Threshold effects on the concanavalin A-mediated agglutination of modified erythrocytes. Bovine erythrocytes, which are not concanavalin A (ConA)-agglutinable, can be rendered so by attaching alpha-D-mannose residues to their outer membrane. The sugars are incorporated by mildly oxidizing the cells with periodate followed by coupling the liberated aldehyde groups with an alpha-thiomannosyl containing hydrazide (I). The rate and extent of ConA-mediated aggregation of the modified cells are not linearly dependent on the amount of sugar incorporated. For example, treatment of the erythrocytes with 0.075 mM periodate for 5 min followed by I led to the introduction of 1.05 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA demonstrated the presence of 66,525 ConA receptors/cell with an average KA = 4.9 X 10(6) M-1 yet the cells failed to aggregate with ConA at concentrations up to 500 microgram ml-1. Treating the cells with 0.1 mM periodate followed by I led to the introduction of 1.42 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA indicated the presence of 78,780 binding sites/cell (KA = 5.9 X 10(6) M-1). These cells were readily aggregated by ConA at concentrations greater than or equal to 64 microgram ml-1. We show here that the sugar incorporation technique is random and that no functional differences were detected in the receptors introduced at the different periodate concentrations. Therefore, the ConA-mediated aggregation of these modified erythrocytes is exquisitely sensitive to small changes in functionally identical receptor densities.


Partial and incomplete oxidation of palmitate by cultured beating cardiac cells from neonatal rats. The discrepancy in the rate of [14C]O2 formation from either [1-14C]- or [16-14C]palmitate is demonstrated and could be explained by the preferential formation of L-(+)-3-hydroxybutyrate from the four carbon atoms at the omega terminus. The identity of this product as L(+)-3-hydroxybutyrate was established and shown to be the major component of the radioactive products in the extracellular medium from palmitate based on (a) ion-exchange chromatographical properties, (b) gas-liquid chromatography, (c) mass spectrometric analysis, (d) stereoisomeric separation, and (e) its very low rate of utilization by the cells. We therefore propose a shunt to the oxidation of palmitate in these cells occurring at the stage of L(+)-hydroxybutyryl-CoA which undergoes deacylation causing the product to be transported outside the cell.


Resonance Raman scattering from uteroferrin, the purple glycoprotein of the porcine uterus. Uteroferrin, a purple-colored, iron-containing glycoprotein, purified from the uterine fluid of progesterone-stimulated pigs, owes its natural purple color to a broad absorption band centered at 545 nm. Laser excitation within the visible absorption band of uteroferrin results in an intense resonance Raman spectrum which bears a striking resemblance to that reported for Fe(III)-transferrin, the iron transport protein of serum. Excitation profiles for the four resonance-enhanced bands of uteroferrin were obtained from 4579 A to 6741 A, using lines from Ar+ and Kr+ lasers. Each of the profiles have maxima near 545 nm. The spectral similarities of uteroferrin and Fe(III)-transferrin lead to the belief that the Fe(III) binding sites of the two proteins must be, at least in some respects, quite similar. In particular, it is concluded that, as in Fe(III)-transferrin, the metal binding site of uteroferrin contains a tyrosine ligand and that the visible absorption spectrum of uteroferrin results from a phenolate to Fe(III) charge transfer.


Mechanism of phosphoacetylglucosamine mutase. Kinetic studies of phosphoacetylglucosamine mutase (EC 2.7.5.2) for the following reactions: 1) Glc-1-P in equilibrium Glc-6-P and 2) GlcNAc-1-P in equilibrium GlcNAc-6-P have been conducted in the presence of Glc-1,6-P2 and GlcNAc-1,6-P2, respectively. In the first reaction, the initial velocity studies at various concentrations of one substrate showed a series of parallel lines in the Line-weaver-Burk plot when the concentrations of the other substrate were changed at several fixed levels. For both reactions, the initial velocity studies performed at fixed ratios of both substrates showed linear lines in the double reciprocal plot. The competitive substrate inhibition pattern was observed in the second reaction. A ping-pong mechanism is proposed for phosphoacetyl-glucosamine mutase. In addition, phosphoacetylglucosamine mutase can be phosphorylated by the addition of Glc-1-[32P]P probably via the reaction of Glc-1-[32P]P with the phosphoenzyme followed by the release of glucose-monophosphate leaving the 32P with the phosphoenzyme. The linkage between the phosphoryl residue and enzyme is stable in acid, but labile in alkali, suggesting phosphoserine (or phosphothreonine) as the phosphorylated amino acid. Biphasic heat denaturation curves suggest the existence of heat-stable and heat-labile forms of this enzyme.


Effect of leucine and metabolites of branched chain amino acids on protein turnover in heart. Leucine, but not isoleucine or valine, inhibited protein degradation and accelerated protein synthesis in hearts perfused with buffer that contained glucose (15 mM) and normal plasma levels of other amino acids, except for the branched chain compounds. Products of leucine, isoleucine, and valine metabolism also inhibited protein degradation and stimulated protein synthesis. These compounds included the transamination and decarboxylation products, as well as acetate, acetoacetate, and propionate. In some, but not all instances, inhibition of degradation and acceleration of synthesis were accompanied by an increase in intracellular leucine. When insulin was added to the perfusate, the rate of degradation was reduced by 40%, but addition of leucine was ineffective in the presence of the hormone. Insulin, leucine (2 mM) and a mixture of branched chain amino acids at normal plasma levels increased latency of cathepsin D in hearts that were perfused with buffer containing glucose. A combination of leucine and insulin increased latency more than either substance alone. These studies indicate that leucine as well as a variety of substrates that are oxidized in the citric acid cycle are involved in regulation of protein turnover in heart muscle.


The covalent differences between bovine alpha- and beta-thrombin. A structural explanation for the changes in catalytic activity. The partial covalent structure of bovine beta-thrombin has been determined by the use of automated Edman degradation and carboxypeptidase digestion of the component polypeptide chains separated by gel filtration following either reduction and carboxymethylation or performic acid oxidation. beta-Thrombin has been found to contain three peptide chains derived by proteolysis of the parent alpha-thrombin molecule. The A chain of alpha-thrombin has been cleaved at two points yielding a peptide (A1 chain) which contains 17 amino acids, beginning with threonine 14 and ending with lysine 30. The B chain of alpha-thrombin has been cleaved at two positions to yield a B1 chain which begins with the NH2-terminal isoleucine and terminates with lysine 65 and a B2 chain which begins with lysine 74 and continues through COOH-terminal serine 259. The A1 chain and B2 chain are linked by a disulfide bridge. Although there is no evidence for a covalent bond between the B1 chain and the B2-A1 chains, the B1 chain is tightly bound to the remainder of the molecule, for separation is achieved only under denaturing conditions.


11-cis-Retinal-binding protein from bovine retina. Isolation and partial characterization. 11-cis-Retinal-binding protein from bovine retina has been purified to apparent homogeneity by gel filtration ion exchange, and hydroxylapatite chromatography. The molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33,000. A value of 36,000 was obtained by gel filtration. With 11-cis-retinal bound to the protein a bleachable spectral peak with maximum absorption at 425 nm was observed. When isolated without addition of exogenous retinal the purified binding protein displayed absorbance maxima at 340 and 425 nm, indicating the occurrence of two ligands in addition to the typical protein absorbance. The endogenous ligands responsible for these maxima were tentatively identified as 11-cis-retinol and 11-cis-retinal, respectively. The binding protein did not cross-react with antibodies prepared against bovine opsin, and its amino acid composition was distinct from that of opsin. The amount of retinal-binding protein extracted was approximately 1 nmol/retina or 1 mol of binding protein/20 mol of opsin. Its specificity and relative abundance suggest that the 11-cis-retinal-binding protein may play a major role in the visual cycle.


The sugar chains of cold-insoluble globulin. A protein related to fibronectin. Cold-insoluble globulin isolated from bovine plasma contains six asparagine-linked sugar chains in 1 molecule (a dimeric form). These sugar chains were released from the polypeptide backbone by hydrazinolysis and labeled by reduction with NaB[3H]4. Most of these sugar chains contain N-acetylneuraminic acid and can be separated by paper electrophoresis. By combination of sequential exoglycosidase digestion and methylation study, their structures were elucidated as Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, NeuAc alpha 2 leads to 6 or 4Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 4 or 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]-Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and NeuAc alpha 2 leads to 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc.


The biosynthesis of cyanogenic glucosides in higher plants. N-Hydroxytyrosine as an intermediate in the biosynthesis of dhurrin by Sorghum bicolor (Linn) Moench. The following compounds were tested as early intermediates in the conversion of tyrosine to p-hydroxymandelonitrile by a microsomal preparation from dark grown sorghum seedlings: p-hydroxyphenylacetamide, 1-nitro-2-p-hydroxyphenylethane, p-hydroxyphenyl-pyruvic acid oxime, tyramine, N-hydroxytyramine, and N-hydroxytyrosine. Of these, only N-hydroxytyrosine was metabolized to p-hydroxymandelonitrile. N-Hydroxytyrosine was produced from L-[U-14C]tyrosine in tracer experiments when unlabeled N-hydroxytyrosine was added as a trap. These data indicate N-hydroxytyrosine as the first intermediate in the biosynthesis of dhurrin, the cyanogenic glucoside of sorghum, and represent the first demonstration of the formation of an alpha-N-hydroxy-amino acid in a biological system. The enzyme system involved in this reaction was partially characterized with respect to substrate specificity and the effect of various inhibitors. The enzyme was shown to have properties different than those reported for the mammalian enzyme system(s) involved in the N-hydroxylation of amine drugs. The possible involvement of N-hydroxyamino acids in the biosynthesis of other secondary plant products is discussed.


Subcellular site and mechanism of synthesis of disaturated phosphatidylcholine in alveolar type II cell adenomas. The site of synthesis of 1,2-disaturated-(diacyl)-sn-glycero-3-phosphocholine (Sat2PC) in mouse alveolar type II cell adenomas has been studied by conducting pulse-chase experiments. Isolation of microsomal and lamellar body fractions from adenomas after a 20-min pulse with [methyl-3H]choline demonstrates that Sat2PC first appears in the microsomal fraction, and after a short lag subsequently appears in the lamellar body fraction. The kinetics of labeling of Sat2PC are consistent with the microsomal membranes functioning as the subcellular site of synthesis for this pulmonary surfactant phospholipid. Short term labeling experiments with [9,10-3H]palmitate demonstrate that this fatty acid is incorporated into the sn-2 position of Sat2PC at a faster rate than its incorporation into the sn-1 position. This finding indicates that the synthesis of Sat2PC occurs by a deacylation-reacylation mechanism.


Purification of a high molecular weight human terminal deoxynucleotidyl transferase. Terminal deoxynucleotidyltransferase has been purified from lymphoblasts of leukemic patients. The enzyme has a molecular weight of approximately 62,000 as determined by gel filtration and nondenaturing gel electrophoresis and is not dissociated into subunits by sodium dodecyl sulfate. In contrast, the terminal transferase enzyme from calf thymus has a molecular weight of 42,000 as determined by gel filtration, and is dissociated into 2 subunits of Mr 30,000 and 8,000 by sodium dodecyl sulfate. The enzyme has an isoelectric point of 8.2 and kinetic characteristics which are similar to those of calf thymus terminal transferase. The apparent Km for purine nucleotide polymerization at saturating initiator concentration with Mg2+ is 0.2 mM and with Mn2+ is 0.05 mM. Like calf terminal transferase, the reaction velocity is higher in the presence of Mg2+ than Mn2+. ATP inhibits the reaction catalyzed by terminal transferase isolated from human lymphoblasts due to mutual recognition of ATP and dATP by a common site on the enzyme. Preliminary experiments indicate that human terminal transferase may contain a small amount of carbohydrate. This report represents the first purification to near homogeneity of terminal transferase from a tissue source other than calf thymus.


Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid. The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column.


Abdominal biopsy with ultrasound guidance. The value of aspiration biopsy under ultrasound guidance has been demonstrated in 51 patients. The confirmation of malignancy, particularly, has allowed many patients the value of chemotherapy or a quiet demise, without the necessity for diagnostic laparotomy.


Percutaneous puncture of abdominal cystic masses in children. A technique of percutaneous puncture and opacification of cystic abdominal masses is outlined, and its diagnostic and therapeutic potential demonstrated in a series of 16 masses in 15 children. It is suggested as an alternative to ultrasound and computed tomography in certain situations.


Percutaneous pulmonary aspiration biopsy using the Chiba needle. The Chiba needle was used for percutaneous needle aspiration of lung lesions in 35 patients. Two separate needles were used for each patient at the same sitting. The aspirate was true positive for malignancy in 23 patients and true negative for malignancy in eight patients. Two aspirates were false negative for malignancy and two aspirates, negative for malignancy, were classified as inconclusive due to inadequate patient follow-up. The overall accuracy rate was 94%. Six patients had small pneumothoraces but only one required a chest tube. Appreciable hemoptysis did not occur. The Chiba needle provided an adequate aspirate for cytologic diagnosis. The complication rate using two needles appears to be entirely acceptable.


[Transhepatic percutaneous cholangiography: A comparative study]. A retrospective study of 75 cases (19 with the conventional method and 56 with the Chiba method) of percutaneous transhepatic cholangiography was done. The utilization of the Chiba method is associated with a higher success rate of opacification of the biliary tree: 97% versus 80% for dilated biliary ducts and 36% versus 0% for non-dilated biliary ducts. No serious complications were encountered with the Chiba method. With the utilization of the "skinny" Chiba needle, percutaneous transhepatic cholangiography has become a simple, safe, and efficient method in the investigation of hepatobiliary disorders.


Hydatidiform mole with co-existent fetus. Co-existence of hydatidiform mole and fetus is a rare condition, but one in which the radiologist, through ultrasound, can play an important role in diagnosis. The typical patient, with no pre-existing hypertension or renal disease, develops severe pre-eclampsia in the second trimester with elevated gonadotropin levels. Ultrasound shows a large placenta containing varying numbers of sonolucent spaces, associated with a live or dead fetus. This condition appears to be a separate entity from classical hydatidiform mole both pathologically and prognostically. It should also be differentiated from simple hydatidiform swelling which does not have the associated clinical picture.


Misleading skeletal surveys of prostatic carcinoma. Bone sclerosis can be a manifestation of regressing metastatic bone disease of carcinoma of the prostate and should not be misconstrued as further dissemination. In eight of our patients the skeletal survey radiographs were misleading, whereas the bone scan provided an accurate assessment of metastatic activity.


Pulmonary embolism with unilateral lung scan defects and matching infiltrates. Ten patients with matching ventilation-perfusion lung scan defects and corresponding pulmonary infiltrates were evaluated with segmental pulmonary angiography. All ten patients presented with sudden onset of pleuritic chest pain and fever. Pulmonary emboli were documented in three of the ten patients (30%). The remaining seven patients had pneumonia or atelectasis. The findings emphasize the non-diagnostic nature of lung scans which show only matching ventilation and perfusion defects in regions of pulmonary infiltrates. Segmental pulmonary angiography is recommended for differentiating pulmonary embolism from atelectasis or pneumonia in these patients.


Gallium-67 citrate in detecting recurrent rectal carcinoma after abdomino-perineal resection. In a retrospective study we have found gallium-67 citrate imaging of the abdomen to be a value in detecting pelvic recurrence of carcinoma of the rectum after abdomino-perineal excision. In this context the technique proved to have an accuracy of 83%, sensitivity of 82% and specificity of 83%.


Astrocytoma in children. A review of 79 cases--1960-1976. Seventy-nine children with histologically proven astrocytomas, 32 cerebral, 30 cerebellar, 9 thalamic, 6 optic nerve, and 2 in the 4th ventricular floor were reviewed retrospectively. Seventy-one per cent of the astrocytomas were well differentiated. Unlike adult astrocytomas, there was slight female predominance in our group. Cerebral astrocytomas were more frequent beyond nine years of age. The overall five-year survival rate of cerebral astrocytomas was 95% (19/20) for well-differentiated, and 19% (2/12) for poorly-differentiated tumors. In cerebellar astrocytomas the survival rates were 75% (18/24) and 33% (2/6) respectively.


Pitfalls to avoid: spasm in ulcerative colitis masquerading as carcinoma. A barium enema on a patient with chronic ulcerative colitis demonstrated a stricture with the radiographic characteristics of an annular carcinoma. The "lesion" disappeared following administration of glucagon.


Technical note: oral water and intravenous glucagon--to aid ultrasonic visualization of the common bile duct. Gas in the stomach, duodenum and hepatic flexure frequently prevents ultrasonic visualization of the distal common bile duct. By filling the stomach and duodenum with water, enhanced by temporary paralysis with glucagon, the gas is displaced and improved visualization of the distal common bile duct is obtained.


Congenital lipoid nephrosis with left renal vein thrombosis and Chiari's syndrome. An infant with primary congenital lipoid nephrosis then developed left renal vein thrombosis and secondary hepatic vein obstruction. This was shown by inferior venacavography. The thrombus detached subsequently, and the child died from massive pulmonary embolism.


Arteriographic demonstration of extension of renal carcinoma into renal vein and vena cava. On rare occasions, arteriography of a renal cell carcinoma shows tumor vessels following the course of the renal vein and vena cava. In our cases, surgical exploration and microscopic examination showed that this appearance  was due to tumor vessels arising from within the renal tumor and extending intraluminally within the venous system to supply the tumor.


Legionnaires' disease in Vancouver.

Legionnaires' disease in Vancouver. A female patient presented at the end of a holiday cruise with the pneumonitis of Legionnaires' disease. The radiographic appearance was indistinguishable from any other cause of air-space consolidation.


Ultrasound findings in small bowel infarction. A patient with small bowel infarction was studied using grey-scale ultrasound. The appearance was that of a lobulated abdominal mass consisting of smaller masses with a central sonolucent area and a peripheral echogenic rind. This is the reverse of what has been described in intramural intestinal hematoma, intussusception, and inflammatory and neoplastic bowel masses.


Abdominal hematomas in unusual locations--contribution of ultrasound. The ultrasonic findings in two patients with abdominal hematomas in unusual locations, gallbladder and mesocolon, are described. When integrated with radiologic imaging methods, ultrasound played a vital role.


Microsomal mediated metabolism of dialkylaryltriazenes. I. Demethylation of ring halogenated 3,3-dimethyl-1-phenyltriazenes. The oxidative N-demethylation was investigated for a series of 3,3-dimethyl-l-phenyl-triazenes. Triazenes, deactivated with halogene atoms in the phenylring, were expected to be better demethylated. The results do indicate a good trend that substitution of the ring with deactivating atoms and extent of demethylation compare well. The percentages of demethylation were: For 3,3-dimethyl-l-phenyltriazene, 45%; for 3,3-dimethyl-l (4-chlor-phenyl)-triazene, 92%; for 3,3-dimethyl-l(4-bromophenyl)triazene, 89%; for 3,3-dimethyl-l-(2,4,6-trichlorophenyl)triazene, 122%; and for 3,3-dimethyl-l-l-(2,4,6-tribromophenyl)triazene, 85%.


Microsomal mediated metabolism of dialkylaryltriazenes. II. Isolation and identification of metabolites of 3,3-dimethyl-1-phenyltriazene. After incubating 3,3-dimethyl-1-phenyltriazene with rat liver microsomes, acetanilid and derivatives of aniline and possibly of 3-methyl-1-phenyltriazene were found as metabolites and identified by mass spectrometry. This is the first time that directly formed metabolites (other than formaldehyde) of a dialkyltriazene were identified. The isolation of a derivative of 3-methyl-1-phenyltriazene as 3-acetyl-3-methyl-1-phenyltriazene supports other evidence that the triazenes are enzymically demethylated. Indication for the formation of phenylhydrazine was also obtained. In addition, hydrolysates of the polar fractions of the incubation mixture contained aniline and 4-hydroxy-aniline as a aglycones.


[Fluorometric determination of "activated" cyclophosphamide and ifosfamide in blood (author's transl)]. "Activated" N-(2-Chloroethyl)amido-oxazaphosphorines like 4-hydroxycyclophosphamide, 4-hydroperoxycyclophosphamide, 4-hydroxyifosfamide, and 4-hydroperoxyifosfamide can be determined fluorometrically by condensation of liberated acrolein with m-aminophenol yielding 7-hydroxychinolin. The method permits determination of 10(-10) mol and is specific for "activated" N-(2-Chloroethyl)amido-oxazaphosphorine metabolites which liberate acrolein under conditions of the test. Neither cyclophosphamide nor ifosfamide or other metabolites of this cytostatics interfere with the test. Blood levels of free 4-hydroxycyclophosphamide and 4-hydroxyifosfamide were determined after injection of cyclophosphamide and ifosfamide into mice.


Malignant progression of angioimmunoblastic lymphadenopathy. In a 79-year-old woman, the progression of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) to malignant lymphoma was observed within one year after diagnosis. Three biopsy specimens from lymph nodes and one tonsil, obtained at intervals of several months, showed an increasing destruction of the tissue architecture and the development of histological criteria for a lymphoid neoplasm, which at autopsy was confirmed as a malignant non-Hodgkin's lymphoma. The demonstration of a chromosomally abnormal clone in lymph node derived and the laboratory findings were in agreement with the histological changes and the sequential clinical deterioation. Initially, a symptom-free interval of eight months was achieved with prednisone therapy. However, this treatment failed after the malignant transformation had become evident.


Morphometric and cytophotometric investigations of lobular neoplasia of the breast with ductal involvement. The two different types, A and B, of in situ lobular neoplasia of the breast can be distinguished by cytomorphological means. DNA-measurements of type A are preponderantly diploid while those of type B vary from diploidy to hyperteraploidy with a maximum in the hyperdiploid range. The results indicate a higher potential of malignancy in type B. Homogenous ductular cell proliferations in the immediate neighbourhood of a lobular neoplasia display virtually the same distribution of DNA-values. Such evidence suggests that ductular changes and concomitant intralobular lesions have a common genesis, showing also a similar tendency towards malignant changes.


Tumor host relations. I. Increased plasma cortisol in tumor-bearing humans compared with patients with benign surgical diseases. Plasma cortisol was measured by a protein binding technique in 81 patients with malignant tumors of different extent and various sites and in 82 patients with benign surgical diseases. The mean value of the tumor patients (x +/- s = 165 +/- 69 micrograms cortisol/l plasma) was increased significantly compared with the benign surgical disorders (100 +/- 45 micrograms/l). Within the group of patients with benign surgical disorders there was little variation by the type of disease (cortisol mean values given in brackets): benign breast tumors (95), gall stones (107), ulcer of the stomach and duodenum (96), hernia (78), appendicitis acuta (112), and struma (90). The results are in accordance with the hypothesis that glucocorticoids are involved in the increased protein catabolism of skeletal muscles and other signs of cachectic tumor patients.


Tumor host relations. II. Influence of tumor extent and tumor site on plasma cortisol of patients with malignant diseases. The increase of plasma cortisol in patients with tumors of five different sites compared with a control group of patients with benign surgical diseases amounted to: +39% (breast), +34% (stomach), +86% (intestine), +60% (skin) and +194% (gall bladder). The first detectable increase of cortisol occurred in patients with tumors classified T 2 according to the TNM scheme (+27% above the control). Highly significant increases were observed for T 3 (+82%) and T 4 (+77%) patients. Patients with palpable lymph nodes showed a most significantly increased cortisol mean value compared with patients without palpable lymph nodes. Similarly, the cortisol mean value of patients with distant metastases was significantly higher than the corresponding value of tumor patients without distant metastases. The question remains open, whether the primary site, the extent of the tumor or the occurrence of metastases is the main determinant for the cortisol increase.


The incidence of gastro-intestinal cancer in North Baden (West Germany) 1971--1977. The incidences of the cancer of the oesophagus, stomach, small intestines, colon, and rectum for the years 1971--1977 in the regional district of North Baden, Federal Republic of Germany are presented and their relation to environmental factors are discussed briefly. The age specific incidence as well as the age standardized incidence and the sex ratios show remarkable differences due to the specific tumor localizations. The colon cancer incidence in the female population increases steeply during the period considered. The risk of developing rectal cancer also increases. The results of the pathoanatomic registry are well comparable with the data from clinical registries.


Promoting action of croton oil on gastrocarcinogenesis by N-methyl-N'-nitro-N-nitrosoguanidine in rats. The promoting effect of croton oil on gastrocarcinogenesis by MNNG was examined in male Wistar rats. Gastric carcinomas were found in five of 10 rats given 83 micrograms/ml MNNG for three months and then 0.02% croton oil with 0.5% Tween 60 as solvent for nine months. No gastric carcinomas were found in rats given MNNG for three months and then Tween 60 only for nine months. The incidence of gastric carcinomas in these two groups was significantly different (p less than 0.05). No tumors were found in rats given only croton oil with Tween 60.


Comparative studies of neoplastic response to a single dose of nitroso compounds. 6. The effect of diethylnitrosamine in Syrian golden hamsters. The effect of a single subcutaneous injection of different DEN doses was examined in adult Syrian hamsters observed for life. The minimal effective dose (threshold dose) for neoplastic response, reflected by induction of papillary polyps in the larynx and/or trachea, was 1.03 mg DEN/kg body weight. No tumors were found which could be related to treatment in other segments of the respiratory epithelium nor in other tissues.


Implantation of cigarette smoke condensate in the lungs of Syrian golden hamsters. Different doses of benzo(a)pyrene, cigarette smoke condensate (CSC) and its nitromethane fraction (NMF), in wax pellets, were directly implanted into the lungs of Syrian golden hamsters. Pleomorphic sarcomas were induced in the B(a)P treated animals. Several epithelial proliferative lesions and a few bronchiogenic adenomas were induced in CSC and NMF treated hamsters. A species-specific effect has been observed in this investigation.


Effect of liposome-entrapped methotrexate on Ehrlich ascites tumor cells and uptake in primary liver cell tumor. A therapeutically useful concentration (0,5 mg/ml) of Methotrexate was prepared in negatively charged artificial liposomes (lecithine: cholesterol: dicetylphosphate=5:5:1 molar ratios). Entrapment yield after separation on Sepharose 6B is nearly 100%. In contrast to free Methotrexate the liposome entrapped folic acid antagonist is eliminated only slowly by the kidneys and after intravenous application it is unevenly distributed in the body of mice, the highest concentrations being found in liver and spleen. Daily injections (7.5 mg/kg/day) of entrapped Methotrexate for 5 days into the tail vein of Ehrlich ascites tumor bearing mice reduced both tumor cell count and the production of ascites fluid about fourfold as compared to mice receiving the same dose of Methotrexate in the free form. Six hours after intravenous application of liposome entrapped Methotrexate the tissue concentration in normal liver is ca. 20-fold higher than when the same amount is applied in the free state. On the other hand, only a little uptake of liposome entrapped Methotrexate was detected in the tissue of nitrosamine-induced primary liver tumor when compared to normal liver tissue of rats.


Age-dependent change of the effect of a cytostatic drug on the proliferation kinetics of a solid tumor of the mouse. The aim of the present investigations was to study the effectiveness of a cytostatic drug (VCR) during different phases of tumor growth (1st, 7th, 14th days a.t.) and at the periphery and at the centre of the tumor (on the 14th day a.t.). Furthermore the inducement of a partial synchronization in the proliferation of tumor cells was attempted. It was found that the intensity of the cytostatic effect significantly decreased both with the passage of time after transplantation and within the tumor from the periphery to the centre. The changing cytostatic sensitivity probably is due to the diminishing vascular supply and the decreased rate of cell proliferation; especially the decline of the growth fraction. A partial synchronization of the proliferation of tumor cells could not be demonstrated.


Brain tumor chemotherapy using a rat glioma model. The antitumor agent chloroethylcyclohexyl-nitrosourea (CCNU) was examined using a rat glioma model. A i.p. administration of three times 40 mg/kg CCNU was highly effective and increased the life-span of tumor-bearing rats from 53 to 86%. A total quantity of CCNU amounting to less than 30 mg/kg was not effective. On the other hand, large dosages exceeding LD10 proved to be toxic. The depression of platelets and white blood cells was mild after a single dosage of 40 mg/kg CCNU which recovered on about the 6th day. In the CCNU-treated animals there was an increase of the extent of necrobiosis. Ballooning of tumor cells with nuclear pyknoses and a lack of mitotic features occurred. Microcystic changes appeared sometimes to be more frequent than in control groups.


Influences of inducers and inhibitors of the microsomal monooxygenase system on the alkylating intensity of dimethylnitrosamine in mice. Male mice were pretreated with various drugs, which are known either induce or to inhibit cytochrome P 450 function. Five hours after the administration of 14C-dimethylnitrosamine (10 mg/kg) in pretreated and control mice, total and specific radioactivities of cellular macromolecules of the liver were determined. Various types of nucleic acids were extracted and hydrolyzed, and the bases and nucleotides separated by ion-exchange-chromatography. Radioactivity resulting from incorporation and alkylation was measured. Pretreatment of mice with the inducers phenobarbital and 3-Methylcholanthrene decreased the alkylation rates, while administration of the inhibitors SKF 525 A and CFT 1201 caused an increase. These results appear to contradict the accepted activation mechanism of nitrosamines if DMN demethylation as catalyzed by cyt. P 450 is influenced in the same way as other cyt. P 450 dependent reactions.


Antitumor activity of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-(4-methyl-cyclohexyl)-1-nitrosourea (MeCCNU) on Yoshida sarcoma ascites cells implanted into the colon wall of rats. Three thousand Yoshida sarcoma cells were inoculated into the wall of the descending colon of each of 120 male Sprague-Dawley rats. On day 8 after the tumor implantation, the animals were at random divided into four groups of 30 rats each. The effect of cyclophosphamide (70 mg/kg), BCNU (25 mg/kg), and methyl-CCNU (45 mg/kg) after single i.p. application was investigated. The Yoshida sarcoma transplanted into the colon is sensitive to all three chemotherapeutic drugs. At the doses given cyclophosphamide showed the best results. The two nitrosoureas had a comparable antitumor activity but methyl-CCNU showed a more distinct toxic effect. The introduction of this model for testing new cytostatics in animal experiments is discussed.


DNA damage and repair induced by diazoacetyl derivatives of amino acids with different mechanism of cytotoxicity. Correlations with mutagenicity and carcinogenicity. Eight synthetic N-diazoacetyl amino acids, prepared by inserting a diazoacetyl group onto the alpha-nitrogen of a natural amino acid, and two natural diazoazetyl amino acids, azaserine (9-diazoacetyl-L-serine) and DON (6-diazo-5-oxo-L-norleucine), have been studied by autoradiography for their capacity to induce DNA repair synthesis in mouse cells cultivated "in vitro". Dose-dependent unscheduled DNA synthesis was present in cells treated with the eight N-diazoacetyl derivatives, and was absent in cells exposed to approximately equitoxic concentrations of azaserine and DON. Azaserine and DON, unlike N-diazoacetyl derivatives, did not alkylate gamma-(4-nitrobenzyl) pyridine at an appreciable extent. When DNA damage (single stranded breaks or weak points in alkali) was measured by the sensitive technique of alkaline elution, DGA was found about 4 times as potent as azaserine and about 12 times as DON on a molar basis, but about 800 and 17,000 times as potent as azaserine and DON respectively by extrapolating to equitoxic concentrations. Carcinogenicity and mutagenicity seem to follow mainly the capability of inducing DNA damage.


Use of radioactive gold in the treatment of pleural effusions caused by metastatic cancer. This paper presents a series of 12 patients suffering from advanced gynecological cancer with the occurrence of pleural effusions. All patients received at least one intrapleural dose of radioactive gold (100 m Ci Au198) when the necessity for thoracentesis became more than once every three weeks. Three showed no recurrence of fluid, five a significant slowing down of effusion, and four did not show any response. Severe complications were minimal, and were comparable to those resulting from thoracentesis in general.


Methylcholanthrene-induced tumors in resistant and sensitive Jensen sarcoma rats. i. effect of fresh prepared MC emulsion. By two subcutaneous or intramuscular injections of 20-methylcholanthrene (MC), 5 mg/animal in vegetal oil, followed after 74 days by 10 mg of MC emulsion, sarcomata were obtained in 63.6% of Jensen sarcoma-resistant rats (R) and in 36.8% of Jensen sarcoma-sensitive rats (S). One of the induces sarcomas, transplanted from R to R and then to S rats, presented a very high antigenicity: it strongly immunised receptive rats against the induced sarcoma and also, in a lower degree, against the Jensen sarcoma. Antigenicity was maintained up to the tenth passage.


Carcinogenicity of chlorinated nitrosotrialkylureas in rats. Four chlorinated derivatives of nitrosotrialkylureas were tested for carcinogenicity by oral administration by gavage to Sprague-Dawley rats. All of the compounds were very toxic, and all were carcinogenic, inducing tumors of the non-glandular stomach and of the lungs. The commonest tumors were carcinomas and papillomas of the non-glandular stomach and alveolar cell adenomas of the lungs. Nitrosochloroethyldimethylurea induced most tumors, but it was possible to administer higher doses of this compound than of the others, which were nitrosochloroethydiethylurea, nitrosomethylbis-(chloroethyl)-urea and nitrosotris-(chloroethyl)-urea. The last of these was a particularly toxic compound so that the maximum total dose given was only 0.07 millimole per rat.


Ascites tumors in CBA mice. Characterization of two new tumors, a carcinoma and a sarcoma in solid and ascites form, with regard to cell surface properties and transplantability. Two ascites tumors in syngeneic CBA mice are described, viz., MCB 21-AA and MCB 31-AA, with their solid progenitors: A sarcoma (MCB 21-SS) and a squamous cell carcinoma (MCB 31-SC), induced by gastric feeding of 20-methylcholanthrene. The ascites tumor cells have certain characteristics in common, which they do not share with either cells from the solid tumors or even with cells from solid ascites tumors (-21-AS and -31-AS=ascites tumor transplanted s.c.). Presumably some of these differences, for instance, in PAS stainability, electrophoretic mobility and lectin agglutinability, are due to enzyme treatment required to bring solid tumors into suspension. Between the two ascites tumors there are certain differences in cell size, aggregability, and growth rate. They are similar, however, in requiring large cell doses for transplantation in syngeneic animals, which is also true for the solid  (SS and SC) tumors. MCB 21 and -AA even required fewer cells for transplantation in allogeneic A mice than in syngeneic CBA mice. MCB 31-AA is also allotransplantable. The pattern of spread, after i.v. cell injection, is almost exclusively to the lungs for all tumor lines.


Intestinal metaplasia and adenocarcinoma induced in the stomach of rats by N-propyl-N'-nitro-N-nitrosoguanidine. PNNG, the propyl derivative of MNNG, was administered to Wistar rats by a concentration of 59.5 micrograms/ml in the drinking water for 4, 8, and 12 months and the rats were killed in the 15th month. Intestinal metaplasia was induced in the glandular stomachs of 25%, 75%, and 83% of the rats treated with PNNG for 4, 8, and 12 months, respectively. Metaplastic glands were found in the pyloric region, especially near the pyloric ring. These glands contained goblet cells and columnar cells with striated borders. No tumors were found in the stomach of rats after 4-months treatment, but adenomas were found after 8-months treatment, and both adenomas and adenocarcinomas after 12-months treatment.


Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm. Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.


Cytochalasin B inhibition of endothelial proliferation at wound edges in vitro. Cytochalasin B prevents both migration and wound-associated replication when applied to wounded monolayers of bovine endothelium in tissue culture. The normal low background rate of replication in undisturbed areas of the cultures is not inhibited by cytochalasin B. These results suggest that some form of movement may be required for initiation of wound-associated replication by endothelial cells.


Sodium uptake and membrane excitation in Paramecium. Although the phenotypes of many membrane-excitation mutants of Paramecium are best expressed in Na+-containing solutions, little is known about the role of Na+ in membrane excitation in Paramecium. By measuring 22Na fluxes, we have shown that: (a) The total cellular Na+ content is equivalent to a cytoplasmic concentration of 3--4 mM, if the Na+ concentration is uniform throughout the cell. (b) The kinetics of Na+ uptake can be divided into a saturable Na+ uptake with an apparent Km = 0.15 mM and a nonsaturable Na+ uptake seen at higher Na+ concentrations up to 20 mM. (c) The rate of Na+ uptake in high Na+ solutions is correlated with the duration of backward swimming and membrane excitation in wild type Paramecium and the mutants fast-2 and paranoiac. (d) Na+ uptake is inhibited at 4 degrees C. From these results, we postulate that Na+ uptake is faster when the membrane is depolarized than when it is at the resting potential level.


Spermatogenesis in the mouse. I. Autoradiographic studies of nuclear incorporation and loss of 3H-amino acids. Autoradiographic and electron microscope methods were used to correlate changes in nucleoproteins with nuclear fine structure during spermatogenesis in the mouse. Testes were fixed at daily intervals after intratesticular injectionwith labeled amino acid. [3H]Arginine, lysine, valine, and proline were rapidly incorporated into primary spermatocyte nuclei, retained through subsequent spermatocyte divisions and through spermatid differentiation to step 12 of spermiogenesis, but were lost with spermatid differentiation beyond step 12. Arginine and lysine (not valine or proline) also were rapidly incorporated into certain elongated spermatid nuclei but differed strikingly in their distribution and fate. Nuclei of late step-12 through step-15 spermatids were initially labeled with arginine. This label was retained through subsequent spermatid differentiation and sperm maturation in the epididymis. By contrast, lysine was initially incorporated only into late step-12 and step-13 spermatid nuclei, and was retained only to early step 14 of spermiogenesis. Spermatid incorporation of lysine coincided with the initiation of chromatin condensation in late step-12 nuclei, and loss of lysine coincided with the completion of condensation in step-14 nuclei.


Membrane events involved in myoblast fusion. Myoblast fusion has been studied in cultures of chick embryonic muscle utilizing ultrastructural techniques. The multinucleated muscle cells (myotubes) are generated by the fusion of two plasma membranes from adjacent cells, apparently by forming a single bilayer that is particle-free in freeze-fracture replicas. This single bilayer subsequently collapses, and cytoplasmic continuity is established between the cells. The fusion between the two plasma membranes appears to take place primarily within particle-free domains (probably phospholipid enriched), and cytoplasmic unilamellar, particle-free vesicles are occasionally associated with these regions. These vesicles structurally resemble phospholipid vesicles (liposomes). They are present in normal myoblasts, but they are absent in certain fusion-arrested myoblast popluations, such as those treated with either 5-bromo-deoxyuridine (BUdR), cycloheximide (CHX), or pospholipase C (PLC). The unilamellar, particle-free vesicles are present in close proximity to the plasma membranes, and physical contact is observed frequently between the vesicle membrane and the plasma membrane. The regions of vesicle membrane-plasma membrane interaction are characteristically free of intramembrane particles. A model for myoblast fusion is presented that is based onan interpretation of these observations. This model suggests that the cytoplasmic vesicles initiate the generation of particle-depleted membrane domains, both being essential components in the fusion process.


Effects of colchicine on endocytosis and cellular inactivation of horseradish peroxidase in cultured chondrocytes. Horseradish peroxidase (HRP) was used as a marker to study the effects of microtubule-disruptive drugs on uptake and cellular inactivation of exogenous material in cultures of embryonic chick chondrocytes. HRP was ingested by fluid endocytosis, and intracellular enzyme activity subsequently diminished exponentially with time. Cytochemically, reaction product for HRP was found in vesicles often located close to the dictyosomes of the Golgi complex. Colchicine and vinblastine caused disappearance of cytoplasmic microtubules and disorganization of the Golgi complex with concomitant reduction in the cellular uptake of HRP to about half of that in the controls. Lumicolchicine, on the other hand, left cell fine structure and HRP uptake unaffected. These results indicate that microtubules are of considerable importance in the process of fluid endocytosis in cultured chondrocytes although the exact mechanism remains to be elucidated. The rate of intracellular inactivation of ingested HRP was not affected by colchicine or vinblastine. Double-labeling experiments with colloidal thorium dioxide and HRP likewise indicated that fusion of endocytic vesicles and lysosomes is not dependent on intact microtubules. The total specific activities of the three lysosomal enzymes examined were weakly or not at all changed by treatment of the cultures with colchicine or vinblastine. It therefore seems unlikely that microtubular organization plays an important role in the production or degradation of lysosomal enzymes in cultured chondrocytes.


Modulation of cell membrane area in renal collecting tubules by corticosteroid hormones. Isolated renal cortical collecting tubules obtained from rabbits treated chronically with desoxycorticosterone acetate (DOCA) have been found to possess elevated transepithelial potential differences and a greatly increased capacity for ion transport. Structural exmination of tubules from rabbits exposed to either DOCA or dexamethasone for 11--18 d reveals a marked increase in basolateral cell membrane area in these tubules. Morphometric analysis shows that this effect is specifically on the basolateral membrane area of only one of the two cell types found in this nephron segment. Increases of greater than 140% and 90% are found for the basolateral membrane area of the principal cells for DOCA and dexamethasone, respectively, but no change could be detected in the basolateral membrane area of the intercalated cells found in this nephron segment. No siginificant changes were found in luminal membrane area, cell number, or cell volume for either cell type. These observations demonstrate that significant changes in membrane area can occur in differentiated epithelia and suggest that this may be an important mechanism for modulating epithelial transport capacity.


Incorporation of membrane proteins into large single bilayer vesicles. Application to rhodopsin. A general procedure to incorporate membrane proteins in a native state into large single bilayer vesicles is described. The results obtained with rhodopsin from vertebrate and invertebrate retinas are presented. The technique involves: (a) the direct transfer of rhodopsin-lipid complexes from native membranes into ether or pentane, and (b) the sonication of the complex in apolar solvent with aqueous buffer followed by solvent evaporation under reduced pressure. The spectral properties of rhodopsin in the large vesicles are similar to those of rhodopsin in photoreceptors; furthermore, bleached bovine rhodopsin is chemically regenerable with 9-cis retinal. These results establish the presence of photochemically functional rhodopsin in the large vesicles. Freeze-fracture replicas of the vesicles reveal that both internal and external leaflets contain numerous particles approximately 80 A in diameter, indicating that rhodopsin is symmetrically distributed within the bilayer. More than 75% of the membrane area is incorporated into vesicles larger than 0.5 micron and approximately 40% into vesicles larger than 1 micron.


Nucleosome packing in interphase chromatin. Higher-order chromatin fibers (200--300 A in diameter) are reproducibly released from nuclei after lysis in the absence of formalin and/or detergent. Electron microscope analysis of these fibers shows that they are composed of a continuous array of closely apposed nucleosomes which display several distinct packing patterns. Analysis of the organization of nucleosomes within these arrays and their distribution along long stretches of chromatin suggest that the basic 100-A chromatin fiber is not packed into discrete superbeads and is not folded into a uniform solenoid within the native 250-A fiber. Furthermore, because similar higher-order fibers have been visualized in metaphase chromosomes, the existence of this fiber class appears to be independent of the degree of in vivo chromatin condensation.


Enhancement of chemotactic response and microtubule assembly in human leukocytes by ascorbic acid. The incubation of human leukocytes with ascorbic acid increased chemotaxis of the cells. In addition, ascorbic acid promoted the assembly of intracellular polymorphonuclear leukocyte (PMN) with colchicine blocked the effect of ascorbic acid on promoting microtubule assembly. Not only did ascorbic acid promote the assembly of microtubules in vivo, but it enhanced the assembly of bovine brain tubulin into microtubules in vitro as quantitated by a glass-fiber filtration assay and by promotion of viscosity changes. The enhancement in leukocyte mobility by ascorbate at concentrations achievable in normal tissues correlates with its ability to assemble microtubule organelles.


Transformed cells have lost control of ribosome number through their growth cycle. Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus-transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in a post-mitotic pre-DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability. The rate of protein synthesis in the stationary phase of the transformed cells fell to 25-30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribisome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L-73 and LR-73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells. A model for growth control of normal cells and its derangement in transformed cells is presented.


Enhanced sterol synthesis in concanavalin A-stimulated lymphocytes: correlation with phospholipid synthesis and DNA synthesis. Incorporation of (14C)choline and (3H)myo-inositol into the total lipid fraction, incorporation of (14C)acetate into the sterol fraction and incorporation of (3H)thymidine into DNA were studied in human lymphocyte cultures. Concanavalin A induced an increase in the incorporation of these labels with the following features: (a) Phospholipid synthesis was increased promptly. The lag time for the increase in sterol synthesis and DNA synthesis were 5 hours and 27 hours respectively; (b) The increase in phospholipid synthesis and sterol synthesis was proportional to ConA concentration initially. Cells treated with a high concentration of ConA showed very low levels of DNA synthesis; (c) The increase in phospholipid synthesis could be abolished immediately by alpha-Methyl-Mannoside. alpha-Methyl-Mannoside blunted but did not abolish the increase in sterol synthesis. alpha-Methyl-Mannoside enhanced DNA synthesis of those cells which had been treated by a high concentration of ConA; and (d) Selective inhibition of sterol synthesis with 25-hydroxycholesterol did not prevent the increase in phospholipid synthesis, but it blocked the increase in DNA synthesis. Supplement of LDL, HDL or total lipoproteins to lymphocyte cultures was effective in preventing the inhibition of DNA synthesis by 25-hydroxy-cholesterol. These results suggest that in lymphocyte activation by ConA phospholipid synthesis, sterol synthesis and DNA synthesis were sequentially increased. The rate of cellular commitment to mitogenesis was proportional to ConA concentrations. High concentrations of ConA arrested the cell growth at a postcommitment point in the G1 phase. Enhanced phospholipid synthesis was a precommitment event. Enhanced sterol synthesis was a postcommitment event and reflected the requirement of an increased cholesterol supply for the passage of cell growth through G1.


Stimulation of chondroitin sulfate synthesis by beta-D-xyloside in chondrocytes of the proteoglycan deficient mutant nanomelia. The potential of nanomelic chondrocytes to synthesize chondroitin sulfate was investigated by providing the mutant cells with p-nitrophenyl-beta-D-xyloside, a compound which acts as an artificial acceptor for glycosaminoglycan synthesis. Under these conditions the synthesis of chondroitin sulfate in nanomelic and normal chondrocytes is comparable. The chondroitin sulfate synthesized by the mutant is indistinguishable in molecular size and composition from that synthesized by similarly treated normal chondrocytes.


Characteristics of concanavalin A-resistant Chinese hamster ovary cells and certain revertants. Clones of Chinese hamster ovary (CHO) cells were isolated by single-step selection for resistance to killing Concanavalin A (ConA) and certain cellular and membrane properties were examined. The ConA-resistant isolates were only about 2-fold more resistant than wild type cells to the selecting lectin, but exhibited pleiotropic temperature-sensitivity for growth, markedly altered morphology and adherence, and significant difference in susceptibility to other agents such as colchicine. Two revertants to full temperature-resistance were isolated from different ConA-resistant mutants. One revertant clone had reacquired wild type sensitivity to ConA while the other revertant remained ConA-resistant. The two series of wild typed, ConA-resistant, and temperature revertant clones were analyzed for altered mobility of cell surface glycoproteins using lactoperoxidase/125I and galactose oxidase/(3H) borohydride labelling procedures. The ConA-resistant clones showed increased mobility on polyacrylamide gels of three classes of labelled proteins, in the molecular weight ranges 225,000, 200,000, and 130,000 daltons. These changes persisted in the temperature-revertant that remained ConA-resistant, while two of the altered protein closses were restored to wild type mobility in the revertant that regained ConA-sensitivity. Cell hybridization experiments indicated that the temperature-sensitivity phenotypes of different ConA-resistant isolates are recessive and noncomplementing, implying that the same gene is affected in each case. The reversions to temperature resistance appear to be recessive suppressor mutation in different genes.


Erythropoietin responses and physical characterization of erythroid progenitor cells in Rauscher virus infected BALB/c mice. Infection of BALB/c mice with Rauscher leukemia virus (RLV) gives rise to pronounced erythrocytopoiesis manifesting in splenomegaly and is associated with progressive development of anemia. In the spleen erythroid colony forming units (CFU-E) increase exponentially up to 800-fold that of normal levels by the third week of infection. In vitro these CFU-E are dependent on erythropoietin for colony formation, their erythropoietin requirements being higher than that of CFU-E from normal mice. Numbers of CFU-E in spleen and degree of splenomegaly in anemic RLV infected mice were also shown to be modified by red blood cell transfusion, but progression of the disease was not stopped. Erythroid burst forming units (BFU-E) were also responsive to erythropoietin. However, a small proportion of cells also formed BFU-E colonies at concentrations which did not support growth of normal marrow BFU-E. When compared to normal, CFU-E found in RLV-infected spleen have similar velocity sedimentation rates. However, buoyant density separation of leukemic spleen cells indicated that CFU-E were more homogeneous (modal density 1.0695 g/cm3) than CFU-E from normal spleen. Analysis of physical properties of CFU-E and the nonhemoglobinized erythroblast-like cells, which accumulate in the spleen showed that they differed mainly in their distribution of cell diameter. Our findings show that erythroid progenitor cells in RLV infected mice are responsive to erythropoietin in vitro. Also in vivo erythropoiesis appears to be under control of erythropoietin but other factors which lead to progression of RLV disease apparently exist. Most proerythroblast-like cells, which are characteristic of this disease, apparently lack the potential to form colonies and may be more mature than CFU-E.


Bromodeoxyuridine-induced reversion of transformed characteristics in BHK21 cells: changes at the plasma membrane level. When BHK21 cells transformed by hamster sarcoma virus are grown in the presence of 5-Bromedeoxyuridine (BUdr), several in vitro properties of the transformed cells such as morphology, adhesiveness, and alignment, revert towards a state close to that of untransformed cells. We have studied plasma membrane changes associated with this phenotypic reversion by several different biochemical methods. Reversion is accompanied by a reappearance of Fibronectin, an increase in a membrane-associated protein of M.W. 100,000 which is increased in transformed cells and a decrease in Con A-agglutinability. On the other hand, several membrane changes associated with malignant transformation namely, the increase in an integral membrane protein M.W. 177,000, the higher rate of hexose uptake, the increase in high molecular weight surface glycopeptides and, to some extent, the increase in the density of intramembranous particles, did not revert under BUdr treatment. Thus, membrane properties of transformed cells may be dissociated into two main groups by BUdr treatment. In addition, the exposure and glycosylation of a growth-regulated membrane protein M.W. 160,000 was highly sensitive to BUdr.


A problem-oriented analysis of database models. Which is the most convenient database model considering specific applications? The goal of this paper is to try to answer this question by the use of a chemical example. Examples of requests describe the problems of insertion, deletion, and updating; these requests are analyzed for the hierarchical model and are expressed in a relational language defined by the authors and in Socrate for the network model.


[Sarcoma of the body of the uterus. Report of 8 cases (author's transl)]. The authors report 8 cases of sarcoma of the uterus and discuss the special characteristics of this type of lesion. It seems that the frequency of sarcoma of the uterus is more importnat than is usually thought : one case out of 100 fibromas in the authors' series. The pre-operative diagnosis is difficult, in particular the radiological signs described by Musset were only found in 2 cases out of 8. On the other hand, the pre-operative diagnosis was made in 5 cases out of 8. The histological diagnosis is difficult and may only be made later. The only treatment is surgical, according to most authors. Chemotherapy may be of interest in the future. A uterine sarcoma should certainly be considered more often, especially in cases of fibroma which undergo rapid change.


[A case of papillary disinsertion during gastro-duo-denectomy for ulcer of the second part of the duodenum and its repair by a "physiological" reconstruction (author's transl)]. The authors report a personal case of repair of papillary disinsertion. They emphasise the risk of retrograde dissection of an old stenosing duodenal ulcer, the importance of peroperative recognition of this accident by cholangiography, the interest of sphincterotomy to attempt to prevent secondary stenosis of the papilla, the absolute necessity of an anastomosis preventing anastomotic ulcer by contact between the mouth of the gastro-enterostomy and the alkaline secretions. Vagotomy seems to them a useful complementary procedure but, associated with a drainage procedure, they believe it might have avoided this severe complication.


[A case of thymolipoma in a child (author's transl)]. A new case of asymptomatic thymolipoma in a 4.5 years old girl is reported. It was discovered during systematic medical investigation. Its radiological characteristics and the lack of evolutivity made the authors suspect the diagnosis before operating. A total surgical extraction of the 300 g weighing tumor was performed through a right thoracotomy. No complication occurred in the post-operative time. Others authors already indicated the scarcity of this affection (less than 10 cases in children) as well as the special radiological characteristics of this tumor and the noticeable clinical tolerance often despite a large bulk. Histologically the tumor is made of an infiltrating of the normal thymic tissue by proliferating adipose adulte cells, the benignity of which is demonstrated by the evolution. In this study a differential diagnosis between thymolipoma and other mediastinal tumors is made and also some pathogenic assessments. Surgery always is required for treatment.


[Use of electrolyte solutions in surgery (author's transl)]. The use of isotonic saline and Ringer's lactate in surgery has become widespread following the work of Shires on the sequestration of extra-cellular fluid in hemorrhagic shock. Their use both in military and civilian practice and during hemodilutions in cardiac surgery have shown their efficacy. During intensive care, the risk of pulmonary edema is insignificant when less than 4 liters are given daily. Apart from cases of severe renal failure, they increase diuresis and reduce post-operative renal failure. Their use in appropriate cases permits economies in blood transfusion.


[Surgery of acute aortic dissection by use of a biological glue. Experimental study (author's transl)]. The improvement in surgical technic in the treatment of dissecting aneurysm of the aorta should permit a reduction in mortality and secondary complications. With this object, an attempt to stick together the two parts of the aneurysm with biological glue, was submitted to an experimental study. The operative protocol included on the one hand, creation of a dissecting aneurysm in the thorax of the dog, and secondly, repair of the latter with biological glue of variable composition. 27 dogs were thus operated on : -- 4 dissections were treated by simple suture (control group); -- in 23 cases, the two parts of the dissecting aneurysm were stuck together with gelatin-resorcin glue which was polymerised either by formaldehyde alone, or by a mixture of glutaraldehyde-glycerinaldehyde or by a mixture formaldehyde-glutaraldehyde. The best results of adhesiveness and tissue tolerance were obtained with the latter mixture.


[G2 technic : a simplified procedure of reconstitution of the angle of His, applied to the treatment of hiatus hernia and malposition of the greater tuberosity (242 cases) (author's transl)]. A simple procedure of reconstitution of the angle of His applied to the treatment of hiatus hernia and malposition of the greater tuberosity is reported. Applied to 242 cases, it presents compared with the classical procedure several advantages : easy to carry out, including no complex manoevre, it does not endanger the vagus nerve and maintains firmly the anastomosis. Radiological control and, above all, the clinical results were excellent or good in 96% of cases, suggesting that this new procedure is perfectly valid.


[Surgery of urethral stenosis, 162 cases (author's transl)]. The authors report a series of 194 operations for urethral stenosis, carried out in 162 patients between 1971 and 1977, and give the indications for various operative procedures. In the absence of chronic prostatitis and changes in the corpora spongiosa, limited stenosis should now be treated by endoscopic urethrotomy. Extensive stenosis or penoscrotal stenosis should be treated by vaginal urethroplasty, the other cases by urethroplasty in two stages. The quality of the results which may and ought to be satisfactory in 8 cases out of 10 depends on the judicious choice of the operative procedure, care in execution and the quality of the postoperative supervision.


[Acute pancreatitis and post-traumatic pseudocysts of the pancreas. Report of 12 cases (author's transl)]. The authors report 12 personal cases and a bibliographic study showing the authenticity of acute pancreatitis and pancreatic pseudo-cyst following trauma. The clinico-pathological aspects, the course and the treatment are not specific. The medico-legal aspect is the most important for the secondary or late manifestations raise the problem of the imputability to the accident. Surgeons and judicial experts should be aware of the post-traumatic risk.


A novel procedure for the determination of tricyclic antidepressants in plasma by use of high performance liquid chromatography. A novel method for the determination of the antidepressant 3-(1-chloro-5-H-dibenzo [a,d] cycloheptene-5-ylidene)-N,N-dimethylpropylamine-N-oxide hydrochloride and its metabolites by use of high performance liquid chromatography was developed. The procedure is applicable to the assay of other similar drugs in biological samples. The method involves extraction of the unchanged drug and its metabolites from plasma, back-extraction into diluted phosphoric acid and re-extraction into an organic phase. Separation is performed on a silica gel column with an acidic mobile phase, containing sodium dodecyl sulfate as ion-pairing agent. The quantitation is carried out by UV detection. The procedure allows the determination of plasma levels down to about 5 ng/ml of the unchanged drug and its metabolites, respectively, when 1 ml of plasma is used. The plasma levels of two volunteers were determined after a single oral dose of the drug.


Short term growth in boys with delayed puberty after diagnostic human chorionic gonadotropin administration. Using a sensitive measuring device, 3-day hCG administration (Pregnyl; 1500 IU daily) was shown to temporarily increase ulnar growth velocity from prepubertal (0.40 +/- 0.35 mm/3 weeks to pubertal values (1.1 +/- 0.64 mm/3 weeks) in 10 boys with delayed puberty. This growth-promoting effect of diagnostic hCG administration, which was demonstrable for 3--9 weeks, was associated with an overt rise in plasma testosterone from 129 +/- 126 to 818 +/- 419 ng/100 ml and an approximate doubling of the serum alkaline phosphatase activities from 193 +/- 46 to 376 +/- 115 U/liter, suggesting an initiated growth spurt.


Male pseudohermaphroditism secondary to 17 beta-hydroxysteroid dehydrogenase deficiency: gender role change with puberty. A 31-yr-old male pseudohermaphrodite is reported with 17 beta-hydroxysteroid dehydrogenase deficiency. Laboratory data revealed a plasma testosterone of 228 ng/100 ml, a plasma androstenedione of 620 ng/100 ml, and an abnormal androstenedione to testosterone ratio. Plasma estradiol was 4.6 ng/100 ml and plasma estrone was 22 ng/100 ml. This subject was born in a hospital, incontrovertibly declared to be a female, and unambiguously raised as a girl by his parents for the first 17 yr of his life. At age 14 yr, he was able to change to a male gender role with ease. As an adult, he is a well adjusted, happily married man with a successful professional career. Surgical correction of bilateral cryptorchidism and hypospadias was carried out at age 14 yr. At age 30 yr, he developed a teratocarcinoma-seminoma of the right testis with retroperitoneal node metastases. After orchiectomy and retroperitoneal node dissection, he was placed on chemotherapy and is presently free of metastases.


Evidence that testosterone can suppress pituitary gonadotropin secretion independently of peripheral aromatization. Testosterone (T) was given to normal men with and without the concomitant administration of the aromatase inhibitor, delta 1-testolactone (Teslac), to examine the role of peripheral aromatization of T in gonadotropin regulation. When T was administered alone by continuous iv infusion (15 mg/day for 4 days), serum T increased 3-fold (P less than 0.01) and estradiol (E) increased by 50% (P less than 0.01). These changes were associated with a 50% decrease in serum LH and FSH concentrations (P less than 0.01). When T was infused into men taking Teslac (2000 mg/day), serum T levels doubled (P less than 0.01), but E levels did not change (13.4 +/- 1.5 vs. 13.5 +/- 1.0 pg/ml; P = NS). This pattern of plasma steroids, increased T and unchanged E, was also associated with significantly decreased serum LH and FSH concentrations (14.5 +/- 0.4 vs. 8.0 + 0.4 mIU/ml and 9.9 +/- 2.5 vs. 5.8 +/- 0.1 mIU/ml, respectively; P less than 0.01). These data support the hypothesis that T or one of its metabolites can modulate LH and FSH secretion independently of peripheral aromatization to E.


Alterations in serum cortisol and its binding characteristics in anorexia nervosa. The binding capacity and apparent binding affinity of corticosteroid-binding globulin (CBG) for cortisol was studied in 20 women hospitalized for anorexia nervosa. The binding capacity for cortisol, as determined kinetically at 0 C by the method of Scatchard, was similar in anorectic patients and control subjects, with mean values of 26.9 and 21.9 microgram/100 ml, respectively. The mean apparent affinity constant of CBG for cortisol was found to be significantly (P less than 0.01) lower (14.1 X 10(8) M-1) than that in control subjects (22.3 X 10(8) M-1) and was similar in blood drawn at 0830 and 2000 h. Mean morning total serum cortisol concentrations were higher in anorectic women (23.3 microgram/100 ml) than in controls (10.0 microgram/100 ml; P less than 0.001) and frequently exceeded the binding capacity of CBG. Diurnal variation was present. Mean serum 17 beta-estradiol levels were low (40.0 pg/ml) and serum progesterone was essentially absent, but mean serum testosterone levels were within the normal range. Eight patients who were retested after weight gain, when they were between 76--100% of ideal body weight, exhibited no significant changes in serum steroids or in the binding capacity or apparent affinity of CBG for cortisol.


Oxytocin in maternal circulation and amniotic fluid during pregnancy. Oxytocin (OT) was measured by a specific and sensitive RIA in plasma and amniotic fluid throughout pregnancy. OT was detectable in 84.5% of 362 maternal plasma samples and showed a slow and fluctuating increase towards term with a significant sharp peak at 39 weeks of gestation. There was a highly significant correlation between mean plasma OT and the week of gestation (r = 0.5419, P less than 0.005). The minute to minute variability in plasma OT during pregnancy and labor in 7 subjects showed episodic release of OT with two or three spurts per 10 min, with the amplitude of the spurts being greater during labor. Serial maternal plasma OT throughout pregnancy in 10 patients revealed good concentrations of OT (greater than 10 pg/ml) in patients who subsequently had spontaneous labor and no intrapartum uterine dysfunction. Poor (less than 10 pg/ml) or undetectable OT levels were found in patients who subsequently developed intrapartum uterine dysfunction which necessitated cesarean section. OT was detectable in 89.7% or 87 amniotic fluid samples, with a mean +/- SE of 7.8 +/- 3.6 pg/ml at 14--15 weeks, 43.9 +/- 14.7 pg/ml at 40 weeks, and 30.8 +/- 10.5 pg/ml at 41--42 weeks. The significance of these findings is discussed in relation to the role and origin of OT in pregnancy and parturition.


Calcitonin in nonthyroidal cancer.

Calcitonin in nonthyroidal cancer. A blind prospective study was undertaken to determine the use of calcitonin (CT) as a tumor marker. After final diagnosis, results revealed elevated plasma CT (greater than 150 pg/ml) in common cancers as follows: lung, 38%; colon, 24%; breast, 38%; pancreas, 42%; and gastric, 30%. Fifty-eight percent of oat cell carcinomas were associated with elevated plasma CT. CT immunoreactivity was detected in 14% of tumor extracts and was not detectable in normal tissue other than thyroid. Hypercalcemia was not the cause of hypercalcitonemia. Incubation studies of [125I]human CT in cancer plasma and tumor extracts demonstrated that measurements were not an artifact of label degradation. In a survey of control patients with nonneoplastic disease, elevated CT was noted in renal failure, acute gastrointestinal bleeding, and in some patients with chronic obstructive lung disease. In conclusion, plasma CT is elevated in a substantial proportion of common neoplasms and is useful as a tumor marker.


Calcium homeostasis in diabetes mellitus. Experimentally diabetic rats have low serum 1,25-dihydroxyvitamin D, intestinal malabsorption of calcium, secondary hyperparathyroidism, and bone loss. To examine the hypothesis that abnormalities similar to those in the diabetic rat might explain human diabetic osteopenia, we studied calcium metabolism in 40 healthy control and 82 diabetic patients aged 18--75 yr [47 untreated: fasting plasma glucose (mean +/- SE), 267 +/- 8 mg/dl; 19 treated but hyperglycemic: glucose 305 +/- 24 mg/dl; 16 treated and in better control: glucose, 146 +/- 8 mg/dl]. Serum total calcium, ionic calcium, immunoreactive parathyroid hormone (Arnaud method, GP-1M and CH-12M antisera), 25-hydroxyvitamin D (Haddad method), and 1,25-dihydroxyvitamin D (Lambert method) concentrations were normal in all 3 groups of diabetics and were not significantly different from values in the control group. We determined absorption of calcium from the intestine by a double isotope method (100 mg Ca carrier; normal range, 40--80%) in 11 control and 13 untreated, uncontrolled diabetics (mean plasma glucose, 285 +/- 17 mg/dl). Absorption of calcium in controls was 60 +/- 3% and in diabetics was 56 +/- 3% (not significantly different). We have found no derangement of calcium metabolism in adults with insulin-requiring juvenile- and adult-onset diabetes regardless of treatment status. The experimental diabetic rat model does not appear to be useful for determining the pathogenesis of adult human diabetic osteopenia.


Effect of flutamide on estradiol metabolism. The effect of flutamide, a potent nonsteroidal antiandrogen, on the metabolism of iv tracers of [3H]estradiol was studied in five patients with advanced prostate cancer. The drug produced no change in the percentage of the injected radioactivity recovered in urine or in the glucuronide or nonglucuronide conjugate fractions. Of the five individual metabolites that were quantitated, estrone, estradiol, and estriol were unaffected by flutamide, but the drug caused striking decreases in conversion of estradiol to 2-hydroxyestrone (4.0% vs. 7.4%) (P less than 0.005) and 2-methoxyestrone (1.1% vs. 2.6%; P less than 0.05); every one of the patients showed a marked fall in recovery of both of these compounds. This depression of the formation of 2-oxygenated metabolites is reminiscent of the findings in liver disease; the same abnormality occurs regularly in cirrhosis and frequently in extrahepatic biliary obstruction. Taken together with our previous studies of the effects of flutamide on testosterone and cortisol metabolism, this study demonstrates that flutamide produces multiple functional, reversible, cirrhosis-like disturbances of steroid metabolism. Because these disturbances are universal in the patients studied regardless of whether they had clinical responses to flutamide, we doubt that the steroid metabolic changes play a role in the therapeutic effect of the drug.


Corpus luteum--conceptus--follicle relationships during the fertile cycle in rhesus monkeys: pregnancy maintenance despite early luteal removal. To determine how early in pregnancy cyclic follicle growth is interrupted, 6 pregnant monkeys were luteectomized (CLX) on day 29 or 30 of the cycle (approximately 1 week after implantation) to abbreviate the fertile cycle to approximately the length of nonfertile cycles. Unexpectedly, 3 of 6 monkeys remained pregnant despite early removal of luteal support. In the remainder, which aborted, the next ovulation was delayed beyond the interval typically observed after CLX in nonfertile cycles. Two of 4 other monkeys ovariectomized on day 29 also maintained their pregnancies. Our findings demonstrate that: 1) in 5 of 10 monkeys, the conceptus could survive without luteal support within a week after luteal rescue, and 2) secretion(s) of the conceptus also contribute to an arrest of cyclic follicle growth early in pregnancy.


Specific radioimmunoassay of human beta-endorphin in unextracted plasma. With an antiserum against human beta-endorphin (beta-EP) crossreacting less than 2% with human beta-lipotropin (beta-LPH) by weight we have developed a radioimmunoassay that can detect 1 pg beta-EP in diluted raw plasma. In a.m. fasting plasma of 14 normal subjects beta-EP ranged from less than 5 to 45 pg/ml. beta-EP was elevated in untreated, but normal in successfully treated Cushing's disease; undetectable in a patient with adrenal adenoma; extremely high in Nelson's syndrome; and elevated in a patient with bronchogenic carcinoma before, but undetectable after tumor resection. In subjects with intact hypothalamic-pituitary-adrenal axis, beta-EP was undetectable after dexamethasone and increased after metyrapone administration and insulin-induced hypoglycemia. beta-EP concentration was considerably lower in serum than in simultaneously collected plasma, but increased in serum left unfrozen for several hours after clot removal. Thus, beta-EP behaves like a hormone responding to the same stimuli as ACTH and beta-LPH and blood appears to contain enzymes both generating and destroying immunoreactive beta-EP.


Acromegaly and vitamin D metabolism: effect of bromocriptine treatment. Fifteen acromegalic subjects were found to have elevated plasma levels of both 1,25(OH)2-vitamin D, 65 +/- 23 (SD) pg/ml [normal 33 + 15 pg/ml (SD)] and 24,25(OH)2-vitamin D, 6.8 +/- 1.6 (SD) ng/ml [normal 3.4 + 1.2 ng/ml (SD)]. Treatment with bromocriptine for 6 months reduced the plasma 1,25(OH)2-vitamin D3 level to 40 +/- 13 (SD) pg/ml, p less than 0.01 and the 24,25(OH)2-vitamin D level to 5.4 +/- 1.7 (SD) ng/ml, p less than 0.05.


Plasma acetate turnover and oxidation.

Plasma acetate turnover and oxidation. Plasma acetate turnover and oxidation were determined in 11 healthy subjects by the constant infusion of a trace amount of [1-14C]acetate for 6 h. The subjects ages ranged from 22 to 57 yr. There was a positive correlation (P less than 0.001) between plasma acetate concentration and turnover rate, and a negative correlation (P less than 0.001) between turnover and age. The plasma acetate concentration in the subjects 22--28 yr old was 0.17 vs. 0.13 mM (P less than 0.02) in subjects 40--57 yr old. The plasma acetate turnover rate was also greater in the younger age group (8.23 +/- 0.66 vs. 4.98 +/- 0.64 mumol/min . kg, P less than 0.01). Approximately 90% of the plasma acetate turnover was immediately oxidized to CO2 in both age groups, however, 13.2 +/- 0.89% of the CO2 output in the younger group was derived from plasma acetate oxidation compared to 7.9 +/- 0.94% in the older group (P less than 0.01). The mean plasma acetate concentration, turnover, and oxidation in six cancer patients 47--63 yr old were similar to the values observed in the age-matched healthy subjects. Uptake or output of acetate by various tissues was measured by arterial-venous plasma acetate concentration differences. In seven of eight subjects undergoing elective surgery, the arterial-portal venous concentration difference was negative, which indicated that the gastrointestinal tract can contribute to plasma acetate production. Uptake of plasma acetate by both the leg and liver appeared to be dictated by the arterial acetate concentration. Net production of acetate by both the leg and liver was most often observed at arterial plasma acetate concentrations less than 0.08 mM.


Properdin factor D: effects on thrombin-induced platelet aggregation. Factor D, when preincubated with platelet suspensions, at concentrations as low as 1.2 micrograms/ml, inhibited thrombin-induced platelet aggregation. No inhibition of collagen or arachidonic acid-induced platelet aggregation was found. Inhibition occurred, but to a lesser extent, when thrombin and factor D were added to platelets at the same time. No inhibition occurred when factor D was added after thrombin. Thrombin was able to overcome inhibition by factor D by increasing its concentration. Diisopropyl-phosphorofluoridate-inactivated factor D also inhibited thrombin-induced platelet aggregation so that enzymatic activity of factor D was not required for inhibition. Factor D absorbed with hirudin coupled to Sepharose 6B showed no decrease in inhibitory capacity. 125I-Factor D bound to platelets in a manner suggesting an equilibrium reaction similar to thrombin. At low factor D input, binding was linear, whereas at higher input, binding began to approach saturation. Binding of 125I-labeled thrombin to platelets was inhibited by factor D. Analysis of these data show that factor D does not alter the total number of thrombin molecules which bind to the platelet surface at saturation. However, the dissociation constant for thrombin is altered from 2.78 to 6.90 nM in the presence of factor D (20 micrograms/ml). Factor D is thus a competitive inhibitor of thrombin binding, although the affinity of factor D for the platelet thrombin receptor is much less than that of thrombin. These phenomena occur at physiologic concentrations of factor D. Therefore, factor D may function in vivo as an inhibitor of platelet aggregation.


Intestinal calcium absorption and serum vitamin D metabolites in normal subjects and osteoporotic patients: effect of age and dietary calcium. Intestinal calcium absorption assessed by a double-isotope method, decreased significantly with aging in 94 normal subjects (r = -0.22, P < 0.025). In 52 untreated patients with postmenopausal osteoporosis, calcium absorption was significantly lower than normal when either age or habitual calcium intake was used as a covariable (P < 0.001). Serum 25-hydroxyvitamin D (25-OH-D) and 1,25-dihydroxyvitamin D (1,25(OH)(2)D) were measured in 44 normal subjects and 27 osteoporotic patients. For all normals, calcium absorption and serum 1,25(OH)(2)D were positively correlated (r = 0.50, P < 0.001). In nonelderly normal subjects (ages 30-65 yr), dietary calcium intake correlated inversely with both calcium absorption (r = -0.39, P < 0.01) and with serum 1,25(OH)(2)D (r = -0.50, P < 0.01). Both osteoporotic patients and elderly normal subjects (ages 65-90 yr) differed from nonelderly normals in that these correlations were not present. In addition although serum 25-OH-D was normal, serum 1,25(OH)(2)D was significantly decreased in both osteoporotic patients and elderly normals (P < 0.001). In osteoporotic patients, calcium absorption increased significantly (P < 0.001) after 7 d administration of a small dose (0.4 mug/d) of synthetic 1,25(OH)(2)D(3). In osteoporotics mean serum immunoreactive parathyroid hormone was either normal (COOH-terminal assay) or low (NH(2)-terminal assay) relative to age-matched controls, and mean serum phosphate was increased. The data suggest that inadequate metabolism of 25-OH-D to 1,25(OH)(2)D contributes significantly to decreased calcium absorption and adaptation in both osteoporotics and elderly normal subjects. In patients with osteoporosis this abnormality could result from a decrease in factors that normally stimulate 1,25(OH)(2)D production, such as the decreased parathyroid hormone secretion and increased serum phosphate demonstrated in this group. In elderly subjects a primary abnormality in metabolism of 25-OH-D to 1,25(OH)(2)D, analagous to that seen in aging rats, cannot be excluded.


Measurement, characterization, and source of somatostatin-like immunoreactivity in human amniotic fluid. Somatostatin-like immunoreactivity (SLI) is widely distributed in tissues and biological fluids. To determine whether SLI is also present in amniotic fluid, samples obtained by amniocentesis from 30 normal and 27 abnormal pregnancies were studied by radioimmunoassay. Direct incubation of [(125)I-Tyr(1)]tetradecapeptide somatostatin (SRIF) with amniotic fluid resulted in 89% tracer degradation. Damage was reduced to <5% when samples were acidified and boiled before the assay. With this technique, SLI was detectable in all normal amniotic fluid samples; the mean level at 15-20 wk of gestation (320+/-55 pg/ml, n = 15) being 4.5 times higher than the mean at 32-43 wk (70+/-12 pg/ml, n = 15) (P < 0.001). In cases of preeclampsia (n = 6), gestational diabetes (n = 5), anencephaly (n = 1), and meningomyelocele (n = 1), SLI values were in the normal range, but in one juvenile diabetic and one patient with chronic renal failure, SLI was undetectable (<10 pg/ml). In a pair of monochorionic diamniotic twins, SLI levels were very different (33 and 197 pg/ml), which suggests that fetal factors are more important than materno-placental ones in determining amniotic fluid SLI. Serial dilutions of amniotic fluid showed parallelism with standard SRIF. When concentrates of pooled amniotic fluid were chromatographed on Sephadex G-25 columns, all SLI eluted in the void volume ahead of SRIF even after treatment with 8 M urea and dithiothreitol. This "big" SLI incubated in amniotic fluid showed 100% stability over 24 h at 37 degrees C, whereas SRIF was rapidly inactivated (t((1/2)) congruent with 7 min). Extracts of placenta and fetal membranes contained no SLI, but small amounts (6-20% of total amniotic fluid SLI) were found in cells from fresh fluid. Radioimmunoassay of SLI in extracts of seven paired cord arterial and venous plasma samples showed no arteriovenous gradient consistent with fetal origin of cord blood SLI. It is concluded that (a) amniotic fluid contains SLI which is of fetal origin and (b) normal levels vary with gestational age. The SLI has a higher molecular weight (>/=5,000) and is more stable in amniotic fluid than SRIF.


Heterogeneity of DNA fragments associated with the sickle-globin gene. We have examined the genetic polymorphism previously reported to be associated with the sickle-cell (beta s) gene. The polymorphism involves an alteration of the DNA sequence 3' to the beta-globin gene as detected with the restriction endonuclease, Hpa I. In normal individuals, the beta-globin gene is contained within a DNA fragment of 7.6 kilobases (kb), whereas 87% of individuals with sickle-cell anemia have been reported to have the beta s-gene associated with a 13.0-kb Hpa I fragment. We have studied this polymorphism in 31 New York Black individuals homozygous for sickle-cell anemia to ascertain its genetic and biochemical significance and to evaluate its potential use in the prenatal diagnosis of sickle-cell disease. Our results show only a 58% association of the beta s-gene and the 13.0-kb Hpa I fragment, as well as the presence of additional variants involving the Hpa I site. In addition, the 13.0-kb fragment is also found associated with the beta c- and beta A-genes. Thus, the Hpa I polymorphism probably represents a change in DNA not specifically associated with the beta s-gene, and appears to antedate the beta s-and beta c-mutations.


Elevated cholesterol and bile acid synthesis in a young patient with homozygous familial hypercholesterolemia. Cholesterol balance studies were carried out twice in a young male patient with homozygous familial hypercholesterolemia. At 13 mo, cholesterol balance in this patient averaged 31.3 mg/kg per d, and bile acid excretion was 12.0 mg/kg per d; at 3 yr, results were similar, 27.3 and 15.5 mg/kg per d for cholesterol balance and bile acids, respectively. A normal boy of 3 yr was also studied for comparison with the second study in our patient. Cholesterol balance and bile acid outputs in the normal child were 11.5 and 3.3 mg/kg per d, respectively. Thus, in comparison with the normal child, the patient with homozygous familial hypercholesterolemia had a marked increase in synthesis of cholesterol and bile acids. Although synthesis of bile acids was high in this patient, the fraction of newly synthesized cholesterol converted into bile acids (40-56%) was in the normal range; this suggests that the enhanced output of bile acids was secondary to an increased synthesis of cholesterol and not to malabsorption of bile acids, which likely would have produced a higher fractional conversion. Although our patient has been studied at a younger age than any reported in the literature, two similar children 5 and 6 yr of age were also observed to have elevated cholesterol synthesis. This finding contrasts with those in older children with the homozygous as well as heterozygous forms of this disease who appear to have normal synthesis of cholesterol and bile acids. Therefore, increased synthesis of cholesterol seems to be characteristic of early homozygous familial hypercholesterolemia, and may be a manifestation of a loss of feedback inhibition of cholesterol synthesis secondary to an absence of specific cell-surface receptors for low density lipoproteins. However, as children with this disease grow older, other mechanisms may come into play to restore cholesterol synthesis to normal levels.


Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (M(r) = 62,000) contains 23% carbohydrate and is composed of a light chain (M(r) = 21,000) and a heavy chain (M(r) = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human alpha-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity.


Relationship between alveolar PO2 and the rate of p-nitroanisole O-demethylation by the cytochrome P-450 pathway in isolated rabbit lungs. The relationship between alveolar PO2 and the rate of O-demethylation of p-nitroanisole, a model substrate for cytochrome P-450 -linked mixed-function oxidation, was evaluated in the isolated rabbit lung perfused with Krebs-Ringer bicarbonate buffer. The appearance of the product, p-nitrophenol, in the pulmonary perfusate was measured spectrophotometrically, The PO2 of the ventilating gas was varied with an accurate gas mixing pump and measured with an electrochemical O2 analyzer. In control lungs ventilated with 5% CO2 in air, the rate of p-nitrophenol production was approximately equal to 3.1 +/- 0.04 (mean +/- SE; n = 9) mumol/h per g dry wt. p-Nitrophenol production was unaltered when O2 in the ventilating gas was decreased to 1%, but it was depressed reversibly when alveolar O2 WAS 0.1% OR LESS AND WAS ABOLISHED DURING VENTILATION WITH 0.005% O2. The rate of the reaction was inhibited by 50% when alveolar PO2 was 0.3 mm Hg representing and intracellular [O2] OF approximately equal to muM. In the presence of metyrapone (0.1--1 mM), an inhibitor of cytochrome P-450-dependent reactions, p-nitrophenol production was 0.07--0.17 mumol/h per g dry wt. Ventilation of lungs with varying CO concentration in 20% O2 resulted in 50% inhibition of p-nitrophenol production when CO concentration was 10% (CO/O2 = 0.5). These results indicated that O-demethylation of p-nitroanisole by the lung is a cytochrome P-450-dependent reaction and that its rate is not affected until alveolar PO2 is less than 1 mm Hg.