the alteration of membrane structure necessary for calcium absorption. It is not yet unequivocally established whether calcium absorption is an energy-dependent active transport process or is a passive carrier-mediated or simple diffusion process. 5. The exact action of vitamin D on bone metabolism is not as well established, but the primary effect of the vitamin is likely to mediate bone resorption. The vitamin D-dependent activities of the cell in both the intestine and bone are to absorb calcium and transfer it to the blood. 6. No direct effects of vitamin D on intestinal absorption of phosphate have been found. Furthermore the validity of a vitamin D-mediated renal reabsorption of phosphate is questioned, for the major effects of vitamin D are cation oriented. If the renal effects of vitamin D are true, it is postulated that the mechanism of action of the vitamin here on the anion, phosphate, is fundamentally different from its cation oriented mechanism. 7. There is a lag in the action of vitamin D on the vitamin mediated: (a) transport of calcium both in vivo in rats and chicks, and in vitro with everted intestinal slices; (b) the apparent increased permeability of intestinal mucosa; (c) increased levels of citric acid in serum or bone; (d) the increased incorporation of radioactive inorganic phosphorus into intestinal mucosa phospholipids. As shown by the use of radioactive vitamin D, this lag is not due to a lack of the vitamin in the target organs. 8. Whereas large, unphysiological doses of radioactive vitamin D localize in all tissues and all subcellular fractions, small physiological doses of radioactive vitamin D localize predominantly in the nucleus of the intestinal mucosa. The amount of vitamin D localized in the nucleus would appear to be too low for the vitamin to function as a cofactor, and is more indicative of an interaction on or with deoxy-ribonucleic acid. 9. Actinomycin D, an inhibitor of DNA-directed RNA synthesis, inhibits the action of vitamin D in mediating intestinal calcium absorption and bone resorption. Vitamin D also stimulates messenger-RNA synthesis in intestinal mucosa within 1/2 hr. of vitamin treatment. Vitamin D may play a crucial role, along with parathyroid hormone and calcium, in a DNA, gene-dependent, homeostatic control mechanism for cal, ium metabolism. In this system the vitamin D molecule has certain very specific structural requirements which are probably a reflection of the specificity of its receptor molecule, rather than structural requirements for a cofactor-enzyme relationship.     

THE ROLE OF ANTIBIOTICS IN THE DECOMPOSITION OF SAWDUST: I. INHIBITION OF THE GROWTH OF CELLULOSE-DECOMPOSING BACTERIA

It has been shown that 'deal' sawdust contains substances which inhibit the growth of cellulose-decomposing bacteria of the genera Sporocytophaga and Cellulomonas , the former being the more sensitive. The substances can be extracted with water or a mildly alkaline solution of inorganic salts, the latter being rather more effective. The extracted material is acidic but still exhibits activity even after neutralization, especially towards Sporocytophaga. Sawdust which has been extracted with water or the alkaline solution, or neutralized by admixture with calcium carbonate, no longer prevents the growth of Cellulomonas on added filter paper cellulose, although there is a delay before the attack becomes evident, but Sporocytophaga is still completely inhibited under all three conditions.     

THE DISTRIBUTION OF ACETYL-CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO-ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE1

A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl-CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl-CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane-chloroform wash followed by an ether extraction. In the acetyl-CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl-CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of [Me-¹⁴C]choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The [¹⁴C]ACh formed from acetyl-CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the [¹⁴C]ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl-CoA levels in rat whole brain when killed by the near-freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl-CoA was the same whether the rats were killed by the near-freezing method or by total freezing in liquid nitrogen. The levels of acetyl-CoA did not change with time after death when the tissue was maintained at a temperature of ?10°C. In the same tissue extracts from rat whole brain killed by the near-freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl-CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl-CoA in heart, liver and kidney are presented.     

REGIONAL DISTRIBUTION OF GLUTAMIC ACID DECARBOXYLASE IN THE DEVELOPING BRAIN OF THE PYRIDOXINE-DEFICIENT RAT

—Glutamic acid decarboxylase was determined in seven brain regions: hypo-thalamus; midbrain; thalamus; corpus striatum; cerebral cortex-hippocampus; medulla-pons; and cerebellum, of suckling rats subjected to Vitamin B₆ deficiency for 2 weeks from birth; of adult rats subjected to the deficiency for 5 weeks and of their respective controls. Large regional variations in the enzyme activity were found in brains of both adult and suckling control rats. The activity of the enzyme (assayed without pyridoxal phosphate) and its saturation with endogenous cofactor were markedly reduced in all brain regions of both suckling and adult pyridoxine-deficient rats. The apoenzyme (activity assayed with pyridoxal phosphate), in adult rat brain, showed no change with the deficiency in all regions except in the cerebellum where it increased slightly. In pyridoxine-deficient suckling rat brain, the apoenzyme increased substantially in all regions suggesting a process of enzyme induction. The increase in apoenzyme varied from region to region.     

纳米羟基磷灰石/胶原复合材料制备方法研究

研究了在脱钙骨基质内原位沉积纳米羟基磷灰石的电化学方法,探讨了影响沉积的实验因素和条件.并利用红外光谱和X衍射表征无机相的组成,透射电子显微镜观测晶体的形态和尺寸,光学显微镜观察无机相分布,灰化法测定无机成分含量.结果表明,电化学方法可以制备出纳米羟基磷灰石/胶原复合材料,其无机成分为53 9±3.2%,并且无机相的组成、分布、性质与自然骨非常一致,是纳米复合材料.     

ELEVATED CONCENTRATIONS OF HISTAMINE IN THE RAT BRAIN

—Whole brain concentrations of histamine (Hm) in the rat were measured with a sensitive and specific single-isotope enzymatic microassay and found to be elevated about 2-fold in rats killed by focused microwave irradiation compared with rats killed by decapitation. However, regardless of the method of killing, whole brain Hm concentrations were elevated about 4-fold when the brains were homogenized in 12.5 vol vs 2.5 vol of water or phosphate buffer. Homogenization of brain regions in 12.5 vol of water resulted in Hm concentrations that were about 10 times greater than previously reported values (except in the hypothalamus). Since (1) the‘extra’Hm could be extracted from pellets of low volume homogenates, and (2) synthesis of Hm is not likely to occur through an increase in the volume of water or buffer used for homogenization, we suggest that the elevated concentrations of Hm seen after high volume homogenization of brain tissue are due to a more complete extraction of Hm from brain tissue, and not to an artifactual production of this amine from its precursor (histidine) or some other compound(s).     

A STUDY OF THE ADENOSINE TRIPHOSPHATASE ACTIVITY OF MYOSIN AND ACTOMYOSIN

1. An experimental study was made on the adenosine triphosphatase action of crystalline myosin and actomyosin preparations under different conditions. 2. No enzymatic activity was found in the absence of salts. Activation was given by KCl and CaCl₂, whereas MgCl₂ in the presence of other ions inhibited. 3. The effect of pH is complex. In stabilizing buffers or at low temperature, there are two optima (pH 6.2 to 6.5 and pH 9.2) provided Ca is present. Without Ca only the acid optimum is found. The highest activities are reached in glycine buffer at pH 9.2 in the presence of Ca. 4. The study of the Mg-Ca antagonism revealed that the inhibition due to Mg is fully developed with Mg:Ca ratios less than 1, the inhibition usually exceeding 90 per cent. 5. It is shown that in the muscle the myosin-ATPase is most probably also subjected to the inhibitory action of the Mg ions. 6. From data in the literature it is calculated that the liberation of inorganic phosphate during muscular activity takes place at a rate of at least 0.200 mg. P per mg. myosin per minute. 7. From the results of the present study it is found that the myosin in the muscle can liberate inorganic phosphate from ATP at a rate of at most 0.003 mg. P per mg. myosin per minute. 8. It is concluded therefore that myosin-ATPase cannot be responsible for the liberation of the main part of the phosphate in contracting muscle, and therefore cannot have the rôle in muscular metabolism ascribed to it in recent hypotheses and discussions.     

THE FINE STRUCTURE OF OSTEOBLASTS IN THE METAPHYSIS OF THE TIBIA OF THE YOUNG RAT

The appearance of osteoblasts after fixation with OsO₄ is described in this paper. They have the basic structures found in other types of cells. The most striking feature is the array of rough-surfaced membranes of the endoplasmic reticulum; this feature is in keeping with the osteoblast's function of producing collagen as the bone grows. The sacs formed by these membranes probably represent the protein-containing granules described by other workers using the light microscope. They contain fine fibrillary material, and similar fibrils are to be found free in the cytoplasm. These fibrils could be tropocollagen units, although fibrils recognizable as collagen by their structure are found only outside the cell. The arrangement of the cell organelles does not seem to be related to the formation of collagen, but correlation of the fine structures of the cells with the histochemical and cytochemical findings in these cells reported by other workers leaves no doubt that they are directly concerned in the production of the organic matrix. It has not been possible to show that osteoblasts influence the passage of calcium or phosphate ions from the blood to the bone matrix.     

THE EFFECT OF ORGANOPHOSPHORUS INSECTICIDAL SPRAYS ON THE GROWTH AND PHOSPHORUS CONTENT OF BRUSSELS SPROUTS

No evidence was found that the insecticides schradan or demeton directly increase the growth of plants. The dry weight of Brussels sprouts plants grown in soil supplying little phosphorus was unaffected by spraying six times with schradan or demeton, or with sodium phosphate Solution supplying the same amount of phosphorus as the insecticides. The dry weight of larger plants grown in soil supplying more phosphorus was unaffected by spraying with demeton or inorganic phosphorus Solutions; schradan spray decreased it in one experiment but not in the other.
Spraying with schradan, or with the equivalent amount of phosphorus as sodium phosphate, in one experiment increased the phosphorus content of plants grown in soil supplying little phosphorus. With high phosphorus supply in the same experiment, sodium phosphate Solution, but not schradan, increased phosphorus content of the plants slightly, but not significantly. Phosphorus content was not affected either by schradan sprays in the other experiment or by demeton sprays in both experiments.     

The Phosphate Source Influences Gene Expression and Quality of Mineralization during In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells

For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of β-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost- and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design.     

Fractionation of sediment phosphate with chelating compounds: a simplification, and comparison with other methods

The NTA/EDTA fractionation of sediment-bound, inorganic phosphate was improved and made more effective. The iron-bound phosphate is now extracted with Ca-EDTA plus dithionite and followed by an extraction of calcium-bound phosphate by Nat-EDTA at pH = 4.5. A comparison is made with two other sequential extraction procedures: the SEDEX and the Hieltjes & Lijklema extractions. The EDTA extractions have an advantage over the other two methods.     

A second moment NMR method to study phase changes in bone

A second moment NMR method is presented to study changes involving phosphorus in intact bone. On the assumption that the inorganic calcium phosphate system in bone is a mixture of hydroxyapatite and tricalcium phosphate, the percentage of each is determined for cow bone and two human samples. Comparing this method with an X-ray radial distribution function method gives promising results.