Effects of potassium azide on soil microbial populations and soil enzymatic activities.

Preplant applications of potassium azide (KN3) to pine nursery beds were evaluated for effect on the soil microflora and on soil enzyme activity where either plastic-sealing or water-sealing techniques were used. Two weeks after incorporation of azide (0-224 kg/hs), soil samplings revealed reduced populations of bacteria and fungi and a corresponding decline in invertase and amylase activities. These effects were proportionate to the amount of azide used and were more pronounced in plastic-sealed plots. Phosphatase activity was little affected. Five weeks after azide application, bacterial populations were higher in treated plots than in controls. Greater numbers of bacteria were recorded from plastic-sealed plots and highest populations coincided with plots receiving the highest rates of azide, regardless of the sealing technique. Fungal populations at this sampling were generally less in treated plots than in the controls, but were higher under plastic seal. At this time, changes in invertase and amylase activities did not correspond to increased microbial numbers. Sixteen weeks after applications of KN3, bacterial populations in treated plots did not differ significantly from controls, but remained higher in plastic-sealed than water-sealed plots. Fungal populations under plastic seal had changed little and remained significantly lower in treated water-sealed plots than in controls. The earlier recorded reduction in invertase and amylase activities was still evident at the final sampling;


Dissimilation of the lignin model compound veratrylglycerol-beta-(o-methoxyphenyl) ether by Pseudomonas acidovorans: initial transformations.

The initial transformadomonas acidovorans are demethylation of the 4-methoxyl group of the 3,4-dimethoxyphenyl portion of the molecule, and a NAD+-linked dehydrogenation of the benzyl alcohol group. Based on these findings and those described before, an overall degradation scheme is postulated.


Glomus jugulare tumors: effects of radiotherapy.

Twenty glomus jugulare tumors which received x-ray therapy are analyzed. Postirradiation angiography showed persistent tumor vessels and abnormal channels in eight patients. Histologic study of five postirradiation specimens showed that tumor cells persisted after treatment in a significant proportion of the patients. Vascular response was nonuniform and consisted of endothelial hyperplasia and subendothelial hyalinization. Radiotherapy is not always effective in the permanent eradication of the tumor when used as the only modality of treatment in patients with large glomus jugulare tumors. Because of the frequent recurrence after irradiation or surgical resection, a combination of both modalities offers the patient the best probability of cure.


Studies on ectopic ACTH-producing tumors. II. Clinical and biochemical features of 30 cases.

This report describes the clinical and biochemical features of 30 cases of ectopic ACTH-producing tumors diagnosed by the detection of ACTH in the tumor tissues. Several uncommon tumors, such as tumors of the esophagus, stomach, and larynx, were included in this series. None of the patients with bronchogenic carcinoma showed signs of classical Cushing's syndrome, whereas 7 of the remaining 13 patients with other tumors were Cushingoid in appearance. Adrenocortical hyperfunction was present in 61 percent at the first examination and developed during the course of the disease in 18 percent more. In the remaining patients (21 percent), adrenocortical function remained within normal limits. These results indicate that there exist ectopic ACTH-producing tumors without clinical and biochemical sequelae of excess hormone. In some of the tumor extracts studied, MSH and CRF-like activities and serotonin were detected. This suggests that multiple hormone production is not uncommon in ectopic ACTH-producing tumors.


Biochemical characterization of carcinogen-induced mammary hyperplastic aveolar nodule and tumor in the rat.

Biochemical parameters characterizing growth, functional activity, and hormone dependence were compared in the normal mammary gland, the 7,12-dimethylbenz(a)anthracene-induced hyperplastic aveolar nodule (HAN), and the mammary tumor in the rat. The rate of DNA synthesis in HAN was significantly lower than the rates in either the normal mammary gland cells or the mammary tumor cells cultured in medium containing identical hormone supplement for the same duration of time. The rate of casein synthesis in the explants of pregnant rat mammary gland and HAN was greatly stimulated when cultured in medium containing insulin, cortisol, and prolactin. The mammary tumor, however, failed to produce an increase in casein synthesis under the same experimental conditions. The specific estradiol-binding proteins were either absent or present in very low concentrations in HAN. In contrast, mammary tumor cytosol contained high concentrations of specific estrogen-binding proteins. The normal mammary gland, in spite of its low cellularity, had twice as many estradiol-binding macromolecules as did HAN. Altogether, these results show that HAN cell populations differ from normal mammary gland and mammary tumor cells by their nonresponsiveness to hormonal stimulation for growth and by their lack of specific receptors for extradiol. They retain, however, the functional capacity to synthesize casein, a biochemical property that the mammary tumor does not posses.


S-adenosylmethionine:protein methyltransferases in hepatomas.

Protein methylase III (S-adenosylmethionine:proteinlysine methyltransferase; ED 2.1.1.25) and protein methylase I (S-adenosylmethionine:protein-arginine methyltransferase; EC 2.1.1.23) activities were examined in isolated nuclei and cytosol fraction, respectively, from various hepatomas with different growth rates. The enzyme activities of both enzymes paralleled the rates of tumor growth in fast- and moderately growing hepatomas. The parallelism was more evident with protein methylase I than with protein methylase III. While protein methylase III activity was elevated in the fast- to moderately growing hepatomas, the enzyme that is responsible for demethylating proteins, epsilon-alkyllysinase (epsilon-slkyl-L-lysine:oxygen oxidoreductase; EC 1.5.3.4), had an inverse relationship to the rate of tumor growth, thus suggesting a possible physiological antagonism. When isolated rat liver nuclei were methylated in vitro with S-adenosyl-L-[methyl-14 C]methionine as methyl donor, H2SO4-insoluble protein and histones had almost equal amounts of methyl-14 C incorporated. However, amino acid analysis revealed that methylated arginines are the predominant form of radioactivity in the H2SO4-insoluble protein (product of protein methylase I), while methylated lysines are the major methylated amino acids in the histones (product of protein methylase III). Furthermore, the hydrolysate of the H2SO4-insoluble protein showed four unknown radioactivity peaks on the amino acid analyzer in addition to the known methylated arginine and lysine derivatives.


The covalent binding of daunomycin and adriamycin to antibodies, with retention of both drug and antibody activities.

Daunomycin and adriamycin, two potent cancer chemotherapeutic agents, were linked to immunoglobulins, making use of various covalent cross-linking methods. The most suitable method for binding of the drugs to the antibodies, which retained both antibody and drug activity, was periodate oxidation of the drug, followed by the linking of the oxidized drug to the immunoglobulin and subsequent reduction of the product with sodium borohydride. The activity of the drug-antibody conjugates was tested in vitro on tumor and normal cell cultures and was found to be similar to that of the free drug. A significant amount of antibody activity was retained, as found both with anti-bovine serum albumin antibodies, assayed by chemically modified bacteriophage, and with anti-mouse tumor antibodies, assayed by C'-dependent cytotoxicity.


The specific cytotoxic effects of daunomycin conjugated to antitumor antibodies.

Daunomycin was covalently bound to immunoglobulins by periodate oxidation as described in the preceding paper. Conjugates were prepared with immunoglobulins directed against either of two mouse lymphoid tumors or with nonspecific immunoglobulins. These conjugates were tested for their toxic effects on various tumor target cells as measured either by their inhibition of RNA synthesis or by their reduction of the growth of the tumor cells after transplantation. We found that the drug preferentially affected the target cells that the antibody to which it was attached could recognize. These daunomycin-antibody conjugates are therefore sufficiently toxic and selective in their effects to be potentially useful in in vivo therapeutic studies.


Specific depression of the antitumor cellular immune response with autologous tumor homogenate.

A momogenate of an SV40-transformed firbosarcoma of BALB/c mice (E4 tumor) injected i.p. into E4, tumor-immune syngeneic mice specifically depressed their cell-mediated immune responses to autologous tumor cells, as measured by a radioisotopic foot pad assay. The fraction of the tumor homogenate that brought about this depression was present in the high-speed supernatant and pellet of a 3 M KCl extract of the tumor. The specificity of the depression was shown in three ways: (a) the serum of E4 tumor-immune mice, but not of normal mice, given injections of E4 tumor homogenate 24 hr previously, suppressed antitumor immunity in vitro, as measured by the release of 51Cr from labeled E4 tumor cells incubated with spleen cells from tumor-immune animals; (b) the i.p. inoculation of E4 tumor homogenate did not alter the cellular immune response of tuberculin-sensitized mice to tuberculin; and (c) the i.p. injection of a homogenate of antigenically unrelated tumor did not depress the cellular immune response of E4 tumor-immune mice to E4 tumor cells.


Chromosome analysis of a simian virus 40-transformed mouse cell line and two variant sublines that are resistant to cytochalasin B1.

The chromosomes of an SV40-transformed mouse cell line, SVT2, were analyzed by the acetic-saline-Giemsa banding technique. By contrast to most established mouse lines, SVT2 cells possess a remarkably homogeneous chromosome complement and contain two copies of most chromosomes. However, trisomy for chromosome 3 is a distinct feature of this cell line. Chromosomes 1, 3, 14, and 19 have given rise to biarmed markers. Two Cytochalasin B-resistant sublines derived from SVT2 are also essentially diploid for all autosomes but contain only one chromosome X; they display an even greater homogeneity than does the SVT2 parental cell line. Each of the Cytochalasin B-resistant cell lines has lost one or several of the biarmed markers from SVT2 and new ones have appeared. Both cell lines have lost one copy of chromosome 3 and one chromosome X. The results presented illustrate the advantages of cells of defined chromosome constitution, like STV2 cells and derived sublines, to study specific interactions between transforming virus and host chromosomes. The possible role of SV40 in the maintenance of the pseudodiploid karyotype of SVT2 is also discussed.


Amino- and carboxyl-terminal analyses of hepatoma lactate dehydrogenase isozymes.

The M4 isozyme of lactate dehydrogenase was purified to homogeneity from normal rat liver and from two Morris hepatomas (7777 and 7793). Amino-terminal analyses with fluorodinitrobenzene failed to detect the presence of free amino-terminal residues in each enzyme studied. Each enzyme contained between 3.7 and 4.1 moles of protein-bound acetyl groups per mole of enzyme. The amino-terminal peptide, characterized as N-acetylalanylalanine, was isolated from Pronase digests of each isozyme preparation, and quantitative recovery experiments indicated that all acetyl residues were bound at the amino termini. Carboxylterminal analyses demonstrated phenylalanine to be the carboxyl-terminal residue in each enzyme studied. These data indicate no differences in either amino- or carboxyl-terminal regions of the hepatoma M4 isozymes compared to normal liver M4 isozyme.


Histochemical observations on uptake of L-dopa into endocrine cells of the rat pituitary gland during the postnatal development.

The uptake of L-dopa into the cells of the adenohypophysis of the rat was studied during the postnatal development and at adult age using the formaldehyde-induced fluorescence method (FIF). The cells taking up L-dopa were classified by Alcian blue-PAS-Orange G staining. The correlation between the cells taking up L-dopa and those containing tryptophyl-peptide was estimated during the postnatal period and in adult rats. The cells containing tryptophyl-peptide were demonstrated using fluorescence induced by treatment with combined formaldehyde and acetyl chloride vapour. The following observations were made: 1) Great majority of the cells taking up L-dopa did not contain tryptophyl-peptide. Thus the accumulation of L-dopa into the cells of pars distalis is not due to accumulation of L-dopa into the cells by the same transport mechanism as the amino acids for tryptophyl-peptide. 2) Of the cells taking up L-dopa in the adult rats 96% were chromophobes, 2.0% acidophilic cells (somatotrophs and cells producing prolactin), 0.9% R-mucoid cells (corticotrophs), and 1.2% S1- and S2-mucoid cells (gonadotrophs and thyrotrophs). At 10 and 25 days' age the relative numbers of the cells taking up L-dopa were about the same. 3) Pretreatment with nialamide caused only a slight increase in the number of the cells taking up L-dopa. The decrease in the number of the cells uptaking L-dopa of the pars distalis, which takes place after 5 weeks' age is thus not caused by the increased MAO-activity. 4) Strongly chromophilic cells did not take up L-dopa. At the light of our results it seems evident that L-dopa is taken up by the chromophobic cells when these differentiate into chromophilic cells. The accumulation of L-dopa may be a sign of an active transport of amino acids into the cells. The accumulation of L-dopa into the chromophobic stellate and follicular cells may reflect their metabolic activity. These cells probably have an important role in the production of the hormones of the pars distalis.


Intranuclear and cytoplasmic annulate lamellae in grasshopper spermatocytes (genus Melanoplus).

Intranuclear and cytoplasmic annulate lamellae were studied in grasshopper spermatocytes (Melanoplus) with the electron microscope. Although cytoplasmic annulate lamellae were observed in all three species examined, intranuclear annulate lamellae were found in only one species. The intranuclear annulate lamellae encompass certain nuclear material adjacent to the nuclear envelope forming a vesicle that is extruded into the spermatocyte cytoplasm. In this same species, cytoplasmic annulate lamellae are seen contiguous with granular masses of varying size. These structures were noted as being morphologically indistinguishable from the "yolk nuclei" of dragonfly oocytes (Kessel and Beams, 1969; Kessel, 1973).


The fine structure and phosphatase cytochemistry of the golgi complex and associated structures in the sertoli cells of Syrian hamsters.

The Golgi complex in the Sertoli cell of the Syrian hamster is well developed and consists of stacks of cisternae and associated vesicles. The inner- and outermost cisternae of the Golgi stacks are usually moderately dilated and exhibit numerous fenestrations. The middle portions of the intermediate cisternae are greatly flattened and not fenestrated, but toward the periphery these cisternae gradually become dilated and show a few fenestrations. On the inner aspect of the Golgi stacks the following structures are seen frequently: (1) one or two series of linearly arrayed circular profiles some of which are interconnected by tubules; (2) networks of anastomosing tubules with circular or oval meshes (800 to 1200 A in diameter); and/or (3) irregularly disposed tubules. The circular profiles and tubules are approximately 450 A in diameter. Acid phosphatase activity was localized in these anastomosing tubules when the tissues were incubated for more than one hour in a modified Gomori's medium (Barka and Anderson, 1963). Strong thiamine pyrophosphatase activity was demonstrated in the inner one to three cisternae of the Golgi stacks but not in the associated tubules. The system of the Golgi associated tubules is morphologically and histochemically distinct from the Golgi stacks and is probably equivalent to the Golgi-endoplasmic reticulum-lysosome system (GERL) in other cell types. The three dimensional aspects of the GERL-equivalent system are discussed.


Accumulation of collagen-containing vacuoles in osteoblasts after administration of colchicine.

Embryonic mouse radii were cultured in the presence of colchicine or vinblastine and subsequently examined in the electron microscope. As a result of the administration of these drugs large numbers of vacuoles accumulated in the cytoplasm of the osteoblasts. The electron-dense contents of at least many of the vacuoles most probably consisted of collagen. This conclusion was based on the results of special staining methods and on the similarity of the vacuoles to collagen vacuoles described in the literature. The observations made in this study provide morphological evidence that colchicine and vinblastine disturb the secretion of collagen.


SV40-transformed cells with temperature-dependent serum requirements.

We have isolated temperature-sensitive SV40-transformed 3T3 cells which are unable to grow in low or depleted serum at the nonpermissive temperature. At 39 degrees C, these cells do not grow in 1 percent serum, but they grow if the serum concentration is raised to 10 percent. At 32 degrees they grow in both serum concentrations. This phenotype seems to be due to a cellular mutation, as the virus rescued from these cells is wild-type. We tested whether other characteristics of transformed cells were expressed in a temperature sensitive way. While high saturation density is ts in these cells, other parameters of transformation are expressed at both temperatures. In addition, when these cells are incubated in low serum at 39 degrees C, they keep synthesizing DNA and lose viability very fast, while under the same conditions normal 3T3 cells remain viable for long times and are unable to initiate DNA synthesis. These cells therefore do not appear to revert to a normal phenotype at the high temperature, and they are more likely to represent transformed cell variants with a temperature-dependent serum requirement.


The interaction of estradiol-receptor protein with the genome: an argument for the existence of undetected specific sites.

In extracts from rat and calf uterus, the steroid hormone 17 beta-estradiol stimulates the binding of its specific receptor protein to DNA. This interaction appears to be of low affinity (half of the estradiol-activated, 5S receptor bound at 300-400 mug/ml DNA) and nonspecific with respect to DNA base sequence. No binding to double-stranded RNA is observed. These findings are consistent with several in vivo observations. In particular, when the cytoplasmic receptor protein binds hormone, it migrates to the cell nucleus to an extent consistent with its affinity for DNA in vitro, and this in vivo nuclear binding is uniform and nonsaturable in the testable range (to greater than 3 times 10-4 sites per cell). The level of biological response appears to parallel the hormone dose up to these high levels of receptor binding. How are these observations to be reconciled with the prevalent view of steroid receptors as gene control proteins regulating transcription at specific loci on the genome? Our model is based on an analogy with the DNA binding properties of the E. coli lac repressor protein. We believe that the estradiol receptor exerts its effect by binding to a small number of high affinity sites on the genome, while also having a finite low affinity for nonspecific DNA sequences. These nonspecific loci, because of their vast number, completely mask the presence of the high affinity sites. We estimate that up to 10-3 specific sites, with affinities in the range 10- minus 8 minus 10- minus 10 M, could exist without being detected by bulk binding assays currently in use. However, alternative approaches should allow detection of these sites, and some of these are suggested.


Chicken leukosis virus genome sequences in DNA from normal chick cells and virus-induced bursal lymphomas.

Genome sequences of two recent field isolates of avian leukosis viruses in the DNA of normal and neoplastic chicken cells were studied by DNA-RNA hybridization under conditions of DNA excess. Comparisons were made between 60-70S RNA from these viruses and that of a chicken endogenous type C virus (RAV-0), and of a series of "laboratory" leukosis and sarcoma viruses, by competitive hybridization analysis. A minimum of 18% of the genome sequences of both ALV isolates detected in DNA from lymphomas they induced were not detected in normal chicken DNA. The vast majority of the fraction of RNA sequences from ALV which do form hybrids with normal chick DNA appear to be reacting with the endogenous provirus of RAV-0. The genomic representation of a variety of avian leukosis and sarcoma viruses in normal chicken cells could not be distinguished by these methods (except that 13% of the RAV-0 genome was not shared with any of the other viruses). In contrast, the portion of the ALV genome exogenous to the normal chicken geome showed significant divergence from that of two sarcoma viruses (Pr RSV-C and B-77). The increased hybridization of ALV RNA with lymphoma DNA was used to detect the appearance of ALV specific sequences in the bursa of Fabricius following infection.increased hybridization was correlated with both the time after infection and the extent of replacement of the bursa by lymphoma. About one half of the increase in hybridization preceded histologic evidence of transformation.


Distribution of murine type B and type C viral nucleic acid sequences in template active and template inactive chromatin.

Template active chromatin and template inactive chromatin have been fractionated from mouse cells infected with the Moloney strain of murine leukemia virus. In vivo the cells produce abundant Rna homologous to Moloney leukemia virus, but do not produce either globin mRNA or RNA homologous to type B mouse mammary tumor virus. The DNA extracted from the template active chromatin or template inactive chromatin contained equal amounts of sequences homologous to Moloney type C virus, to type B virus, or to globin mRNA. The results are discussed with regard to the in vivo structure of chromatin and the difficulties in fractionating chromatin in vitro.


Methylated nucleotides block 5' terminus of HeLa cell messenger RNA.

Polyadenylylated [poly(A)+] mRNA from HeLa cells that were labeled with [3H-methyl]-methionine and 14C-uridine was isolated by poly(U)-Sepharose chromatography. The presence of approximately two methyl groups per 1000 nucleotides of poly(A)+ RNA was calculated from the 3H/14C ratios and known degrees of methylation of 18S and 28S ribosomal RNAs. All four 2'-O-methylribonucleosides, but only two base-methylated derivatives, 7-methylguanosine (7MeG) and 6-methyladenosine (6MeA), were identified. 6MeA was the major component accounting for approximately 50% of the total methyl-labeled ribonucleosides. 7MeG, comprising about 10% of the total, was present exclusively at the 5' terminus of the poly(A)+ RNA and could be removed by periodate oxidation and beta elimination. Evidence for a 5' to 5' linkage of 7MeG to adjacent 2'-O-methylribonucleosides through at least two and probably three phosphates to give structures of the type 7MeG5'ppp5pNMep- and 7MeG5'ppp5'NMepNmep- was presented. The previous finding of similar sequences of methylated nucleotides in mRNA synthesized in vitro by enzymes associated with virus cores indicates that blocked 5' termini may be a characteristic feature of mRNAs that function in eucaryotic cells.


Organ-specific effects of DNA methylation by alkylating agents in the inbred Swiss mouse.

Young adult inbred Swiss mice given single or repeated equitoxic doses of N-methyl-N-nitrosourea (MNUA) or methyl methanesulphonate (MMS) develop thymomas and pulmonary adenomas only following MNUA in spite of nearly identical overall alkylation of DNA of tumour target tissues by both agents due mainly to the biologically ineffective product 7-methylguanine. The main difference in DNA alkylation was the production of O6-methylguinine, a known pre-mutagenic product, by MNUA in amounts 10 or more times as large as following MMS. This supports the possibility that somatic mutations are a part of the process of carcinogenesis.


Fluorometry of citrate in serum, with use of citrate (pro-3S)-lyase.

We describe a procedure for enzymatic assay of citrate in human serum. The citrate is degraded to acetate and oxaloacetate with citrate oxaloacetate-lyase (pro-3S-CH2-COO- yields acetate) (EC 4.1.3.6). Some oxaloacetate loses CO2 to form pyruvate. Addition of malate and lactate dehydrogenases (EC 1.1.1.37 and 1.1.1.27) permits determination of the oxaloacetate and pyruvate generated, and thus of the citrate concentration. The decrease in NADH concentration is measured fluorometrically. Results obtained for 30 consecutive human sera by this procedure were compared to the procedure in which the citrate is converted to pentabromoacetone. There was no statistically significant difference in values obtained by the two procedures. The range of values (mean plus or minus 2 SD) found for sera from 25 blood donors by this procedure was 12.8-27.2 mg/liter (mean, 19.0 mg/liter). Serum citrate as measured by both procedures during a glucose tolerance test was decreased from initial values under the influence of administered glucose (and endogenous insulin). Insulin concentrations were also measured during these glucose-tolerance tests. Citrate concentrations remain subnormal after the glucose and insulin concentrations return to their initial values. This accords with published reports.


Hyperlipoproteinaemia in ponies: mechanisms and response to therapy.

(I) The lipid and lipoprotein concentrations in sera of 4 healthy and 9 hyperlipaemic patients were determined. From the results of the analyses, it is suggested that three types of hyperlipoproteinaemia can be distinguished in ponies: (a) Type 1, characterised by a very pronounced increase in only the very low density lipoproteins (VLDL); (b) Type 2, in which there is a pronounced increase in the chylomicrons (Chylo) and the very low density lipoproteins (VLDL); (c) Type 3, which can be the result of fasting and is characterised by a moderate increase in the concentration of chylomicrons and a greater increase in very low density lipoproteins. It is suggested that hyperlipoproteinaemia in ponies can be classified on the basis of electrophoretic patterns. (II) To gather more information on the metabolism of extra offered lipids, blood from ponies suffering from two different types of hyperlipoproteinaemia was transfused into normal ponies and the changes in lipid and lipoprotein concentrations in the blood serum were followed. The reactions were different. When heparin was used as anticoagulant a great increase in chylomicrons and a significant increase in very low density lipoproteins in a healthy pony was observed. When sodium citrate was used as anticoagulant a clear increase in the VLDL fraction only was noticed.


The formation of 2-hydroxybutyric acid in experimental animals.

1. The formation of 2-hydroxybutyric acid (2-HB) has been studied by animal experiments. 2. 2-HB was excreted in the urine together with lactic acid following intravenous administration of huge amounts of 3-hydroxybutyric acid (sodium salt) to a dog. 3. Rats made diabetic by an injection of streptozotocin were found to excrete large quantities of 2HB in the urine (up to 300 mumol/24 h) together with the development of ketoacidosis. 4. The use of 14-C-labelled precursors clearly showed that the amino acids methionine, threonine and homoserine can be converted to 2-HB. 5. 2-Aminobutyric acid is also converted to 2-HB, with 2-oxobutyric acid as an intermediate metabolite. The ratio between the urinary concentrations of 2-HB and 2-oxobutyric acid was increased by the ingestion of ethanol. 6. In normal rats neither a prolonged fasting period nor loading with large doses of methionine, threonine and homoserine resulted in the excretion of 2-HB. 7. The mechanisms behind the formation of 2-HB are discussed, and it is concluded that an increased NADH2/NAD ratio in the cytoplasma is the most important factor.


The quantitation of cyclophosphamide in human blood and urine by mass spectrometry-stable isotope dilution.

The levels of cyclophosphamide in the blood and the urine of patients have been monitored by direct insertion, electron impact mass spectrometry using the principle of stable isotope dilution. When a tetradeuterated analogue of the drug was added to a sample of blood or urine the concentration of cyclophosphamide could be determined from the ratio of the intensities of the signals (M-CH2 C1) characteristic for cyclophosphamide and the tetradeuterated analogue present in the mass spectrum of a chloroform extract. The procedure is highly specific for cyclophosphamide and obviates the need to use radioactively labelled cyclophosphamide for quantitation of the drug in man.


A longitudinal assessment of lipid ratios in the diagnosis of type III hyperlipoproteinaemia.

Type III hyperlipoproteinaemia (HLP) has been overdiagnosed in the past, but still may go undiagnosed because of technical difficulties. This longitudinal study of 19 ultracentrifugally proven cases of Type III HLP shows that 2 lipid ratios can be used to supplement the findings of a floating beta band. These patients have been followed for 3 months to 2.5 years whilst on dietary and/or drug treatment. A group composed of more than 100 Type IIB, Type IV and Type IIB-IV hyperlipoproteinaemic patients has been used for comparison purposes. From a total of 310 ultracentrifugal analyses, it was found that the following 2 ratios have more than a 90 percent predictability for Type III HLP: supernatant cholesterol/supernatant triglyceride greater than or equal to 0.35 (Ratio 2); supernatant cholesterol/whole plasma triglyceride greater than or equal to 0.25 (Ratio 3). These ratios are particularly valuable in Type III HLP when the plasma is grossly hyperlipaemic or when the lipid levels are low because of successful treatment.


Serum pseudocholinesterase and ceruloplasmin in various types of hyperlipoproteinemia.

Starting from previous observations emphasizing an increased pseudocholinesterase (PCE) activity in obese and hyperlipemic subjects, the behaviour of this enzyme and of ceruloplasmin was studied in connection with changes of serum lipids and lipoproteins in various types of hyperlipoproteinemia. When compared to values detected in 67 middle-aged normal weight normolipemic subjects, PCE activity was found to be significantly greater (smaller than 0.001) in the 49 overweight subjects without obvious hyperlipemia but presenting a moderate increase of the prebeta electrophoretic fraction. PCE activity was much higher in lean or overweight subjects with endogenous hypertriglyceridemia (68 patients with type IV and 86 patients with mixed hyperlipemia). The slight increase of mean values of PCE activity in the 53 subjects with type II-a was due mainly to overweight subjects, while this enzyme's activity was not significantly changed in lean subjects with pure hypercholesterolemia. PCE activity was positively correlated with serum triglyceride (r equals 0.540; p smaller than 0.001) and the prebeta electrophoretic fraction (r equals 610; p smaller than 0.001). The correlation with beta-lipoproteins was not significant. Ceruloplasmin levels were not significantly changed. It is suggested that elevation of PCE activity could be connected to mechanisms leading to an increased secretion rate of lipoproteins.


Studies on changes in lipid profiles of the rat gastric mucosa with stress ulcers.

Gastric mucosal lipid patterns were studied in normal and in restrained rats which developed mucosal erosions. Organic solvent extraction, silicic acid column, and thin-layer chromatography were employed to separate and gas-liquid chromatography and spectrophotometry to quantitize the individual lipid components. Chemical analysis revealed profound alterations in the mucosal lipid profiles of rats with gastric mucosal erosions. Cholesterol esters were markedly elevated, cholesterol, free fatty acids and phosphatidylethanolamine were markedly lower in the extracts of ulcerated tissue, and the titer of lysophospholipids was about eight times higher than in the controls. Lipid phosphorus was elevated in stressed rats. The amount of neutral glycolipids was similar for both groups, but tetra- and oligohexoside ceramides, which were present in the gastric mucosa of the control rats, were virtually absent from the ulcerated mucosa. In contrast to the alterations in lipid profile mentioned above, the total lipid weight per tissue preparation was very similar for the ulcerated and normal control groups.


Immunoreactive ACTH and cortisol plasma levels during pregnancy. Detection and partial purification of corticotrophin-like placental hormone: the human chorionic corticotrophin (HCC).

The high plasma cortisol and ACTH levels present in pregnant women as well as the non-parrallelism of their plasma extract dilution curves in comparison with the standard curve in the ACTH radioimmunoassay, are evidence for the presence of an ACTH-like substance during pregnancy which would interfere with the assay. Placental extracts were obtained by acid-acetone extraction, followed by partial purification with oxycellulose and by extraction with porous glass powder. A substance was detected which partially cross-reacted with synthetic human ACTH in the radioimmunoassay and which showed biological activity using the assay procedure described by Liscomb & Nelson. The data sustain the existence of an ACTH-like placental hormone: human chorionic corticotrophin (HCC).


Stature and longitudinal growth in thalassemia major. A study of 229 Greek patients.

A high percentage of Greek children with homozygous b-thalassemia were short and had a lag in growth, though some severely affected attained normal height. Bone age lagged significantly behind the chronologic age, even in those with normal height for their age. A longitudinal study demonstrated an initial normal growth velocity which decreased after the age of six for males and eight for females. No patient had a spurt of growth at adolescence. Height gain continued up to the age of 21 and then ceased. Statistical analysis with a multiple regression equation showed a significant relationship between the age and height of the patients. Low hemoglobin levels and severity of the disease seemed to hinder normal growth, but neither relationship was statistically significant.


Effects of clofibrate and a fat-modified diet on serum lipids.

The combined effect of clofibrate and a fat-modified diet was determined in 17 hyperlipidemic patients: 7 type IIA, 7 type IV, 2 type VI(IIB), and 1 type III. Control serum lipid levels and lipoprotein patterns and their alteration with a fat-modified diet had been determined previously 1/2 to 2 years for 3 patients, and 6 to 10 years for 14 patients. Two grams of clofibrate a day (0.5 gm four times daily) was taken along with the fat-modified diet for 2 to 6 months by 5 patients and for 2 years by 13 patients. The effect of clofibrate and a fat-controlled diet was also determined in 10 normolipidemic men who were subjects of an 18-day test in which the polyunsaturated fat diet was quantitatively prepared and eaten along with 2 gm clofibrate a day (0.5 gm four times daily). The effect of clofibrate on serum cholesterol levels was a further mean reduction in type IIA patients by 19 %, in type III by 23%, in type IV by 12%, in type VI by 7%, and in normolipidemic subjects by 8%. The extent of the additional serum cholesterol reduction with clofibrate in individual hyperlipidemic patients varied from +10% to minus 44% and was not related directly to the type of hyperlipidemia. The extent of reduction appeared related directly to the level of minus S 40-70 (similar to Sf 12-20) lipoprotein fraction in the control serum sample. Serum triglyceride levels were unaffected in type IIA and normolipidemic subjects. Serum triglyceride levels did not change consistently in the 2 type VI patients, rising by 11% in 1 and dropping by 31% in the other. Serum triglyceride levels were significantly (p = 0.001) and consistently reduced by 39% only in type IV patients.


Long-term therapy of viramin D-resistant richets with 25-hydroxycholecalciferol.

The long-term effects of the vitamin D metabolite, 25-hydroxycholecalciferol (25-HCC), were evaluated in 2 children with hypophosphatemic vitamin D-resistant rickets. Serial total balance studies demonstrated an apparent lack of correlation between the effects of the vitamin on intestinal absorption of calcium and phosphorus and both the onset of healing in 1 of the 2 patients treated with 5,000 to 7,500 u of the metabolite and the absence of demonstrable radiologic improvement in another patient in whom the final dosage was 20,000 u. per day. At first, the metabolite induced a positive calcium balance in both patients resulting largely from a reduction in intestinal calcium excretion. Despite a continued positive calcium balance, 1 of the 2 patients did not demonstrate further healing, while in the other patient healing was noted even when total calcium balance was negative. Serum phosphate levels did not return to normal in either patient, nor was phosphate excretion altered by 25-HCC. Serum alkaline phosphatase remained elevated in both. Serum immunoassayable parathyroid hormone levels were consistently normal to high-normal in the 2 patients throughout more than 24 months of observation. No instances of hypercalcemia and only occasional hypercalciuric episodes were noted.


Effects of high dosage delta-9-tetrahydrocannabinol on sleep patterns in man.

Electroencephalographic readings and eye movement were recorded in experienced marijuana users under placebo and tetrahydrocannabinol (THC). Four subjects were studied for 3 baseline nights, 3 nights under initial dosage of 70 mg/day, the last 3 nights of a 2-wk period of 210 mg/day, and the first 3 nights of withdrawal. Three other subjects were studied only during the latter 2 conditions. Administration of THC significantly reduced eye movement activity during sleep with rapid eye movements (REM) and, to a lesser extent, the duration of REM itself. Withdrawal led to increases above baseline in both measures but the "rebound" effect was greater for eye movement. Stage 4 sleep tended to increase on drug, but this effect was not statistically significant. On withdrawal, stage 4 sleep decreased significantly; this change was marked only on the first withdrawal night. The functional or biological significance of these changes is unclear. Nevertheless, these are the most marked effects of THC on brain electrical activity demonstrated thus far. Since its pattern of effects on sleep appears unique to THC, this drug may prove to be a valuable tool in the elucidation of the pharmacology of sleep. Possible relations between effects on sleep pattern and on behavior are discussed.


Bone metastases in Wilms' tumour.

In a series of 81 patients with a diagnosis of Wilms' tumour seen at the Hospital for Sick Children, Great Ormond Street, between 1960-1972, two patients developed histologically proven bone metastases. In one patient a lump on the head was the presenting symptom of his Wilms' tumour. In the second patient an isolated metastasis developed in the iliac bone 2 3/4 years after complete removal of an encapsulated Wilms' tumour. The bone lesion was outside the previously irradiated volume. Response to treatment was poor and both patients died with further widespread disease. A review of other series confirms the low incidence of bone metastases from Wilms' tumour and the poor prognosis of this complication. This contrasts with a high incidence of pulmonary metastases in which the response to treatment and subsequent survival is good. The value of skeletal surveys and scans in detecting this relatively rare site of metastases from Wilms' tumour is discussed.


Some urographic aspects of the horseshoe kidney anomaly-a review of 59 cases.

The urographic findings in 59 cases of horseshoe kidney are reviewed. Significant degrees of rotation are commoner and more pronounced in the left kidney in these cases than in the right kidney. This factor, rather than obliquity of the kidneys, is more likely to play a part in the development of upper urinary tract obstruction. Reversal of the normal direction of obliquity is not as common as is generally thought. The horseshoe kidney is, more often than not, mildly ectopic. Gross failure of ascent of the kidneys in the abdomen is uncommon. Evidence of pelvi-ureteric obstruction, especially hydronephrosis, occurred in just under half the cases. The reasons for this are discussed. The occurrence of other associated urinary tract anomalies, as well as acquired renal conditions, is considered.


Calcification in undifferentiated abdominal malignancies.

Four cases of undifferentiated abdominal malignancy are reported with a pattern of bizarre dystrophic calcifications. All of the cases had confusing histology and were long-term survivors; two being still alive. We feel that the radial, conglomerate masses of calcification, which do not conform to any organ, should suggest the diagnosis of an undifferentiated malignancy. The differential diagnosis and distinction from other causes of abdominal calcification is discussed.


Subcutaneous bone formation in the naevoid basal-cell carcinoma syndrome: normal urinary cyclic AMP response to parathyroid hormone infusion.

A patient with the naevoid basal-cell carcinoma syndrome had subcutaneous bone formation in the areas other than those occupied by the carcinomata. The patient did not show a significant phosphaturic response after parathyroid hormone infusion, but this extract produced a normal excretion of cyclic AMP. This proves that the subcutaneous bone does not signify an affinity to pseudo-hypoparathyroidism.


[Malignant epithelial liver tumours in children (author's transl)].

Ten cases of malignant epithelial liver tumours in children, observed during a 12-year period (1962 to 1974), are described. They represent one of the largest groups of this neoplasm reported in the German-language medical literature. All histological types of malignant epithelial tumours were found among the ten cases. The prognosis of these tumours in children is poor, but it can be improved by modern therapeutic methods. Once the diagnosis has been established histologically (which may be difficult), radical surgery is essential, but to be successful early diagnosis is needed. Judged by optimal results of treatment this personal series constitutes a negative selection.


[Long-term treatment of hyperlipoproteinaemia, types IIa and IIb, with etiroxate (author's transl)].

A synthetic thyroxine derviative, etiroxate (DL-alpha-methyl-thyroxine-ethylester-CG 635) was administered to 40 patients with type IIa and 19 with type IIb hyperlipoproteinaemia for up to 390 days, 40 mg daily. It achieved a constant decrease in cholesterol of 83 mg/dl (23%) in type IIa and 64 mg/dl (18%) in types the alpha-and pre bata-lipoproteins fell when the concentration was greater than or equal to 200 mg/dl and greater than or equal to 140 mg/dl, respectively, both fractions rising when the initial concentration was below the mentioned figures. There were only slight side effects, such as gastric and autonomic nervous system symptoms. No statistically significant increase in anginal symptoms was found, even in patients with known coronary insufficiency.


[Activity of enzymes of galactose metabolism in so-called congenital cataract (author's transl)].

Activity of galactokinase (69 subjects) and galactose-1-phosphate uridyl transferase (92 subjects) were measured in haemolysed blood from children (predominantly of school age) with congenital cataract. chi2 tests, gene-frequency determination and metabolic-kinetic studies indicated that the changes in the lens in congenital cataract are partly due to a manifest or latent disorder of galactose metabolism, in particular a glactose-1-phosphate uridyl transferase defect.


Formation of steroids by the pregnant mare. V. Metabolism of 14C-isopentenylpyrophosphate and 3H-dehydroisoandrosterone injected into the fetus.

A mixture of 1-14C-isopentenylpyrophosphate and 3H-dehydroisoandrosterone was injected into a horse fetus intramuscularly during laparotomy, after which maternal urine was collected for 4 days. Steroid conjugates in the urine were extracted with Amberlite XAD-2 resin, hydrolysed and separated into phenolic and neutral fractions. From the phenolic fraction estrone, 17alpha-estradiol, equilin and equilenin were isolated. Only estrone and 17alpha-estradiol contained both 3H and 14C, while the ring B unsaturated estrogens contained only 14C. From the neutral fraction 14C-labeled 3beta-hydroxy-5alpha-pregnan-20-one, 5alpha-pregnane-3beta,20beta-diol and 5alpha-pregnan-3beta, 20alpha-diol were isolated. These results demonstrate that the route of biosynthesis of both the ring B saturated and unsaturated estrogens is the same up to the stage of isopentenylpyrophosphate. Thus, the bifurcation in the classical pathway of steroid biosynthesis reported previously by us is occurring at a point after the formation of isopentenylpyrophosphate and prior to the formation of squalene.


Effect of PGE1 and TSH on cAMP-dependent protein kinase activity in the thyroid.

PGE1, like TSH, can increase cAMP-dependent protein kinase activity in calf thyroid slices. The intracellular levels of cAMP produced by either of these agents alone appeared to correlate well with the degree of kinase activation. PG synthesis did not appear to be necessary for TSH action in this system, since indomethacin, and inhibitor of prostaglandin synthesis, did not alter either cAMP levels or kinase activity in slices incubated with TSH. Both the cAMP level and kinase activity rose when a submaximally effective dose of TSH was added to a maximal dose of PGE1. However, neither the cAMP levels nor the kinase activity produced by a maximal dose of TSH was affected by the addition of PGE1.


Effect of ACTH and histamine stress on serum corticosterone and adrenal cyclic AMP levels in immature rats.

Adrenal cAMP and plasma corticosterone levels were determined in pre-weanling rats subjected to treatment with either ACTH (50 mU/rat) or histamine dihydrochloride (0.2 mg/g body wt). ACTH injection elevated both serum corticosterone and adrenal cAMP levels on all days tested. However, the ACTH-induced elevation of adrenal cAMP and serum corticosterone both diminished steadily from day 2 to day 8 and then increased from day 8 to day 16. Histamine injection resulted in elevated serum corticosterone levels in a pattern similar to that of the corticosterone response to ACTH. However, histamine injection did not result in any significant increase in adrenal cAMP from day 2 to day 10. From day 12 to day 16 the adrenal cAMP concentration rose steadily in parallel with ther serum corticosterone levels. These results indicate: (1) that a functional, ACTH-sensitive adenyl cyclase system is present in the adrenal gland of the immature rat, (2) that the responsiveness of this system diminishes during the first postnatal week before returning to its previous 2-day-old capacity by day 16, and (3) that during the first few days after birth, histamine stress results in elevated serum corticosterone levels without elevating adrenal cAMP levels.


The hypothalamo-neurohypophysial complex in organ culture: effects of metabolic inhibitors, biologic and pharmacologic agents.

Organ cultures of the guinea pig hypothalamo-neurohypophysial complex synthesize the octapeptide hormone, vasopressin, a specific product of the neurosecretory cells of the supraoptic nucleus. Inhibitors of both protein and RNA synthesis (cycloheximide and bromotubercidin respectively) were found to block vasopressin biosynthesis. In the presence of bromotubercidin, the apparent half-time of decline in the rate of hormone biosynthesis was about 28 h. Colchicine inhibited the distal transport of vasopressin into the posterior pituitary. Ultrastructural studies on colchicine-treated cultures indicated the neuronal stalks were intact and that neurotubules were still present. The narcotic drug, levorphanol at 10-7 M and 10-9 M was found to inhibit RNA synthesis by 20 percent. At these concentrations it had no demonstrable effect on vasopressin synthesis. Cultures established from animals that had been rendered tolerant to narcotics also had no observable alterations in vasopressin biosynthesis, although the initial pituitary vasopressin content of these cultures was reduced by about 35 percent. Various pharmacologic and biologic compounds were tested for their effects on vasopressin biosynthesis in organ cultures. Dibutyryl cyclic AMP, estradiol-17beta, nicotine, nerve growth factor (NGF), and pineal extract all had no effects under the present experimental regimen. Medium conditioned by the presence of fetal hypothalami of 40-55 days gestation produced a 2-4 fold increase in vasopressin biosynthesis in cultures established from adult animals. Medium conditioned by fetal cerebral cortex, liver, or hypothalamic tissue from fetuses of less than 33 days gestation did not have this stimulatory effect.


Biochemical comparison of estrogen receptors of the hamster hypothalamus and uterus.

Comparison of the soluble estradiol receptor proteins in the hypothalamus and uterus of the golden Syrian hamster revealed that both have sedimentation coefficients of 8S in a low ionic strength buffer and migrate similarly on analytical disc-gel electrophoresis in both 5% and 7% acrylamide gels. The competition of [3H]-17beta-estradiol binding by unlabeled estrogenic as well as nonestrogenic compounds was similar with the receptors present in the two tissues. The affinity constants for the hypothalamic and uterine receptors were 4.3 X 10-9M-1 and 1.6 X 10-10M-1, respectively; the number of binding sites when expressed on an equivalent protein basis (i.e. 1 mg/ml) was 0.18 X 10-10M and 3.1 X 10-10M, respectively. Identical amounts of soluble receptors with similar sedimentation characteristics were found in hypothalami from male and female animals gonadectomized 64 h prior to use. A titration of binding sites in the uteri of intact and ovariectomized animals revealed decreased binding in the supernatant preparation from intact animals, probably due to the presence of bound endogenous unlabeled hormone.


Role of adrenal 5alpha-reductase activity in determining the responsiveness of hypophysectomized rats to ACTH.

Studies were conducted to examine the contribution of adrenal 5alpha-reductase to the phenomenon of diminished adrenal "responsiveness" to ACTH after hypophysectomy in rats. Rats hypophysectomized for 1 week secreted small amounts of corticosterone (B), 5alpha-dihydrocorticosterone (DHB) and 3beta,5alpha-tetrahydrocorticosterone (R) acutely after ACTH. Adrenal reductase activity in vitro at that time was high. After replacement with ACTH for 24 h, B,DBH, and R secretion increased slightly. Reductase activity remained high. Treatment with ACTH for 2 days further stimulated secretion of DHB  and R but not B. Reductase activity was unaffected. Enzyme activity declined at 3 days concomitant with a proportionately greater increase in B than DHB and R secretion. Only after 7 days of ACTH did B secretion exceed DHB and R output. At that time, reductase activity was still lower. The results establish the functional significance of 5alpha-reductase activity as a regulatory site for the action of ACTH in determining the composition of adrenocortical secretory products in hypophysectomized rats.


Insulin, prostaglandin E1, PHENYLISOPROPYLADENOSINE AND NICOTINIC ACID AS REGULATORS OF FAT CELL METABOLISM.

Insulin-stimulated glucose oxidation was enhanced by the addition of N6 (phenylisopropyl)adenosine (PIA), prostaglandin E1 (PGE1) or nicotinic acid during a 1-h incubation of small amounts  5-8 mg/ml) of rat fat cells. Basal lipolysis was appreciable if low numbers of fat cells were incubated per ml. Insulin inhibited basal lipolysis at 20 to 50 mugU/ml and abolished lipolysis of 100 mugU/ml was present over a l-h period. However PIA, PGE1 or nicotinic acid potentiated glucose oxidation due to the 100 mugU/ml dose of insulin indicating that these agents are not increasing glucose oxidation solely as a result of an inhibition of lipolysis. PIA, PGE1 and nicotinic acid acted synergistically with insulin in stimulating glucose oxidation and inhibiting lipolysis in the presence of norepinephrine. Insulin was unable to decrease basal cyclic AMP accumulation or the increase in cyclic AMP seen with norepinephrine and theophylline after various time periods (2 to 60 min) but PIA, PGE1 and nicotinic acid were able to inhibit cyclic AMP accumulation at all times tested.


Cyclic 3', 5'-adenosine monophosphate levels in separated bone cells.

It has been shown that both parathyroid hormone (PTH) and calcitonin (CT) increase the concentration of cyclic 3',5'-adenosine monophosphate (cyclic AMP) in skeletal tissue. Since these two hormones have opposing actions on bone resorption, it has been postulated that the cell types upon which the hormones act in skeletal tissue may be different. We have previously shown that osteoblasts and osteocytes contain adenylate cyclase responsive to both PTH and CT whereas the enzyme prepared from periosteum and marrow cells did not respond to either. To further examine the postulate, the effect of these hormones on the concentration of cyclic AMP was determined. Bovine PTH (5U/ml equal to 4.2 times 10-7M) significantly increased the concentration of cyclic AMP/mug DNA in periosteum, osteoblasts, and osteocytes but not in marrow cells. Porcine CT (0.5 U/ml equal to 8.0 mug/ml approximately equal to 3.0 times 10-6M) significantly increased the concentration of cyclic AMP/mug DNA in periosteum, osteoblasts, osteocytes, and marrow cells. Adenylate cyclase activity prepared from periosteum and marrow cells was re-examined using EGTA and DMSO to improve enzyme stability and activity. Wth these additions PTH  (5 U/70 mul) increased activity of the preparation from periosteum, and CT (0.5 U/70 mul) increased activity from marrow cells and periosteum. These studies provide evidence that: 1) periosteum, osteoblasts, and osteocytes respond directly to both PTH and CT and 2) marrow cells respond to CT and not PTH.


Effects of thyroid and ovaries on prolactin binding activity in rat liver.

125I-radiolabeled ovine prolactin (oPRL) binding activity was measured in microsomal membranes of liver tissue from intact, ovariectomized, ovariectomized-thyroidectomized, and ovariectomized-thyroidectomized rats injected with thyroxine (T4) or estradiol benzoate (EB). Thyroidectomy and ovariectomy each reduced PRL binding activity in liver tissue significantly. The combination of ovariectomy and thyroidectomy decreased PRL binding activity more than thyroidectomy or ovariectomy alone. Doses of 2.5 mug or 10 mug T4/100 g BW daily returned PRL binding activity in the thyroidectomized rats to intact control values, and in the ovariectomized-thyroidectomized rats to the ovariectomized values. A dose of 2 mug EB/rat increased PRL binding activity significantly in ovariectomized-thyroidectomized rats, and a combination of 2 mug EB and 2.5 mug T4/100E that of intact controls. Scatchard analysis showed that ovariectomy and thyroidectomy decreased the number of PRL binding sites in the liver as compared to those in intact controls or in ovariectomized-thyroidectomized rats treated with EB and T4. It is concluded that the thyroid and ovaries are important regulators of PRL binding activity in the liver of the rat.


Soluble and membrane-bound enzyme-active antigens of rat-liver lysosomes.

Secondary lysosomes were isolated from rat liver and separated into a soluble and a membrane fraction. Plasma membranes and microsomes were also isolated and antisera against the various fractions were prepared in rabbits. Lysosomal content and detergent-solubilized membrane fractions were analysed in two-dimensional immunoelectrophoresis (crossed immunoelectrophoresis). The immunoprecipitates were stained by histochemical procedures for different enzyme activities such as phosphatases, non-specific esterase, arylsulphatase, glycosidases and L-leucyl-beta-naphthylamidase. When lysosomal content was tested against its corresponding antiserum, 17 different precipitates could be seen. Most of the enzyme activities tested were shown to reside separately in one or a few precipitates each. In contrast, when the membrane extracts were investigated, a more polymorphic pattern of enzyme-active precipitates appeared. Thus, when lysosomal membrane extracts were reacted with homologous antiserum 11 precipitates with acid phosphatase activity were obtained. Several of the antigens were electrophoretically different and immunologically non-identical. As expected from the biology of secondary lysosomes, many of their antigens were also found in microsomes and/or plasma membranes, but several antigens unique for lysosomes were detected concomitantly. Closer analysis of these results indicated that several seemingly identical enzyme-active proteins occurred both in soluble and membrane-associated forms. However, while many of the membrane antigens expressed 2-4 different enzyme activities, only one activity was detected in individual precipitates of the lysosomal content. Thus, acid phosphatase activity was found together with esterase activity in three membrane-associated antigens. The precipitates formed by two of these also stained for arylsulphatase and nucleoside tri-, di- and monophosphatase activities. L-Leucyl-beta-naphthylamidase activity was found in one additional acid-phosphatase-active precipitate.


Crystalline adenylate kinase from carp muscle.

1. The concentration of adenylate kinase in carp muscle is about 0.3 mg/g. An improved isolation procedure makes use of a dilute solution of the substrates, ATP and AMP, to elute the enzyme from a phosphocellulose column in overall yields of 60% before crystallization. By the hexokinase--pH-stat assay the specific activity is 3550 units/mg. The preparation has been found to be essentially homogeneous by dodecylsulfate gel electrophoresis, isoelectrofocusing and gel filtration. 2. The molecular weight has been determined to be 22000 by several methods. The absorbance of a 1% solution at 280 nm is 6.9 and the isoelectric point by electrofocusing is pH 5.9. 3. The crystals of carp adenylate kinase have the space group P4-1-22 or P4-3-22. 4. The amino acid composition has been determined. There is no tryptophan, no cystine. There is one amino acid residue each of cysteine and histidine which are at or close to the catalytic center. 5. Several peptides derived by tryptic hydrolysis have been isolated and identified with corresponding peptides of porcine adenylate kinase. Consideration is given to histidine and cysteine being a part of the active site.


Premature induction of glucokinase in the neonatal rat by thyroid hormone.

1. It was shown that the development of liver glucokinase in the rat coincided with a peak in the levels of circulating thyroid hormone at about the 16th postnatal day. 2. Administration of thyroid inhibitors blocked the development of the enzyme and administration of thyroid hormone restored activity to normal levels. 3. Glucokinase could be induced prematurely as early as the 2nd postnatal day by the administration of thyroid hormone followed by daily injection of glucose (10 mg/g body weight). 4. Glucocorticoids and corticotropin failed to induce glucokinase activity prematurely. 5. The postnatal increase in circulating thyroid hormone levels together with increased intake of carbohydrate at weaning may be the normal physiological stimulus for induction of this enzyme.


The hormonal control of activity of skeletal muscle phosphorylase kinase. Amino-acid sequences at the two sites of action of adenosine-3':5'-monophosphate-dependent protein kinase.

Two tryptic phosphopeptides containing the sites on the alpha and beta subunits of phosphorylase kinase which are phosphorylated by protein kinase, dependent on adenosine 3':5'-monophosphate (cyclic AMP), have been isolated and their amino acid sequences have been determined. 32P-labelled phosphorylase kinase, containing 1.9 mol phosphate per mol enzyme, was digested with an equimolar quantity of trypsin for 2.5 min at pH 7.0, 20 degrees C. This treatment released nearly all the 32P radioactivity associated with the beta subunit as trichloroacetic-acid-soluble material. Only a small proportion of the 32P radioactivity associated with the alpha subunit was solubilised, the remainder being removed in the trichloroacetic acid pellet. The beta-subunit tryptic phosphopeptide was completely resolved from traces of the alpha-subunit phosphopeptide by gel filtration on Sephadex G-25. Further purification by peptide mapping separated the phosphopeptide into four components, each derived from the same nine-amino-acid segment of the betachain, which was found to possess the sequence: Gln-Ser-Gly-Ser(P)-Val-Ile-Tyr-Pro-Leu-Lys. The four components were produced by the partial cyclisation of the N-terminal glutaminyl residue, and by the presence of two alleles for the beta subunit in the rabbit population, which led to a valine-isoleucine ambiguity. The alpha-subunit phosphopeptide was liberated from the trichloroacetic acid pellet by redigestion with trypsin. It was the largest component in the digest which remained soluble in 5% trichloroacetic acid, and obtained in a highly purified form by a single filtration on Sephadex G-50. The peptide comprised 39 amino acids of which nine were serine and three were threonine residues. Only one residue, the serine at position three from the amino terminus, was phosphorylated. The amino-terminal sequence of the peptide was shown to be: Arg-Leu-Ser(P)-Ile-Ser-Thr-Glu-Ser-Glx-Pro-Asx-Gly. The sequences confirm the stoichiometry of the reaction and the absolute specificity of cyclic-AMP-dependent protein kinase for just two of the 200 serine residues in the enzyme. These results and an inspection of the rate of phosphorylation of a number of skeletal muscle proteins, including each enzyme of the glycolytic pathway, lead to the conclusion that cyclic-AMP-dependent protein kinase is an extremely specific enzyme. The molecular basis of this specificity is discussed.


Lipoprotein of the density 1.006-1.020 in the plasma of patients with type III hyperlipoproteinaemia in the postabsorptive state.

Using a rate zonal ultracentrifugal technique in 10 patients with type III hyperlipoproteinaemia, very low density lipoproteins (VLDL) (d less than 1.006, S(f) greater than 20), an intermediate lipoprotein density class with a S(f) range of 15 - 20 (Lp III), and low density lipoproteins (LDL) (S(f) 6 - 8) could be isolated from the plasma. The Lp III was found as a symmetrical peak between VLDL and LDL; its plasma concentration was 248 mg/100 ml plasma (mean), 92 - 469 mg/100 ml plasma (range); the Lp III/LDL ratio was 0.65 (mean), 0.25 - 2.3 (range); the chemical composition w/w was 35.8 percent total cholesterol, 19.3 percent triglycerides, 24.3 percent phospholipids, and 20.4 percent protein (mean). In agarose gel electrophoresis, the Lp III migrated faster than LDL, but slower than VLDL. By means of a double immunodiffusion technique and immunoelectrophoresis, apo-Lp B and apo-Lp C were detectable. In an angle head rotor at 1.4 x 10-8 g. min. Lp III can be separated from VLDL at a solvent density of 1.006, and from LDL at a density of 1.025. In the postabsorptive state in one patient with type I hyperlipoproteinaemia, five with type II and 20 with type IV, and in 50 healthy persons of both sexes, this Lp III peak was not found. The finding of a high concentration of Lp III and a rather low concentration of LDL in the plasma of patients with type III hyperlipoproteinaemia can be regarded as further evidence for an impaired catabolism of VLDL to LDL at Lp III ("intermediate" lipoprotein).


A potent depressant action of adenine derivatives on cerebral cortical neurones.

Adenosine and several adenine nucleotides depress the excitability of cerebral cortical neurones, including identified Betz cells. Cyclic 3',5'-adenosine monophosphate was a less effective depressant than various other adenine nucleotides, including cyclic 2',3'-adenosine monophosphate. Adenine and inosine had only weak depressant activity. An initial excitant action of ATP was observed on several neurones. This was succeeded by a depressant effect when the application of ATP was terminated.


Voltage transients in neuronal dendritic trees.

An analytical method is outlined for calculating the passive voltage transient at each point in an extensively branched neuron model for arbitrary current injection at a single branch. The method is based on a convolution formula that employs the transient response function, the voltage response to an instantaneous pulse of current. For branching that satisfies Rall's equivalent cylinder constraint, the response function is determined explicitly. Voltage transients, for a brief current injected at a branch terminal, are evaluated at several locations to illustrate the attenuation and delay characteristics of passive spread. A comparison with the same transient input terminal input, the fraction of input charge dissipated by various branches in the neuron model is illustrated. These fractions are independent of the input time course. For transient synaptic conductance change at a single branch terminal, a numerical example demonstrates the nonlinear effect of reduced synaptic driving potential. The branch terminal synaptic input is compared with the same synaptic conductance input applied to the soma on the basis of excitatory postsynaptic potential amplitude at the soma and charge delivered to the soma.


Motoneuron dendrites: role in synaptic integration.

Dendrites constitute over 80 per cent of the receptive surface area in cat motoneurons. Calculations based on matched electrical and gemoetrical measurements in these neurons indicate that the specific resistance of dendritic membranes in resting motoneurons is at least 2,000 ohm-cm2. When the specific membrane resistance is this high, even the most distal dendritic synapses can contribute significantly to the depolarization of the soma, and hence influence the rate of action potential generation. However, dendritic membrane resistance depends strongly on the level of background synaptic activity. The conductance changes associated with excitatory synaptic activity on a dendrite can be great enough to reduce significantly both the excitatory synaptic driving potential and the effective membrane resistance on that dendrite, and thus greatly reduce the effectiveness of synapses on the dendrite. Inhibitory synaptic activity produces an even greater reduction in dendritic membrane resistance. Thus the relative effectiveness of dendritic synapses depends on the type, distribution, and intensity of background synaptic activity, as well as on dendritic geometry and resting membrane properties.


Tumour cell destruction by macrophages from conventional animals.

Peritoneal exudate cells (PEC) from conventional donors displayed in vitro the cytotocix effects on the rat sarcoma cell lines LWI3K2, LW13K3, LW13K4 and RSL but they were not cytotoxic to fibroblasts derived from normal rats of the same strains. PEC were also cytotoxic to human urinary bladder carcinoma cells (T-24) grown in vitro. Cytotoxicity was detectable already at the effector to target cell ratio of 1 : 1, and the effector cells from syngeneic, allogeneic and xenogeneic donors were equally effective. The cytotoxic effect was apparently exerted by macrophages because the addition of lymphocytes from PEC donors did not change the target cells. The addition of serum from PEC donors to a mixture of target and effector cells protected the tumour cells from the destruction by macrophages. Cytotoxicity was blocked by serum from all PEC donors tested (syngeneic, allogeneic and xenogeneic with regard to the target cells) but not by foetal calf serum.


Persisting enhanced proinsulin-insulin and protein biosynthesis (3H-leucine incorporation) by pancreatic islets of the rat after glucose exposure.

Pancreatic islets isolated from rats infused with glucose for twenty-four hours incorporated 3H-leucine into protein at higher rates than islets isolated from normoglycemic rats. Incorporation into proinsulin-insulin showed a thirteenfold increase. The effect on other islet proteins was fourfold. Exposure of islets from normoglycemic rats to high glucose in vitro for twenty and ninety minutes and subsequent incubation with 3H-leucine at low glucose showed a twofold and fivefold increase in proinsulin biosynthesis. In the in vitro system pre-exposure of the islets to mannose and dibutyryl-cyclic AMP induced a much smaller increase in proinsulin biosyntheses.


Pyrimidine biosynthetic enzymes in rat intestine after small bowel resection.

After small bowel resection in the rat, mucosal hyperplasia and an increase in nucleic acid synthesis and cell proliferation occur in remaining small intestine. Male Sprague-Dawley rate underwent resection of 50 cm of proximal or distal intestine or sham operation. One month and 6 months after surgery, aspartate transcarbamylase, dihydroorotase, and uridine kinase were assayed in whole mucosa, and in some instances, in crypt mucosa ffrom the remaining intestinal segment. In control bowel, enzyme activity was significantly greater proximal compared with distal segments. One month after proximal or distal resection, mucosal enzyme activity per cm of gut was greater in the remnant bowel compared with controls. There was no such difference at 6 months. Specific enzyme activity of whole mucosa did not increase after resection because the assay was influenced by the disproportionately large contribution of villous protein. Specific enzyme activity (including thymidine kinase) of isolated crypt mucosa was significantly increased 1 month after distal resection. In addition, [3H]thymidine uptake into DNA of crypt mucosa from proximal remnants was also significantly increased. These results indicate that after small bowel resection, the enzymes of pryimidine biosynthesis increase in remaining bowel and parallel the accelerated rate of cell proliferation.


Response of camels to intradermal inoculation with smallpox and camelpox viruses.

Young camels were inoculated intradermally with either camelpox or smallpox virus and the courses of infection, including serological response, were compared. Camelpox virus was highly infectious; generalized disease resulted which was transmitted naturally to contact animals. Smallpox virus produced only transient lesions at the inoculation site and a less marked serological response. Nevertheless, the camels inoculated with smallpox virus subsequently resisted a severe challenge with camelpox virus, and the possibility that limited replication of smallpox virus took place is discussed. The differences demonstrated between the behavior of the vituses is discussed in the light of their otherwise close relationship and the limited information available about camelpox infections in man.


Determination of toxin-induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts.

Human diploid lung fibroblasts were treated with cytolytic bacterial toxins and the nature of the membrane damage was investigated. [3H] uridine was used for differential labeling of cytoplasmic components of small or large molecular size. Two principal size categories were achieved by labeling the fibroblasts in either early growth phase or stationary phase, a high-molecular weight ribonucleic acid label and a low-molecular-weight nucleotide label. The size of the labeled molecules was determined by perchloric acid precipitation and gel chromatography. Leakage of labeled molecules of different size indicated the size of the "functional pores" in the plasma membrane caused by the test substance. The nonionic detergent Triton X-100 produced large functional pores in the fibroblast membrane as evidenced by rapid leakage of both large and small labeled molecules. Theta-toxin from Clostridium perfringens and the polyene antibiotic filipin both gave rise to considerably small functional pores in the plasma membrane. Although small molecules easily passed the treated membrane, large molecules could not escape from the cells even after prolonged treatment with these substances or by increasing their concentration. By the contrast, the leakage profiles obtained with melittin from bee venom or with delta-toxin from Staphylococcus aureus in each case suggested the formation initially of pores of intermediate size that increased upon prolonged incubation or when higher concentrations were used.


Cytomegalovirus infection in a volunteer blood donor population.

Among 223 volunteer blood donors who were studied for evidence of cytomegalovirus (CMV) infection, 58 percent had complement-fixing antibody and 59 percent had indirect hemagglutinating antibody to CMV. No virus was isolated from any donor's washed leukocytes or leukocyte-rich plasma in fibroblast monolayer culture. In seven asymptomatic donors (3 percent), CMV was recovered from urine cultures obtained at the time of blood donation. However, at the time of reexamination, viruria was no longer present and serum antibody titers had not changed. In the three patients studied who received blood from three of the cytomegaloviruric donors, serological evidence of CMV infection developed (fourfold or greater indirect hemagglutinating antibody rise), and one recipient also developed cytomegaloviruria; no illnesses was associated with these infections. Further study is needed to establish that the detection of viruria in donors may identify potentially infective blood.


Lysis of herpesvirus-infected cells by immune spleen cells.

Spleen cells from herpes simplex-infected mice have been shown to lyse 51Cr-labeled virus-infected target cells. The cell-mediated lysis was shown to be antibody dependent but not involving adherent cells. Lysis of infected cells by this mechanism may be one form of host defense in infection by some viruses.


Bovine parainfluenza type 3 virus infection: virus replication in bovine embryonic cell cultures and virion separation by rate-zonal centrifugation.

Replicative sequences of a bovine strain of parainfluenza type 3 virus in bovine embryonic kidney and spleen cell cultures were investigated by light and fluorescence microscopy and by ultrathin section and negative-contrast electron microscopy. Observations from light and fluorescence microscopy showed that intracytoplasmic inclusions were detected as small granules surrounding the nuclei of more than 90 percent of the cell population by day 2 postinoculation. With the increase of postexposure times, these inclusions coalesced into larger bodies which occupied large portions of the cell. Ultrastructurally, the first sign of virus development was the appearance of aggregates of viral nucleocapsids in the vicinity of the nucleus. With the concomitant accumulation of viral nucleocapsids in the cytoplasm, the virus maturation was expressed by budding processes through the cell membrane into round, oval, or elongated forms. Eosinophilic inclusions were demonstrable in many mitotic cells. Ultrastructurally, these cells were observed to produce virus particles by a process identical to that of resting cells. Virions, prepared from infected culture fluid and negatively stained, appeared to be pleomorphic and their diameter ranged from 200 to 600 mm. The virions were separated, by rate-zonal centrifugation, into two subclasses in a sucrose gradient (15 to 60 percent, wt/wt). The slowly sedimenting virions had a density approximately 1.20 gm/cm3 and an average size of 200 nm in diameter, whereas the faster-sedimenting virions had a density of 1.24 gm/cm3 and average diameter of 400 nm.


Bovine parainfluenza type 3 virus infection: ultrastructural aspects of viral pathogenesis in the bovine respiratory tract.

After aerosolization of a bovine strain of parainfluenza type 3 virus, the pathogenesis of the virus was followed from the trachea to the bronchioalveolar compartments of the lung of colostrum-free calves and of conventionally reared calves during a 5- to 12-day postexposure interval.  By tissue titration, plaque assay, and electron microscopy, it was found that virus infection could be established in colostrum-free calves as well as in conventionally reared calves, even though sequential changes of virus replication were observed mainly in the infected colostrum-free calves during the 5- to 6-day postexposure periods. Electron microscopy demonstrations of (i) aggregates of viral nucleocapsids in the cytoplasm, (ii) alterations of cilia and basal bodies, (iii) dissolution of cytoplasmic membranes, and (iv) the shedding of virus into luminal spaces confirmed that epithelial cells of the respiratory tract were the primary target cells for the virus replication leading to cell destruction. These observations revealed further that productive infection was more efficient in the bronchioalveolar regions than in the tracheal regions, although large aggregates of viral nucleocapsids and destructive changes were more pronounced in the tracheal epithelium. The finding that parainfluenza type 3 virus replicates in the alveolar type II cells suggests that changes in surfactant production may occur during the peak of infection of these cells. The demonstration of virus budding through the basement membrane of small bronchioles and the presence of virus particles in the interstitial regions imply that one of the host defense lines, the basement membrane, may be impaired by virus invasion.


Comparative studies of Visna and Maedi viruses as antigens.

Rabbits were immunized with purified visna and maedi viruses, using complete Freund adjuvant, in footpad and intramuscular sites. The resulting antisera and their isolated immunoglobulin G (IgG) and M (IgM) classes were evaluated by tanned-cell passive hemagglutination (PHA), complement fixation, gel diffusion, and virus neutralization tests. Early, intermediate, and late bleedings showed increasingly high antibody activities by the PHA, complement fixation, and gel diffusion tests. The activities were associated mainly with the IgG class, although low, but significant activities were also found in the IgM class, as detected by PHA and complement fixation tests. Both antibody classes appeared at the same time during the course of immunization. The viruses, when tested against specific rabbit anti-visna and anti-maedi sera in gel diffusion tests, showed the presence of one to two precipitin lines depending upon the antibody concentration of the sera. A low amount of neutralizing activity was demonstrated in late bleedings but was not seen in earlier bleedings. The neutralizing activity against visna virus in immunized rabbit sera was found to be associated only with the IgG class. Visna and maedi viruses appeared to be immunologically identical when examined in gel diffusion tests and showed the same degree of inhibition when compared in passive hemagglutination inhibition test using antisera made specific for each virus.


Lanthanum inhibition of Vibrio cholerae and Escherichia coli enterotoxin-induced enterosorption and its effects on intestinal mucosa cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels.

Several trivalent cations, including lanthanum (La3+), inhibited the secretion (enterosorption) induced by the enterotoxins of Vibrio cholerae and Escherichia coli in the rabbit ileum in vivo. High concentrations (greater than 10 mM) of La3+ were required to inhibit cholera enterotoxin (CE)-induced enterosorption, probably because of the adsorption of the La3+ often potentiated the CE-induced enterosorption. If luminal La3+ exposure followed CE exposure, some recovery of the enterosorptive response was observed. The longer the lag between the CE exposure and the La3+ exposure, the greater was the recovery of the enterosorptive response. Lanthanum inhibited HCO3- secretion more than Cl- secretion. By altering the luminal fluid pH at the time of La3+ exposure, it was found that La3+ was adsorbed to negatively charged luminal sites, having an apparent pK between 2.5 and 3.0. Although La3+ antagonized the enterosorptive response to CE, it mimicked rather than antagonized the cyclic adenosine 3',5'-monophosphate elevation and cyclic guanosine 3',5'-monophosphate depression induced by the toxin. It is therefore concluded that the La3+ inhibition of the CE-induced enterosorption must have occurred at a site following the generation of the cyclic nucleotides. Cholera enterotoxin caused complex time-dependent changes in the mucosal cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate levels, as revealed by studying tissue cyclic adenosine 3',5'-monophosphate/cyclic guanosine 3',5'-monophosphate ratios. The possible roles these two cyclic nucleotides may play in the pathogenesis of the cholera diarrhea are discussed.


Characterization of enterotoxin purified from Clostridium perfringens type C.

Enterotoxin produced by a sporulating culture of Clostridium perfringens type C, which had been isolated from a case of severe necrotic enteritis, was purified. The molecular weight was estimated to be 36,000 by gel chromatography on Sephadex G-100 and 33,400 by ultracentrifugation. The sedimentation coefficient S20,W was 2.92. The toxin protein exhibited unusual behavior on sodium dodecyl sulfate gels, and toxin aggregates having molecular weights of 68,000, 85,000, 105,000, and 140,000 were obtained. The purified enterotoxin often separated, apparently due to slight charge differences, into two protein bands on 7% polyacrylamide gels. Electrofocusing of enterotoxin on polyacrylamide gels gave an approximate isoelectric point of 4.3, with the enterotoxin being fractionated into four distinct protein bands. The specific toxicity of the enterotoxin was about 1,900 mouse mean lethal doses per mg of calculated nitrogen. The data obtained indicate that the enterotoxin from C. perfringens type C is identical in most respects to that obtained from type A strains. Whether or not this toxin plays a role in the necrotic enteritis caused by type C strains is unknown at present.


Theiler's virus infection in mice: an unusual biphasic disease process leading to demyelination.

An unusual biphasic central nervous system disease developed in 3-week-old Swiss outbred mice after intracerebral inoculation of the DA strain of Theiler's murine encephalomyelitis virus. Nine to 20 days postinfection 86% of mice became paralyzed, and approximately one-half of these animals survived. During this period neuronal necrosis and microglial proliferation were seen in thalamus, brainstem, and spinal cord. There was an initial phase of virus growth in spinal cord followed by persistent infection at a lower concentration. Virus antigen was readily found in the cytoplasm of neurons by immunofluorescent staining early in the course of infection, whereas after 30 days there was a paucity of cells containing virus antigen which were present only in the spinal cord white matter. Between 1 and 5 months, an intense mononuclear inflammatory cell lesion evolved in the spinal cord leptomeninges and white matter, which coincided with a mild gait disturbance in some surviving mice, and patchy demyelination was found in areas of inflammation. The acute gray matter pathology would appear to be the result of direct virus lytic effect. Although the late white matter lesion culminating in demyelination probably represents a cytocidal infection similar to the situation that exists in certain picornavirus carrier culture systems, a virus-induced immunopathological process merits further study.


In ovo assay for Marek's disease virus and turkey herpesvirus.

Marek's disease virus (MDV) and the turkey herpesvirus (HVT) may be assayed on the chorioallantoic membrane (CAM) of the chicken embryo after intravenous inoculation of chicken embryo fibroblasts (CEF) or chicken blood leukocytes infected with these viruses. Free HVT, MDV associated with Marek's tumor cells, and lymphoblastoid cell lines derived from Marek's tumors, may be assayed in the same way. The intravenous assay is quicker than the yolk sac assay and somewhat more sensitive than in vitro or conventional CAM assay after direct inoculation of the CAM. The optimal time for inoculation was day 10 of embryo incubation; therafter the log-10 CAM lesions decreased as a negative linear function of embryo age at the time of inoculation. The log-10 CAM lesions increased as a positive linear function of the time since inoculation. The optimal time for counts was day 5 after inoculation. The log-10 CAM lesions was a linear function of the log-10 cells in the inoculum; the slope was 1.0. Venous in ovo inoculation caused as increase in the weight of the spleen proportional to the number of CAM lesions. Repression of the splenomegaly, by prior X irradiation of the embryo, did not reduce the number of CAM lesions. Embryols from lines inbred for susceptibility to Marek's disease produced more CAM lesions than embryos from resistant lines. This difference did not depend on prior exposure of the mothers to MDV or HVT.


Mechanism of induction of cell-mediated immunity to virus infections: in vitro inhibition of intracellular multiplication of mouse adenovirus by immune spleen cells.

Mice were protected from lethal infection with mouse adenovirus (M-Ad) by adoptive transfer of immune spleen cells (ISC) that were prepared from mice immunized with M-Ad and not protected by sonicated ISC. However, a similar extent of protection was also observed by passive immunization with anti-M-Ad serum. In contrast, by in vitro experiments ISC were shown to be able to interrupt intracellular multiplication of M-Ad, whereas sonicated ISC, unimmunized mouse spleen cells, or anti-M-Ad serum were unable to do so. ISC were inhibitory in vitro when added within 12 h postinfection but not when added later. The inhibitory activity of ISC was regarded as due to cell killing by ISC, since the trypan blue exclusion test showed that above 80% of infected cells were killed by ISC even when 5'-fluorodeoxyuridine was added to the cells to block viral deoxyribonucleic acid synthesis, under which conditions control infected cells, to which ISC were not added or normal spleen cells were added, were kept alive at least for a few days. Kinetics studies in M-Ad-infected mice showed that the inhibitory activity of ISC became highest at 1 to 2 weeks postinfection and faded away thereafter in a few weeks, whereas serum antibody titer assayed by passive hemagglutination reached its peak level at about 4 weeks postinfection and declined gradually therafter.


Effect of silica on virus infections in mice and mouse tissue culture.

Silica injections of mice have been reported to kill macrophages, thus allowing herpes simplex virus (HSV) to spread rapidly and leading to an increased severity of HSV infection. Thus, silica presumably could be used to eliminate lactic dehydrogenase virus (LDV) (a model for slow viruses), which is known to multiply exclusively in macrophages. Contrary to expectation, it was found that the LDV titers were increased in silica-injected mice as compared to the titers in control mice. Counts of peritoneal cells at different periods after silica injection showed that silica-induced macrophage damage in vivo resulted in proliferation and migration of macrophages, thus providing additional target cells for LDV replication and leading to high LDV titers. In vitro, silica ingestion also damaged the macrophages, but since no replacement of cells could occur by infiltration, decreased LDV titers were found. Similar findings were obtained with HSV. It is suggested that all persistent viruses multiplying in macrophages will show a similar recrudescence under comparable conditions.


Detection of cellular hypersensitivity among multiple sclerosis (MS) patients to 6/94 virus; a parainfluenza type 1 isolate from MS brain tissue.

Multiple sclerosis (MS) patients and normal subjects were tested for cellular hypersensitivity, using the leukocyte migration inhibition test LMI), to a parainfluenza-1-virus (6/94), previously isolated from brain biopsy tissue of an MS patient. The response of MS patients as a group was not greater than that of normals to 6/94, but a significant increase in the response was observed among selected patients in the early active and chronic advanced stages of the disease. MS patients, however, did not differ from normal subjects in their cell response to Sendai virus, a similar parainfluenza type 1 virion used for comparison.


Isolation and characterization of the cyanogen bromide fragments of lobster arginine kinase (homarus vulgaris).

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).


Determination of the active portion of the folic acid molecule in cellular slime mold chemotaxis.

From earlier work it is known that folic acid attracts the amoebae of various species of cellular slime molds (11). Here we have tested a wide variety of pteridines, pyrimidines, and pyrazines to determine what part of the folic acid molecule is chemotactically active. It was shown that the activity lies in the pteridine ring itself. Furthermore, the cell-free supernatants of slime mold amoebae contain an enzyme that renders pterin and folic acid chemotactically inactive, which apparently increases the chemotactic sensitivity of the amoebae to those compounds. Despite the fact that slime mold amoebae secrete small amounts of folic acid-related compounds, there is no evidence that folates are acrasins; rather it is postulated that attraction to folates may be a food-seeking device for the amoebae which prey on folate-secreting bacteria in the soil.


Kynureninase-Type enzymes of Penicillum roqueforti, Aspergillus niger, Rhizopus stolonifer, and Pseudomonas fluorescens: further evidence for distinct kynureninase and hydroxykynureninase activities.

The kynureninase-type enzymes of three fungi and one bacterium were isolated and examined kinetically for their ability to catalyze the hydrolysis of L-kynurenine and L-3-hydroxykynurenine. The phycomycete Rhizopus stolonifer was found to contain a single, constitutive enzyme with Km for L-3-hydroxykynurenine and L-kynurenine of 6.67 times 10-minus 6 and 2.5 times 10-minus 4 M, respectively. The ascomycetes Aspergillus niger and Penicillium roqueforti each contain an enzyme, induced by L-tryptophan, with similar Km for L-3-hydroxykynurenine and L-kynurenine ranging from 5.9 times 10-minus 5 to 14.3 times 10-minus 5 M, as well as a constitutive enzyme with Km for the two substrates of similar to 4 times 10-minus 6 M and 10-minus 4 M. The bacterium Pseudomonas fluorescens has a single, inducible enzyme with Km for L-3-hydroxykynurenine and L-kynurenine of 5 times 10-minus 4 and 7 times 10-minus 5 M. In addition, significant differences in maximal velocities (Vmax) were observed in two cases. The Vmax of the inducible activity from P. fluorescens was 4.5 times greater for L-kynurenine than L-3-hydroxykynurenine, whereas the Vmax of the constitutive activity from R. stolonifer was 2.5 times greater for L-3-hydroxykynurenine. It is concluded (i) that the constitutive activities are hydroxykynureninases involved in the biosynthesis of nicotinamide adenine dinucleotide from L-tryptophan, (ii) that the inducible activities are kynureninases involved in the catabolism of L-tryptophan to anthranilate, and (iii) that R. stolonifer and P. fluorescens, respectively, carry the most specific examples of each type of enzyme.


Amino acid transport in membrane vesicles of obligately anaerobic Veillonella alcalescens.

Membrane vesicles of Veillonella alcalescens, grown in the presence of L-lactate and KNO-3, actively transport amino acids under anaerobic conditions in the presence of several electron donors and the electron acceptor nitrate. The highest initial rates of uptake are obtained with L-lactate, followed by reduced nicotinamide adenine dinucleotide, glycerol-1-phosphate, formate, and L-malate.. The membrane vesicles contain the dehydrogenases for these electron donors, and these enzymes are coupled with nitrate reductase. In membrane vesicles from cells, grown in the presence of nitrate, the dehydrogenases are not coupled with fumarate reducatase, and anaerobic transport of amino acids does not occur with fumarate as electron acceptor. Under aerobic conditions none of the physiological electron donors can energize transport. However, a high rate of uptake is observed with the electron donor system ascorbate-phenazine metho-sulfate. This electron donor system also effectively energizes transport under anaerobicconditions in the presence of the electron acceptor nitrate.


Intergration of the receptor for bacteriophage lambda in the outer membrane of Escherichia coli: coupling with cell division.

Induction of the synthesis of the receptor for phage lambda is obtained by adding maltose and adenosine 3'-5'-cyclic monophosphate to glucose grown cells of Escherichia coli. Bacteria induced for a short period of time were infected with a high multiplicity of phage lambda , and examined under the electron microscope. Only a fraction of the bacteria were seen to have adsorbed a large number of phage particles. The majority of such bacteria had a constriction indicating formation of a septum, and, in this case, the density of adsorbed particles was highest in the vicinity of the constriction. When found on bacteria showing no sign of septum formation, the adsorbed particles were asymmetrically distributed, one pole of the bacteria being more heavily covered with phage particles than the other. Such asymetrically covered bacteria are believed to have originated from cells which divided during the induction period. The results suggest that the receptor for phage lambda, a protein of the outer membrane, is integrated in the cell envelope during the last quarter of each generation and that the integration process is initiated in the vicinity of the forming septum.


Deletion of the Escherichia coli crp gene.

Spontaneous crp mutants Escherichia coli were selected from a strain that does not require 3',5'-cyclic adenosine monophosphate for CAP activity. Several deletions of the crp gene were characterized. The crp gene was not essential for growth of E. coli. crp mutations reduced the donor ability of Hfr strains.


Demonstation that thymine dimers are excised as oligonucleotides from specifically incised ultraviolet-irradiated deoxyribonucleic acid.

The average size of the dimer-containing acid-soluble oligonucleotides after incubation of specifically incised DNA with two dimer excising nuclease activities has been determined to be about 8 nucleotides.