Mammoth albumin.

Serum albumin was detected immunologically in muscle from a mammoth that died about 40,000 years ago. Rabbits injected with ground mammoth muscle produced antibodies that react strongly with elephant albumin, weakly with sea cow albumin, and still more weakly or not at all with other mammalian albumins. Since elephant albumin elicited antibodies with the same specificity, some of the surviving mammoth albumin molecules evidently have antigenic sites identical to those on native elephant albumin. Much of the mammoth albumin has, however, undergone postmortem change. The small amount of soluble albumin extractable from mammoth muscle is heterogeneous in size, charge, and antigenic properties.

Metabolic control of the circulation. Effects of acetate and pyruvate.

Chloralose-anesthetized dogs were infused intravenously with either Tris-acetate or Tris-pyruvate at 0.0375, 0.075, and 0.15 mmol/kg per min successively, each for 20 min. Acetate infusion increased cardiac output, left ventricular dP/dt and dP/dt/P, and coronary blood flow, while pyruvate infusion did not. Infusions of either substance increased arterial blood and skeletal muscle concentrations of citrate and malate, but only acetate infusion increased the tissue AMP content and decreased the ATP:AMP ratio. The increase in cardiac output produced by acetate was accompanied by an increase in total body oxygen consumption and a decrease in the difference between arterial and mixed venous blood oxygen. Myocardial oxygen consumption increased during acetate infusion, but the decrease in myocardial oxygen extraction and the increase in coronary sinus blood oxygen saturation suggest that an active coronary vasodilation which was not a result of the increased cardiac work, occurred. The concentration of hypoxanthine in the coronary sinus and the content of myocardial adenosine increased, which suggests that the increase in coronary blood flow was caused by the vasodilator action of adenosine released from the myocardium, and that adenosine production is not necessarily tied to PO(2). These systemic and coronary hemodynamic changes also occurred when acetate (0.075 mmol/kg per min) was infused into conscious dogs. Acetate infusion also increased blood flow to the gastrointestinal tract, kidneys, intercostal muscle, and diaphragm. These changes were not affected by propranolol pretreatment, but were abolished by pretreatment with fluoroacetate which reduced acetate oxidation. These results suggest that the circulatory stimulation produced by acetate was not caused by increases in tricarboxylic acid cycle intermediates. Instead, it was probably related to the increased cleavage of ATP to AMP that accompanies activation of acetate to acetyl CoA, and was not mediated via beta-adrenergic receptors. It is speculated that hemodynamic changes may occur in patients who undergo hemodialysis with acetate-containing dialysate. Hemodynamic changes of ethanol may also be brought about by acetate, which is one of the intermediates that accumulates during ethanol metabolism.

The purine nucleotide cycle. A pathway for ammonia production in the rat kidney.

Particle-free extracts prepared from kidney cortex of rat catalyze the formation of ammonia via the purine nucleotide cycle. The cycle generates ammonia and fumarate from aspartate, using catalytic amounts of inosine monophosphate, adenylosuccinate, and adenosine monophosphate. The specific activities of the enzymes of the cycle are 1.27+/-0.27 nmol/mg protein per min (SE) for adenoylosuccinate synthetase, 1.38+/-0.16 for adenylosuccinase, and 44.0+/-3.3 for AMP deaminase. Compared with controls, extracts prepared from kidneys of rats fed ammonium chloride for 2 days show a 60% increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity, and a greater and more rapid synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate. Extracts prepared from kidneys of rats fed a potassium-deficient diet show a twofold increase in adenylosuccinate synthetase and a threefold increase in adenylosuccinase activity. In such extracts the rate of synthesis of ammonia and adenine nucleotide from aspartate and inosine monophosphate is also increased. These results show that the reactions of the purine nucleotide cycle are present and can operate in extracts of kidney cortex. The operational capacity of the cycle is accelerated by ammonium chloride feeding and potassium depletion, conditions known to increase renal ammonia excretion. Extracts of kidney cortex convert inosine monophosphate to uric acid. This is prevented by addition of allopurinol of 1-pyrophosphoryl ribose 5-phosphate to the reaction mixture.

Effect of omega 3 and omega 6 fatty acids on transformation of cultured cells by irradiation and transfection.

Mouse embryo fibroblasts (C3H 10T1/2) were exposed to 4 Gy of gamma-rays. The cells yielded 5-8 transformed foci per 10(4) surviving cells. Addition of 100 microM of either eicosapentaenoate or docosahexaenoate to the tissue culture medium reduced the number of transformed foci to 0-1.4. C3H 10T1/2 and NIH 3T3 cells were transfected with plasmid T24 containing the Harvey ras oncogene. C3H 10T1/2 cells yielded 0.85-1.1 foci/ng DNA, while NIH 3T3 cells yielded 0.12-0.14 foci/ng DNA. Foci formation was suppressed 65% in C3H 10T1/2 cells and 93% in NIH 3T3 cells when 100 microM eicosapentaenoate was present in the culture medium. Docosahexaenoate had a similar but somewhat weaker effect. Addition of arachidonate to the medium had little or no effect. Cells grown in the presence of added eicosapentaenoate or docosahexaenoate produced much less prostaglandin E when challenged with calcium ionophore A23187. This is a reflection of changes in arachidonate production or utilization that occur during transformation which are suppressed by the added omega 3 fatty acids. Addition of eicosapentaenoate or docosahexaenoate to the culture medium resulted in extensive remodeling of the molecular species of the four major phospholipid classes that were examined. In its simplest form, omega 3-fatty acid-containing species substantially replaced omega 6-fatty acid-containing species. However, many more subtle changes occurred, and the different phospholipids responded differently to different polyunsaturated fatty acids. A feature of C3H 10T1/2 cells was their preferential accumulation of molecular species of 22-carbon fatty acids such as docosapentaenoate (22:5 omega 3) and docosatetraenoate (22:4 omega 6) in preference to eicosapentaenoate (20:5 omega 3) and eicosapentaenoate (arachidonate, 20:4 omega 6). It is proposed that the protective effect of eicosapentaenoate and docosahexaenoate arises out of the changes in the composition of the fatty acids that are released from one or more phospholipids by the action of phospholipases. The changes consist of a reduced release of arachidonate, the normal substrate of cyclooxygenase and lipoxygenases, and a greatly increased release of eicosapentaenoate and docosahexaenoate, which inhibit one or more of these enzymes, or form oxygenated products which are not as active as the arachidonate-derived products. Other mechanisms are also considered.

Molecular preservation in Late Cretaceous sauropod dinosaur eggshells.

Exceptionally preserved sauropod eggshells discovered in Upper Cretaceous (Campanian) deposits in Patagonia, Argentina, contain skeletal remains and soft tissues of embryonic Titanosaurid dinosaurs. To preserve these labile embryonic remains, the rate of mineral precipitation must have superseded post-mortem degradative processes, resulting in virtually instantaneous mineralization of soft tissues. If so, mineralization may also have been rapid enough to retain fragments of original biomolecules in these specimens. To investigate preservation of biomolecular compounds in these well-preserved sauropod dinosaur eggshells, we applied multiple analytical techniques. Results demonstrate organic compounds and antigenic structures similar to those found in extant eggshells.

A multifunctional vector system for heterologous expression of proteins in Escherichia coli. Expression of native and hexahistidyl fusion proteins, rapid purification of the fusion proteins, and removal of fusion peptide by Kex2 protease.

Vectors have been constructed for the general purpose of expressing foreign proteins in E. coli. These vectors allow the production in high yield of either native proteins or of fusion proteins which contain, at their amino terminus, the peptide Met Gly His6 Ser Gly Leu Phe Lys Arg/, where Leu Phe Lys Arg/ is the recognition site for Kex2 protease which cleaves at the site indicated by /. The His6 sequence is used as a ligand for the one-step affinity purification of the expressed proteins on columns containing Ni or Zn ions chelated to iminodiacetic acid-agarose. After affinity chromatography, the purification peptide is cleaved off with Kex2 protease from Saccharomyces cerevisiae. The vectors also allow site-directed mutagenesis and sequencing of the cloned gene to be expressed without any intermediate subcloning. For practical examples of over-expression, affinity purification, and removal of the purification peptide, we chose a high-molecular-weight protein, phospholipase C gamma 1 (PLC gamma 1, M(r) 148,000) and a low-molecular-weight protein, Hit-1 (M(r) 16,000). Both were obtained pure and in high yield. PLC gamma 1 was fully active; the function of Hit-1 is not known. A set of companion vectors for co-expression of additional proteins has also been developed. These allow expression of proteins which enhance the production or activity of the protein of primary interest and of proteins which exhibit trans-interactions.

Gross cystic disease fluid protein in nipple aspirates of breast fluid of Asian and non-Asian women.

Gross cystic disease fluid protein 15 (GCDFP-15) is universally present in the apocrine metaplastic epithelium of cystic breast disease and breast cancer, but it is rarely found in normal breast epithelium. Therefore GCDFP-15 detected in nipple aspirates of breast fluid (NAF) could serve as a biochemical marker of the presence and possibly extent of apocrine metaplasia within the breast. GCDFP-15 levels were measured in NAF from 37 Asian and 78 non-Asian women using radioimmunoassay. GCDFP-15 (range, 0-81,643 micrograms/ml) was found in the NAF of all but 1 woman and was highly correlated between right and left breasts. Mean concentrations of GCDFP-15 were significantly lower in NAF from Asian compared with non-Asian women. Markedly reduced levels of GCDFP-15 were found in the 17 women who had been parous in the previous 2 years. In women not parous within the prior 2 years, no relationship was found between GCDFP-15 levels and age, weight, age at menarche, first-degree family history of breast cancer, parity, oral contraceptive use, or smoking history. High concentrations of GCDFP-15 were found in the NAF of women with a history of a benign breast biopsy. Because similarly high levels of GCDFP-15 were found in NAF in over 40% of women without a history of benign breast biopsy, and because GCDFP-15 in the breast is produced only by apocrine metaplastic epithelium, we infer that the breasts of these women likely contain a significant degree of apocrine metaplasia.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of different molecular species of sphingomyelin on phospholipase C delta 1 activity.

Bovine brain sphingomyelin was separated into different molecular species using a reverse phase column. PLC delta 1 was inhibited by all molecular species of sphingomyelin. The extent of this inhibition was dependent on the hydrophobicity. Based on fatty acid analysis, we conclude that the inhibition of PLC delta 1 depends on the chain length and degree of unsaturation of the fatty acid moiety of SM. N-palmitoyl-D-sphingomyelin and N-stearoyl-D-sphingomyelin inhibited PLC delta 1 less then N-oleoyl-D-sphingomyelin. In the absence of Ca2+ (1 mM EGTA) all tested molecular species of SM inhibited weakly the enzyme. The sensitivity of PLC delta 1 to inhibition by SM increased with increasing Ca2+ concentration. The shape of calcium curve differed for molecular species with saturated and unsaturated fatty acids. Inhibition of PLC delta 1 by N-palmitoyl-D-sphingomyelin and N-stearoyl-D-sphingomyelin reached a maximum at 0.2 microM Ca2+, while inhibition by N-oleoyl-D-sphingomyelin reached maximum at 2 microM Ca2+. PLC delta 1 is more sensitive to inhibition by SM when it is maximally activated by spermine and calcium and the extent of this inhibition depends on the length and degree of fatty acid unsaturation of the molecular species.

Clinical experience with 99mTc-DMSA (dimercaptosuccinic acid), a new renal-imaging agent.

Results are reported from the clinical evaluation of a new radiopharmaceutical for renal imaging, 99mTc-DMSA (dimercaptosuccinic acid). Sixty-five patients were studied and six of these patients' scintiphotos are illustrated. The physical characteristics of 99mTc and the mercurial-like kinetics of the chelate produced high-resolution scintiphotos of the renal parenchyma in patients of all ages and with a variety of disease entities. The commercial availability of the material in kit form permits its usage in all nuclear medicine facilities.

Effects of adenosine and adenosine analogues on glycogen metabolism in isolated rat hepatocytes.

Adenosine and adenosine analogues were incubated with isolated rat hepatocytes. Adenosine and 5'-deoxy-5'-chloroadenosine stimulated glucose release, glycogen loss, and the conversion of glycogen phosphorylase b to a. The effect was of short duration for adenosine, but of long duration for 5'-deoxy-5'-chloroadenosine. The effects on glucose release and phosphorylase were blocked by theophylline, an R-receptor blocking agent, but not by nitrobenzylthioinosine or dipyridamol which are nucleoside transport inhibitors. A dose-dependent rise in cyclic AMP concentration was observed in hepatocytes 1 min after adding adenosine. It is concluded that adenosine exerts these effects in liver by activating adenylcyclase. Adenosine may be involved in the short-term regulation of hepatic glycogen phosphorylase.

Inhibition of phosphorylation of troponin I in rat heart by adenosine and 5'-chloro-5'-deoxyadenosine.

We have investigated the effects of adenosine on protein phosphorylation in extracts of rat heart. Incubation of a myofibrillar fraction with [gamma-32P]ATP resulted in the phosphorylation of several proteins by endogenous protein kinases. The adenosine analog 5'-chloro-5'-deoxyadenosine inhibited the phosphorylation of a 29 kD protein in this preparation. The protein was identified as cardiac troponin I (cTnI) by two-dimensional gel electrophoresis, using purified cTnI as standard. Addition of the catalytic subunit of cAMP-dependent protein kinase to the myofibrillar fraction increased phosphorylation of cTnI; this increase was inhibited by 5'-chloro-5'-deoxyadenosine and adenosine. Phosphorylation of purified cTnI by the catalytic subunit was also inhibited by 5'-chloro-5'-deoxyadenosine. Under these conditions used, 50% inhibition of phosphorylation by either endogenous or exogenous kinase was observed at approximately 50 microM 5'-chloro-5'-deoxyadenosine or adenosine. The inhibition described here occurred independently of catecholamines. The effects of ADP, AMP, and adenine on cTnI phosphorylation are also described.

Inhibition of phospholipase C delta by hexadecylphosphorylcholine and lysophospholipids with antitumor activity.

The antineoplastic compound hexadecylphosphorylcholine (HPC) was shown to be a highly effective inhibitor of phospholipase C delta (PLC delta 1), with an I50 of about 30 nmol/mL (30 microM) in the presence and absence of 200 microM spermine. A number of lysophospholipids, of which HPC can be considered to be a structural analog, also inhibited PLC. Lysosphingomyelin, lysophosphatidylserine, and lysophosphatidylcholine exhibited I50 values of 15, 10, and 7 nmol/mL, respectively, in the presence of 200 microM spermine. The I50 values were increased to 21-53 nmol/mL in the absence of spermine. N,N-Dimethylsphingosine and N,N,N-trimethylsphingosine, which inhibit the metastatic potential of human and murine tumor cells, were weak activators of PLC delta 1. It is postulated that HPC is more effective as an antineoplastic agent than lysophospholipids because HPC is metabolized slowly, while the lysophospholipids are metabolized rapidly in vivo.

Inhibition of adenylosuccinate lyase by L-alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-AICOR).

L-Alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-AICOR) has been synthesized enzymatically using 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) synthetase in conjunction with 5-aminoimidazole-4-carboxylic acid ribonucleotide and L-2-amino-3-(N-hydroxy-N-nitrosoamino)propionic acid (alanosine). The product was characterized by chromatography, ultraviolet spectrum and NMR spectrum at 300 MHz. Alanosyl-AICOR was not a substrate of adenylosuccinate lyase from rat skeletal muscle, but it was an apparent competitive inhibitor in both of the reactions catalyzed by the enzyme. The KI values for alanosyl-AICOR were approximately 1.5 and 1.3 microM in the SAICAR and adenylosuccinate cleavage reactions respectively. These KI values were essentially the same as the Km values for the two substrates of adenylosuccinate lyase. They compare with an accumulation of 70 microM alanosyl-AICOR in leukemic nodules of mice treated with alanosine [A. K. Tyagi and D. Cooney, Cancer Res. 40, 4390 (1980)]. Thus, inhibition of adenylosuccinate lyase may account for much of the inhibitory effect exerted by alanosyl-AICOR in vivo. We confirmed the previous observation that alanosyl-AICOR is an inhibitor of adenylosuccinate synthetase.

Inhibition of phosphatidylinositol kinase in vascular smooth muscle membranes by adenosine and related compounds.

Adenosine and 5'-chloro-5'-deoxyadenosine inhibited the phosphorylation of phosphatidylinositol in membranes prepared from aortic smooth muscle. The nucleosides did not affect the breakdown of phosphatidylinositol-4-phosphate. Under certain conditions, the membrane-bound phosphatidylinositol kinase phosphorylated exogenous phosphatidylinositol. The nucleosides inhibited the enzyme competitively with respect to magnesium-ATP and non-competitively with respect to phosphatidylinositol. Adenosine analogs modified in the ribose moiety were inhibitors with potencies comparable to that of adenosine, whereas adenine nucleotides and purine-modified adenosine analogs were much weaker inhibitors. Density gradient fractionation studies showed that phosphatidylinositol kinase is primarily associated with the sarcoplasmic reticulum. Vascular smooth muscle contraction is associated with increased phosphatidylinositol turnover. Inhibition of phosphatidylinositol kinase by intracellular adenosine may, therefore, be a factor involved in regulating vasodilation.

An extended method for separating and quantitating molecular species of phospholipids.

An improved and extended method for separating and quantitating molecular species of four phospholipid classes is presented. Crude lipid extract is first separated into phospholipid classes on a silica column. Each phospholipid class is then separated into molecular species without derivatization using high-performance liquid chromatography on columns packed with octadecyl silica. Quantitation of individual species is achieved by measuring absorbance at 205 nm. Factors for converting absorbancies to mol fractions have been determined. Quantitation by absorbance at 205 nm agrees well with quantitation by gas chromatography which is preferred to quantitation by phosphate analysis. One hundred phospholipid species have been identified. A table of relative retention times of molecular species is provided. Examples of quantitative analyses of species composition are presented.

Regulation of soluble 5'-nucleotidase I from rabbit heart.

Rabbit heart contains two soluble 5'-nucleotidases, termed N-I and N-II, which can be separated using phosphocellulose chromatography. N-I prefers AMP over IMP as substrate, in contrast to N-II which prefers IMP over AMP. Both enzymes require Mg2+, but the optimum Mg2+ concentrations for the two enzymes are different. High concentrations of NaCl inhibit N-I and activate N-II. Purified N-I is activated by ADP but not by ATP. According to Itoh et al. (1986), purified N-II is activated by both ADP and ATP. N-I has been purified approximately 1000-fold to a specific activity of approximately 100 mumol/mg protein/min. The properties of N-I suggest that it is the enzyme responsible for the release of adenosine from AMP under conditions of hypoxia or increased work load.