Wilms' tumor: natural history and prognostic factors: a retrospective study of 248 cases treated at the Institut Gustave-Roussy 1952-1967. A statistical analysis of 248 histologically proven cases of Wilms' tumor treated at the Institut Gustave-Roussy, Villejuif, France, from 1952 to 1967, is presented. Two years recurrence-free survival and five years survival have been selected as prognostic criteria. Patients under two years of age have significantly fewer metastases and a better five-year survival rate than those over two. Stage is stronly correlated with recurrences and survival. Influence of tumor size, number of tumor nodules, tumor rupture, adhesions, regional lymph node involvement, and renal vein infiltration is shown. Detailed analysis of histopathologic features indicates that a favorable clinical course is significantly related to the number of different varieties of epithelial differentiation (tubular, glomerular, microcystic) found in any tumor, whatever the abundance of each of them. Presence of striated muscular cells, however abundant, does not influence survival or metastatic rates. Cases where irradiation given prior to surgery resulted in complete destruction of tumor cells, form a distinctive group of very unfavorable prognosis. Relapse of tumor occurs in 54% of cases, 10% presenting with metastases. Involved sites are listed. No correlation is found between date of metastases and other parameters. The three major prognostic factors of age, stage, histologic pattern, bear prognostic value separately. Stage and age are linked, as well as histology and age. Stage and histology are not linked. Age in itself therefore seems less important. It is concluded that these well-defining treatment for each individual case.


The transformation by simian virus 40 of cells from patients with mucopolysaccharidosis and from normal controls. Fibroblasts derived from individuals with mucopolysaccharidosis, an inborn error of metabolism, have been found to be more easily transformed by simian virus 40 than are cells derived from normal individuals. The increased susceptibility does not seem to depend upon changes in glycoprotein at the cell surface. Repeated observations were necessary to demonstrate these differences, and we do not believe that this test is suitable for routine screening for cancer susceptibility.


Characterization of microsomal methyl sterol demethylase in two Morris hepatomas. Previously, we reported that the rate of metabolism of methyl sterol intermediates of cholesterol biosynthesis by broken-cell preparations of Morriss hepatoma 7777 is very slow, whereas the intact tumors are known to synthesize cholesterol quite efficiently. Active preparations have now been obtained by substitution of pyrophosphate for phosphate buffer. Although substitution of pyrophosphate buffer markedly enhances microsomal methyl sterol demethylation rates 3- to 4-fold in hepatoma 7777, other microsomal enzymes and electron carriers in either liver or a more slowly growing hepatoma appear to be unaffected by pyrophosphate. Several properties of the active microsomal methyl sterol demethylase have now been compared for control rat liver, host liver, tumor 7777, and tumor 5123C. Conditions necessary for the assay of initial velocities of enzymic reactions in the tumor microsomes have been established with respect to the amount of protein, time-course, concentrations of cofactors and substrate, pH, and other variables. The K'm and the responses to the variables studied above are very similar for methyl sterol demethylase of microsomes isolated from control liver, host liver, tumor 5123C, and tumor 7777. The multienzymic demethylase in the various preparations has been found to be inhibited similarly by in vitro additions of cyanide, cytochrome c, and bile salts. Thus, the enzymes of the microsomal-bound 4-methyl sterol demethylase of cholesterol biosynthesis appear to be very similar in liver and these 2 Morris hepatomas. When xenobiotic inducers of microsomal oxidases, such as phenobarbital and methylcholanthrene, are administered to normal and tumor-bearing rats, elevated rates of methyl sterol demethylation are observed with isolated liver microsomes obtained from both normal and tumor-bearing rats. Similar increases are not observed in the tumors. Furthermore, daily administration of an intestinal bile acid sequestrant elevates hepatic methyl sterol demethylase, but statistically significant changes were not observed in tumors 7777 and 5123C. Since the enzymes of methyl sterol demethylase appear to be grossly similar in liver and these hepatomas, regulation of the activity of the multienzymic system contained in the tumors may be altered. On the other hand, these agents in vivo simply may not affect liver and the hepatomas similarly, due to a lack of uptake of the foreign substances by the tumor that has been transplanted to the thighs.


Carcinogenic activity of quinoline on rat liver. The effects of prolonged p.o. administration of quinoline or 2-chloroquinoline on rat liver were examined histologically. Hepatocellular carcinomas and hemangioendotheliomas were observed in the livers of rats fed a basal diet containing 0.05, 0.10, or 0.25% quinoline for about 16 to 40 weeks. However, no neoplastic changes were seen in the livers of rats fed a basal diet containing 0.05, 0.10, or 0.25% 2-chloroquinoline for 40 weeks. In groups that received low concentrations of quinoline, the incidences of hepatocellular carcinomas were higher and the incidences of hemangioendotheliomas were lower than in the group that received a high concentretion of quinoline. The liver tumors induced by quinoline were classified histologically as hemangioendotheliomas or hemangiosarcomas and trabecular hepatocellular carcinomas. Typical nodular hyperplasias were occasionally seen in the livers of rats treated with quinoline. 2-Chloroquinoline did not induce any nodular hyperplasia or other neoplastic changes, but it caused diffuse fatty changes of parenchymal cells. Oval cell infiltration and bile duct proliferation were slight or moderate. Cirrhotic changes were rare in the livers of rats treated with either quinoline or 2-chloroquinoline. The serum levels of glutamic oxaloacetic transaminase and alkaline phosphatase were slightly changed in groups treated with quinoline, but no other remarkable changes were detected.


Renal neoplastic response to leukosis virus strains BAI A (avian myeloblastosis virus) and MC29. Previous reports described the induction of avian renal neoplasms by leukosis virus strains BAI A [avian myeloblastosis virus (AMV)] and MC29, and illustrated morphological characteristics of the tumors. Continued studies in this work confirm evidence of the origin of the tumors from embryonal cells residual in the posthatched chick. The work further emphasizes differences in histopathology of the neoplasms caused by the two viruses and reveals differences in the histopathogenesis of the respective growths. Embryonal rests may consist of two types of cells, those of epithelial characteristics and a second element of differentiation between nephroblastema (mesenchyme) and epithelium and designated here as nephromesoblastoma. Infection by AMV induces tumors of epithelial characteristics and, in addition, derivatives of nephromesoblastoma consisting of cartilage, bone, areas of keratinization, and sarcoma. Keratinized structures in the nephroblastoma originate from nephromesoblastoma. In contrast, MC29 virus induces only epithelial growths representing principally aberrant and malformed glomerular and tubular structures with occasional cartilage derived from epithelial cells. MC29 tumors are completely lacking in nephromesoblastoma tissue and contain no bone, sarcoma, or keratinized formations. In MC29 tumors, occasional cartilage was derived from epithelium. Tumors caused by AMV exhibit the complex structure of nephroblastoma with all of the features of the growth in humans (Wilms' tumor). The neoplasms induced by both AMV and MC29 exhibit marked aberration, distortion, and malformation in the differentiation of the cells growing out from the embryonal rests representing rare  manifestations of cell genetic influence inherent in the primordial growth of nephroblastema. The results thus illustrate fundamental differences in cellular composition and capacity to respond to etiologically different leukosis viruses.


Estrogen and prolactin receptor concentrations in rat mammary tumors and response to endocrine ablation. Estrogen and prolactin receptor concentrations were measured in 24 carcinogen-induced rat mammary tumors and correlated with the tumor response to host ovariectomy or hypophysectomy. It was found that essentially all of the tumors contained some specific estrogen receptor, and all but three contained prolactin receptor. The values for each receptor comprised a continuum from very low to relatively high concentrations, suggesting that previous considerations of hormone dependence on the basis of presence or absence of hormone receptors may be oversimplified. The concentration of each receptor tended to be lower in the hormone-independent than in the hormone-dependent tumors, but there were a number of hormone-independent tumors with higher receptor levels than some of the hormone-dependent tumors had. A better correlation of tumor response to endocrine ablation resulted from a combination of the 2 receptor levels than from either receptor concentration alone. These results suggest that there is a complex relationship between mammary tumor response to endocrine ablatin and levels of estrogen and prolactin receptors and that some tumors may be dependent upon 1 or both of these hormones for growth.


Virion interaction in mouse lymphomas.

Virion interaction in mouse lymphomas. Virion expression in filtrates of lymphomas in AKR/J or C3H/HeJ mice was assayed by two techniques: (a) in vitro infectivity (XC assay), or (b) acceleration of oncogenicity in vivo (O assay). The two assays appeared to detect different virus populations or activities. This was shown when the same filtrates were tested in parallel by the XC and O tests and opposite results were obtained on the two assays, e.g., XC+ 0- or XC- O+. It was postulated that two viruses, termed "XC+" and "O" to correspond to the assays used to detect them, were involved in oncogenesis. When lymphomas originally virus induced were passaged by cells in 2- to 3-month-old syngeneic AKR or C3H mice, oncogenic activity of filtrates of these transplanted lymphomas decreased as cell passages increased. These filtrates from C3H lymphomas also had decreased XC activity as cell passages increased. Normal lymphoid tissue from 1- to 5-month-old AKR mice was XC+ O-, while from C3H mice it was XC- O-. Thus, the two strains modified activities lacking in their normal tissues. Filtrates highly oncogenic in XC+ newborn AKR mice were oncogenic in C3H newborn mice only when sufficient XC+ virus was in the inoculum received by the XC- C3H mice. Thus the XC+ virus appeared to play a synergistic role with a postulated O virus in oncogenesis.


Characteristics of a surface-adherent subline derived from Friend erythroleukemia cells in continuous suspension culture. A surface-adherent cell population developed spontaneously from Friend erythroleukemia cells (FLC745) which previously had grown continuously in suspension cultures. The adherent cells have been transferred through more than 60 passages and apparently represent a stable variant, herein designated FLC745-Ad. The cell size, chromosome complement, and tumorigenicity in DBA/2 mice were similar to the parental FLC745 line, although the FLC745-Ad cultures grew at a slightly slower rate and synthesized less hemoglobin in response to dimethyl sulfoxide. Production of C-type particles was abundant in both suspension and adherent cells, but the latter appeared to contain more intracisternal A-type particles. Scanning electron microscopy disclosed that most adherent cells were globoid and nonspreading. They attached to the substrate by a circumferential lamellar skirt of unusual breadth. Adhesion of the FLC745-Ad cells was not influenced by serum concentration, trypsinization, 5-bromodeoxyuridine, or N6, O2'-dibutyryl adenosine 3':5'-monophosphate. Treatment with cytochalasin B increased the cell size and the area of cell attachment.


Amino acid and hexose transport of normal and simian virus 40-transformed human cells. Studies on amino acid and hexose transport were performed on human WI-38 cells and WI-38 SV40-transformed cells (VA13A and VA13-2RA). Depending upon cell line or conditions, either no difference or a relative decrease in initial uptake by transformed cells was found. Under similar growth conditions, transformed hamster cells (PyBHK-21/C13) had increased uptake, compared with the normal hamster cells (BHK-21/C13). The normal and transformed human cells were also similar in sialic acid content and agglutinated when treated with concanavalin A.


Expression of virion and tumor-specific antigens on the surface of chick embryo cells infected with strain MC29 avian leukosis virus. The immunological properties of the surface of chick embryo cells infected with strain MC29 avian leukosis virus were investigated by immune electron microscopy in conjunction with antiviral and anticellular immune sera. When samples were taken at sequential times after infection, it was found that cells stained strongly for viral antigen very early after infection. Staining reached a minimum 6 to 12 hr after infection and then increased to maximum at 15 hr after infection, remaining at this level at later times. Anticellular immune sera showed the presence of a distinct antigen or antigens which appeared concurrently with morphological alteration of the infected chick cells. This antigen was cross-reactive or identical to an antigen appearing on the surface of chick embryo cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus.


Changes in serum putrescine and spermidine levels following local radiation to hepatoma 3924A of the rat. Local irradiation of tumors of rats bearing 3924A hepatomas resulted in more than a doubling of the putrescine levvel and nearly a doubling of spermidine concentration in the serum within 12 hr. Within 24 hr, the putrescine concentration had increased four-fold in the serum, along with a continued increase in the spermidine concentration. The decrease in the spermidine concentration of the tumor paralleled increased levels of spermidine in the serum, whereas the concentrations of polyamines in the liver were unchanged. These changes are similar to previously reported changes in the spermidine concentration in sera and in tumors following the administration of a single dose of 5-fluorouracil to rats with 3924A hepatomas. Since local irradiation was confined to the tumor, we conclude that the increases in putrescine and spermidine detected in the serum are derived from the tumor tissue with no involvement of the host tissues.


Isozyme patterns of glycogen phosphorylase in rat tissues and transplantable hepatomas. Isozyme patterns of glycogen phosphorylase in the Morris and Yoshida hepatomas were compared electrophoretically and immunochemically with those in rat tissues during development. A 3rd phosphorylase isozyme, observed previously in hepatomas and fetal tissues by isoelectric focusing and by immunochemical titration, was also separated by polyacrylamide disc gel electrophoresis. It was observed commonly in various rat hepatomas, together with variable activities of the liver type, depending on the degree of differentiation. This isozyme is nearly the sole type in placenta and early embryo, and in liver and skeletal muscle it is replaced during fetal development with the organ-specific liver and muscle type. In adult rat brain the fetal type is retained at low levels, together with the muscle type. In kidney, spleen, testis, uterus, lung, and stomach, the fetal type is present together with the liver type. This isozyme in hepatomas and adult brain is identical with the fetal-type, as determined by Ouchterlony double diffusion and activity inhibition tests. Thus, it is considered to be a prototype whose appearance in hepatomas is one of many known examples of fetal protein expression in cancer. In some poorly differentiated hepatomas, the liver-type isozyme migrated slightly more slowly in polyacrylamide gel but could not be distinguished from the liver isozyme immunochemically.


Hepatocellular carcinomas in acatalasemic mice treated with nafenopin, a hypolipidemic peroxisome proliferator. The effects of long-term administration of nafenopin, a potent hypolipidemic drug with marked hepatomegalic and peroxisome-proliferative properties, were studied in wild-type (Csa strain) and acatalasemic (Csb strain) mice. Nafenopin was administered in the diet at a concentration of 0.1% during the first 12 months and then at 0.05% until the termination of the experiment at 20 months. By 56 weeks, 100% mortality occurred in both male and female wild-type mice, whereas the mortality rate in acatalasemic mice was approximately 50%. Between 18 and 20 months of the experiment, 9 of 9 male and 12 of 12 female acatalasemic mice that survived chronic nafenopin treatment developed hepatocellular carcinomas, some of which metastasized to the lungs. None of the 15 male and 15 female acatalasemic controls developed liver cancers. Numerous peroxisomes were seen in the lung metastases of these hepatocellular carcinomas on electron microscopic examination; in contrast the number of peroxisomes in primary liver tumor cells varied considerably. The hepatocarcinogenicity of nafenopin strongly suggests the need for long-term studies with other hypolipidemic drugs that cause hepatomegaly and peroxisome proliferation to clarify the role, if any, of peroxisome proliferation in liver carcinogenesis.


Transmission of murine leukemia virus (Scripps) from parent to progeny mice as determined by p30 antigenemia. All mice of C57BL/St, C3H/St, BALB/cSt, NZB/Scr, and NZW/Lac strains developed high levels of p30 antigenemia after inoculation at birth with murine leukemia virus (Scripps). Transmission of virus from neonatally infected parents to their progeny for three successive generations, as evidenced by development of p30 antigenemia, varied among the five strains. Through the three generations, 100% transmission occurred in C3H/St and BALB/cSt mice, 50 to 61% transmission occurred in C57BL/St and NZW/Lac mice, and 11% transmission to the first generation, with no subsequent transmission, occurred in the NZB/Scr mice. Transmission appeared to occur readily via the milk in all strains. Intrauterine events also played a role with evidence of some viral transfer prior to birth in the C3H/St strain or, conversely, the development of resistance to infection prior to birth in C57BL/St mice. The occurrence of litters from infected parents containing both normal offspring and offspring with elevated p30 appeared to be the result of variable resistance in the intact offspring, perhaps as a result of intrauterine events, and not related to cellular resistance observable in tissue culture or to dominant genetic factors.


In vitro growth characteristics of virally transformed murine myeloid cells. Bone marrow from normal BALB/c mice, mice with myeloid leukemia induced by Soule myeloid leukemia virus, and mice with virally induced mammary carcinoma was cultured in semisolid agar. Bone marrow from either leukemic or mammary tumor-bearing mice produces more clones in vitro in the presence of a specific colony-stimulating factor. However, in all cases, the myeloid progenitor cells have similar requirements for the colony-stimulating factor. The optimum condition for growth in all instances is 7% fetal calf serum + 7% horse serum + 7% tryptose phosphate broth. Decrease in the concentration of these three constituents has a less drastic effect on in vitro proliferation of bone marrow cells from leukemic mice. Some cells from Soule virus-induced leukemias even grew in the absence of serum. The combination of suboptimal amounts of serum and colony-stimulating factor is used as a tool for detecting cells with altered growth characteristics in bone marrow of leukemic mice. During the progression of the leukemia, there is an increase in the amount of transformed colony-forming cells per 5 X 10(4) bone marrow cells. The increase is already noticeable 4 weeks after inoculation, when no clinical signs of the leukemia are present, and reaches a maximum of about 20%.


Accelerated regeneration of trypsin-treated surface antigens of simian virus 40-transformed BALB/3T3 cells induced by X-irradiation. The antigens of SV40-transformed BALB/3T3 cells measured by a radioisotopic footpad assay after removal by trypsin treatment regenerated in vitro in 3 to 6 hr. After X-irradiation with 3000 R, however, the antigens were regenerated to normal levels within 1 h. X-ray doses of between 1000 and 5000 R accelerated the regeneration of cell surface antigens, while X-irradiation with the larger dose of 8000 R did not. X-irradiation of nontrypsinized tumor cells was without effect. Possible mechanisms of this phenomenon are discussed.


Occurrence of the malate-aspartate shuttle in various tumor types. The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm.


Alanosine toxicity in Novikoff rat hepatoma cells due to inhibition of the conversion of inosine monophosphate to adenosine monophosphate. 2-Amino-3-(hydroxynitrosoamino)propionic acid (alanosine), at a concentration as low as 2.7 muM, completely inhibits the incorporation of hypoxanthine into adenosine triphosphate by cultured Novikoff rat hepatoma cells. Alanosine inhibits the first step in the conversion of inosine monophosphate to adenosine monophosphate because inosine monophosphate, but not adenylosuccinate, accumulates in treated cells. However, the alanosine inhibition is not prevented by aspartic acid, even at a concentration of 1 mM. Alanosine treatment results in the inhibition of cell division, DNA synthesis, RNA and protein synthesis (in this order), and a depletion of the cells of adenosine triphosphate. Some of the cells accumulate in late G2 or M, but the remainder become arrested in other stages of the cell cycle. All effects are due to the inhibition of adenosine monophosphate synthesis and the consequent depletion of the adenosine triphosphate pool since they are completely prevented or reversed by addition of adenine, but not hypoxanthine, to the medium. Pyrimidine nucleotide synthesis is not significantly inhibited by alanosine, since the uridine triphosphate pool is not affected and uridine fails to reverse the cytotoxicity of alanosine. Alanosine also inhibits the transport of aspartic acid, but has a much lower affinity for this transport system than aspartic acid.


Vincristine (NSC-67574) in the treatment of small-cell anaplastic carcinoma of the lung. In a phase II trial the effect of vincristine as a single agent was evaluated in patients with small-cell anaplastic carcinoma of the lung. The dosage of vincristine was 1.5 mg/m2/week given iv for 4 weeks, followed by 1.5 mg/m2 given every other week with dose modifications according to neurologic tolerance. Nineteen of 27 patients included in the study were evaluable. Objective response was observed in eight patients (42%) including three who were not previously treated and five who were previously treated. The median duration of response was 60 days (range, 21-182 days) with the response always occurring within 4 weeks. This study demonstrates that vincristine is an active agent for small-cell anaplastic carcinoma without cross resistance to CCNU, cyclophosphamide, or methotrexate.


BCNU (NSC-409962) and procarbazine (NSC-77213) treatment for malignant brain tumors. Sixty-five patients with malignant brain tumors were treated with a combination of BCNU (100 mg/m2 qd X 1) and procarbazine (100 mg/m2 qd X 14); the cycle was repeated in 1 month and then on a 6-week schedule with procarbazine being given for 21 days. Forty-five patients had malignant gliomas (glioblastoma multiforme, anaplastic astrocytoma, malignant glioma, or gemistocytic astrocytoma) and were evaluated as a group. All patients had either shown evidence of tumor regrowth after previous surgery and/or radiotherapy, or had deep unbiopsied tumors presumed to be malignant gliomas. Of these 45 patients, 13 of 45 (30%) were judged to be unequivocal responders and an additional eight of 45 (17%) were designated as probable responders. The median duration of clinical response was 34 weeks for responders and 20 weeks for probable responders. The combination of BCNU and procarbazine, therefore, was somewhat inferior to a previous combination of procarbazine, CCNU, and vincristine.


The influence of temperature on the thyroid gland of Chthonerpeton indistinctum (Gymnophiona, Amphibia). The thyroid gland of the tropical amphibian Chthonerpeton is markedly stimulated when the animal is exposed for two days to relatively high temperatures (36 degrees C). Control animals were kept at 20 degrees C. The stimulated gland exhibits collapsed follicles and a very tall follicular epithelium. At the ultrastructural level the activated epithelial cells were characterized by an increased number of apical microvilli, increased amounts of rough endoplasmic reticulum, an extended Golgi apparatus and a high number of various granular and vesicular inclusions. These differences in histology and fine structure were not paralleled by  quantitative results, which were approximately the same in both groups of animals. The values of these determinations were relatively high.


Temperature dependent changes in the thyroid gland of Mertensiella caucasica (Urodela, Amphibia). The influence of temperature on the thyroid gland of the Caucasian salamander, Mertensiella caucasica has been studied (1) with the light- and electron microscopes, (2) with enzyme histochemical and quantitative methods. Animals which had been kept for years at 16 degrees C were exposed for 48 hrs to 4 degrees C or 27 degrees C. At 4 degrees C the follicular epithelium consisted of cuboidal cells with well-developed rough ER, occasional distended cisternae of the rough endoplasmic reticulum (ER) filling most of the cytoplasm, a relatively small Golgi apparatus and numerous round or oval shaped lysosomes. In animals kept at 27 degrees C the follicular wall was composed of markedly taller epithelial cells, the apices of which bulged into the follicular lumen. Compared to the animals at 4 degrees C, there appeared to be a decrease in the rough ER. The Golgi apparatus did not exhibit clear differences. The lysosomes increased in number and showed various shapes among which elongated ones with pointed ends were particularly striking. In the apical cytoplasm colloid droplets appeared indicating hormone release. The apical microvilli increased in number. The measurements of total T4 have shown that the hormone level in animals kept at 27 degrees C was about twice as high as in animals kept at 4 degrees C.


Comparative ultrastructural study of the effects of serum-free medium and dibutyryl-cyclic AMP on newborn rat astroblasts. Modifications of cell shape induced in cultured newborn rat astroblasts by serum deprivation or dibutyryladenosine 3'-5' monophosphate (dBcAMP) are described. Serum suppression modifies the adhesitivity of the cells to the substrate, but this modification is not consistent with a true differentiation. The main ultrastructural feature of dBcAMP-treated astroblasts is the presence of an extensive system of 90 A microfilaments, while control cells are relatively devoid of these structures.


Extracellular cyclic-amp phosphodiesterase regulation in agar plate cultures of Dictyostelium discoideum. Extracellular cyclic-AMP phosphodiesterase and the inhibitor of this enzyme is tested in agar plate cultures of two Dictyostelium discoideum wild-type strains and in a mutant which lacks the inhibitor. Under the conditions used, the phosphodiesterase inhibitor is formed in both wild-type strains either before or in an early stage of cell aggregation. During aggregation of one strain the phosphodiesterase activity is extremely low, excluding a necessary function of the enzyme in the aggregation process.


Expression of differentiative traits in the absence of cell fusion during myogenesis in culture. Fusion of myoblasts is inhibited in cultures at low Ca++ concentration (0.44 mM); yet creatine phosphokinase and myokinase activities as well as myosin synthesis and the appearance of post-mitotic myoblasts do not significantly differ from those of control cultures (grown at 1.04 mM Ca++) which undergo cell fusion. When Ca++ concentration is increased to the control value after the second day of culture, fusion occurs very rapidly and it is not inhibited by actinomycin D or cycloheximide. Treatment with 0.06 mM bromodeoxyuridine strongly inhibits creatine phosphokinase activity and myotubes formation. The study of the kinetics of reversal of cell fusion and of creatine phosphokinase activity after removal of the analog, shows that this process is slower than the decrease of the relative content of bromodeoxyuridine incorporated into DNA. The result obtained support the following conclusions: a) the expression of the differentiative characters examined does not require cell fusion; b) the process of myotube formation seems to imply two subsequent stages consisting first of a slow maturative process, which is followed by the actual fusion of cell membranes; the former is Ca++ independent, the latter is Ca++ dependent and does not require RNA or protein synthesis.


Spin label studies of micellar and pre-micellar aggregates. Micelles of hexadecyl trimethyl ammonium bromide (CTABr) have been investigated with the use of a faty acid spin label and its methyl ester derivative. The esr * spectra provided information about the degree of motion of the probes in the micelles as evaluated from calculation of rotational correlation times. Evidence is presented for the formation of pre-micellar aggregates at concentrations below the cmc. The effect of addition of thiophenoxide on the structure of CTABr micelles was to decrease the rate of motion of the spin probes, probably due to a tighter packing of the hydrophobic core as a consequence of charge neutralization at the micelle surface by the substrate. Decreasing values of the isotropic hyperfine splitting of the spin probe with increasing concentration of thiophenoxide were taken as indicating that the latter causes a decrease of the degree of hydration of the polar head region of the detergent.


Synthesis of 2-n-(hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine-hydroxide, a chromogenic substrate for assaying sphingomyelinase activity. 2-N-(Hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine-hydroxide a compound resembling sphingomyelin is synthesized. It is cleaved by sphingomyelinase to the chromogenic N-acylaminonitrophenyl moiety. Phospholipase C preparations do not hydrolyze this compound. The starting material is 2-amino-4-nitrophenol which when acylated with palmitoyl chloride yields the hexadecananilide. Reaction with beta-bromoethylphosphoryldichloride gives the phosphate which is quaternized with trimethylamine to give the title compound.


[Meiosis: role of a histone kinase in the condensation of ovarian oocyte chromosomes of Xenopus laevis and Ambystoma mexicanum]. By injecting heterologous histone-kinase preparations into ovarian Axolotl oocytes, it has been possible to speed up the progesterone-induced process of chromosome condensation. Moreover, in some instances, this condensation and even complete maturation have been obtained after injection of protein kinase alone, thus in the absence of hormone stimulation. Two different histone kinase preparations have been used: one was prepared from ascites cell chromatin and the other from in vitro ovulated Xenopus oocytes.


[Autoradiographic study of a bacterial colony. Application to the effects of polymyxin on Pseudomonas aeruginosa colonies]. The autoradiograph of a colony of Ps. a. which has been transferred, during growth, on a medium added with polymyxin and tritiate leucin makes it possible to locate an upper zone with a high metabolic activity and a basal zone with no metabolic activity. The latter, which consists of lysed cells, acts probably as a selective filter against the drug.


[Maternal and fetal corticosteroids in the sow at the end of gestation]. Foetal and maternal blood samples are taken on the sow at the end of pregnancy. No variations in maternal cortisol are found, but plasma cortisol levels in the foetus, which are relatively constant between days 70 and 100 of pregnancy (10 to 20 ng/ml), increase suddenly at the end of pregnancy reaching a mean of 210 ng/ml at birth. This high increase of foetal corticosteroids at birth, similar to that found in other species (ewe, goat, etc.), probably plays a role in the mechanisms releasing parturition. It also permits us to understand the action of ACTH which induces premature birth when given to the pig foetus in the last days of pregnancy.


[The mode of action of bacteriocin N5 purified from Clostridium perfringens]. The purified bacteriocin N5 produced by Clostridium perfringens BP6K inhibited simultaneously the syntheses of DNA, RNA and protein in sensitive cells, without DNA degradation. Bacteriocin N5 inhibited the accumulation of leucin and caused the exit of the previously accumulated amino acid. The effects of bacteriocin N5 are very similar to those observed for colicins E1, K, A and I.


[Changes in the level of ACTH in the fetal hypophysis. A study in the rat toward the end of gestation]. The isolated adrenal cell method for bioassay of ACTH was employed in order to determine the ACTH content of the hypophysis of the rat foetus, in late pregnancy. The ACTH concentration decreases between 17 days 8 h and 17 days 15 h, then it increases regularly until the term. This evolution is correlated with the change in the fluorescence intensity of the corticotrophs and with the modification of the adrenocortical activity.


[The effect of an ionophore on the aggregation of Dictyostelium discoideum]. Treatment during starvation of D. discoideum amoebae with micromolar amounts of A23187 causes an enhanced aggregation. Cells develop the properties of differentiated, aggregation competent amoebae earlier than untreated populations. Ionophore increases the release of calcium, and prevents the excretion of the phosphodiesterase ihibitor normally released in the media. A23187 suppresses the morphogenetic block of some aggregates mutants, suggesting that the ionophore either activates cAMP synthesis and excretion, or increases the cellular sensitivity to extracellular cAMP signals. This might result from the enhanced mobilisation of intracellular calcium


Technetium 99m stannous pyrophosphate myocardial imaging in patients with and without left ventricular aneurysm. To further explore the usefulness of technetium 99m pyrophosphate (99mTc-PYP) myocardial imaging and test its validity in the diagnosis of acute myocardial infarction, 99mTc-PYP myocardial scintigrams were performed in 50 patients. Out of 28 patients with acute myocardial infarction, myocardial scintigrams demonstrated localized activity in the 15 patients with transmural, and diffuse activity in the 13 patients with subendocardial myocardial infarction. Twenty-two patients with significant coronary artery disease documented by coronary angiography but without acute myocardial infarction were also studied. Nine of ten patients with clinical evidence of left ventricular aneurysm from previous myocardial infarction and definite left ventricular dyskinesis had positive scintigrams with activity localized to the site of the wall motion abnormality. Two of five patients without definite aneurysm but with left ventricular akinesis also had localized uptake in the involved area of the left ventricle. Seven patients with normal left ventricular wall motion had negative scintigrams. These findings suggest caution in interpreting positive 99mTc-PYP scintigrams as being indicative of acute myocardial infarction when evidence of a left ventricular aneurysm is also present.


Demonstration and semiquantitative determination of complexes between various proteases and human alpha2-macroglobulin. Complex formation in vitro between human alpha2-macroglobulin and the human proteases cationic trypsin, chymotrypsin, plasmin and granulocyte elastase and collagenase was clearly visualized by the use of thin-layer electrofocusing in polyacrylamide gel followed by electrophoresis in agarose gel containing antibodies against human alpha2-macroglobulin. The technique permits semi-quantitative determination of the amount of complex and can demonstrate the formation of complexes between alpha2-macroglobulin and protease in vivo in ascitic fluid in peritonitis.


Neonatal screening for hyperlipoproteinemia. Methods for direct estimation of cord serum VLDL + LDL. The early detection of hyperlipoproteinemia in newborn infants has so far been based upon estimation of cord blood total lipids (cholesterol and triglyceride) and lipoprotein-lipids (VLDL-, LDL- and HDL-cholesterol). To be able to make a direct estimation of cord serum beta-lipoproteins (VLDL + LDL) two quite different methods were modified, one immunological and the other turbidimetric. Good correlations were found to VLDL- + LDL-cholesterol isolated in the ultracentrifuge (r = 0.848 and 0.831, respectively). If neonatal screening for hyperlipoproteinemia is considered, we recommend the very easy and inexpensive turbidimetric method. Furthermore, using cord serum, two conventional precipitation methods with heparin-CaCl2 and heparin-MnCl2 were compared by ultracentrifugation and high correlations were found (r = 0.923 and 0.899, respectively). A clamping study showed that following early clamping of the cord, the concentration of cord serum lipids and lipoproteins did not change markedly within the first five minutes. Storing experiments showed that serum should be separated within the first 12 h to avoid unpredictable changes in the concentration of cord serum lipids and lipoproteins.


Serum alkaline phosphatase, nucleotide pyrophosphatase, 5'-nucleotide and lipoprotein-X in cholestasis. The nature of the stationary band of alkaline phosphatase, which occurs on starch gel electrophoresis of sera from patients with biliary obstruction, has been examined. Stationary alkaline phosphatase was eluted from Sepharose 4-B gel close to the void volume and together with the plasma membrane enzymes, nucleotide pyrophosphatase and 5'-nucleotidase, and with lipoprotein-X. Electron microscopy of concentrates of stationary alkaline phosphatase, prepared by ultracentrifugation and gel filtration, showed large (0.3--1 mum diameter) and small structures (30-70 nm diameter) by negative staining. The activity of the stationary alkaline phosphatase was associated in fixed sections with particles of about 10 nm X 40 nm resembling those of lipoprotein-X. It is suggested that the stationary alkaline phosphatase does not move into starch gel during electrophoresis because it is particulate. In agar electrophoresis the alkaline phosphatase which was stationary on starch gel moved towards the cathode with lipoprotein-X.


A simplified competitive protein-binding assay for 25-hydroxycalciferol. A single and reliable, competitive, protein-binding assay for plasma 25-hydroxycalciferol (25-OH-D), based on a previously described method which omits the chromatographic step, has been tested without the use of beta-lipoproteins. The accuracy is similar in the absence of beta-lipoproteins and in their presence, the recoveries being respectively 110-136% and 74-85%. The intraassay coefficient of variation is 7.23% in the absence versus 12.56% in the presence of beta-lipoproteins. The sensitivity of the described assay is 0.01 ng per assay tube and 1 mug/1 plasma. The mean plasma 25-OH-D level in eighteen normal Swiss volunteers aged 20 to 40 years is 39.5 +/- S.D. 9.3 mug/l. This level is similar to those found in Western Europe and U.S.A. subjects. Ten alcoholic patients without cirrhosis have significantly lower 25-OH-D plasma levels (13.0 +/- S.D. 8.9 mug/l) than appropriate age controls. This precise and accurate method is convenient for clinical studies since both chromatographic and beta-lipoproteins steps are avoided.


Three siblings with Robert's syndrome.

Three siblings with Robert's syndrome. A Yugoslavian family with one female and two male infants with Robert's syndrome is described. The main features were: (1) tetraphocomelia with missing or malformed bones of arms and legs; (2) bilateral cleft lip and palate; (3) ectrodactyly; (4) syndactyly of the digits; (5) hypertelorism with exophthalmos at birth; (6) congenital heart defect; (7) low birth weight and failure to thrive. These observations raise the reported cases of Robert's syndrome--including questionable ones--to a total of 26. The most likely basic etiology is a major single recessive gene mutation.


Immunodeficiency in the chicken. IV. An immunological study of infectious bursal disease. Chickens inoculated orally with infectious bursal disease virus (IBDV) 1 day after hatching subsequently showed a 50% incidence of immunodeficiency but little mortality. Antibody responses against IBDV and to immunization with sheep red blood cells (SRBC) or human serum albumin (HSA) were suppressed. Serum IgG concentration was decreased while IgM occurred exclusively in its 7S monomeric form (mIgM). An allotypic marker of chicken IgM (Mla) was lacking in mIgM derived from IBDV-infected birds. The loss of Mla occurred gradually in several birds between 3 and 12 weeks after perinatal infection. Inoculation of IBDV into chickens 3 weeks after hatching resulted in 50% mortality level but little immunodeficiency. Paradoxically, the serum IgG concentration was elevated, in comparison with normal birds. Histology of the bursa showed permanent hypo- or aplasia of follicles irrespective of the age of infection. The results suggest that bursal but not peripheral B cells are targets for IBDV, and immunodeficiency results from impaired peripheral seeding of B cells in infected juvenile chickens.


Neoplasia in patients with chronic renal failure on long-term dialysis. Out of 67 patients accepted for maintenance dialysis (56 for hemodialysis and 11 for peritoneal dialysis) 47 had not received any form of immunosuppression either in connection with renal transplantation or as part of the therapy for their intrinsic renal disease. Six out of the 47 patients (12.8%) were found to have malignancies which were not directly causing their renal failure. Two patients had carcinomas of the kidney, 2 patients had skin cancers, 1 patient had a carcinoma of the stomach and 1 patient had a carcinoid tumour of the small intestine. This raises the question whether prolongation of uremia by long term dialysis may contribute to the development of malignancy by increasing longevity in the chronically immunosuppressed uremic state.


One-year trials with halofenate, clofibrate, and placebo. The hypolipidemic as well as other laboratory and clinical effects of halofenate, clofibrate, and placebo were compared in 29 patients with type IV hyperlipoproteinemia in a double-blind, controlled, therapeutic trial of 1 yr duration. Plasma drug levels were obtained to monitor compliance. Clofibrate and halofenate lowered serum triglycerides to a similar extent. The hypotriglyceridemic effect of halofenate was significant only when data from noncompliant patients were discarded. Only clofibrate lowered baseline levels of plasma cholesterol. Very low density lipoproteins were decreased and low density lipoproteins were increased by clofibrate but not by halofenate. Halofenate had a marked hypouricemic effect that was greater than that of clofibrate. The hypouricemic effect of halofenate and clofibrate was paralleled by a concomitant decrease in serum bilirubin. Abnormal increases in serum creatine phosphokinase were observed with both drugs primarily in patients who had abnormal initial levels.


The pattern of spread and survival in 596 cases of breast cancer related to clinical staging and histological grade. Carcinoma of the breast in 596 patients diagnosed between 1949 and 1966 at the Reading group of hospitals has been evaluated. All operable cases were treated by simple mastectomy and post-operative radiotherapy to the chest wall and regional nodes. Tumours were graded for malignancy either routinely or retrospectively on all patients with carcinoma simplex or scirrhous carcinoma, and the relation of grading to the subsequent development and distribution of metastases studied. Recurrence developed in 348 patients, 184 of whom suffered local recurrence. Eighty-three per cent of locally recurrent cases developed generalised metastases. The incidence of pulmonary and liver metastases was significantly increased in Grade II and III tumours compared with Grade I. Very significant differences in the survival rates between grades were noted, unfavourable to the higher grade tumours; and the influence of grading on prognosis is discussed.


Metatropic dwarfism, the Kniest syndrome and the pseudoachondroplastic dysplasias. Metatropic dwarfism, the Kniest syndrome, and pseudoachondroplastic dysplasia are specific chondrodystrophic disorders that have in common dysplasia of the metaphyses, epiphyses, and vertebrae. Metatropic dwarfism and the Kniest syndrome have been confused with Morquio's disease and with each other in the past, but can be easily distinguished on the basis of radiographic features, clinical features, and cartilage pathology. The pseudoachondroplastic dysplasias are associated with short limbed dwarfism and have been confused with achondroplastic dwarfism, but can be readily distinguished on the basis of the normal head, delayed age of onset, the typical radiographic features and the cartilage pathology.


Negative contrast electron microscopic diagnosis of viruses of neonatal calf diarrhea. Ninety-one cases of neonatal calf diarrhea were examined for viruses with negative contrast electron microscopy. Viruses were demonstrated in 41% of the cases. Reo-like viruses and corona-like viruses, and mixed virus populations were observed in 12%, 20% and 9%, respectively. Twenty-six of the cases were examined by negative contrast electron microscopy, and by virus isolation or by fluorescent antibody technique. There was an 81% agreement in obtained results. The disagreements resulted from the demonstration of a viral agent by negative contrast electron microscopy while the other techniques did not indicate a virus. The results suggest that negative contrast electron microscopy is a more sensitive diagnostic tool for demonstration of viruses associated with neonatal calf diarrhea than are viral isolation or the fluorescent antibody technique.


Serological comparison of French WD-42 enterovirus isolate with bovine winter dysentery in New York State. Forty-eight acute and convalescent paired serums from 10 dairy cattle herds naturally infected with winter dysentery and 10 paired serums from laboratory infected heifers were tested for antibody to bovine enterovirus WD-42 which reportedly reproduced the disease syndrome in France. Only 3 naturally infected and 1 experimentally infected animals had 2-fold antibody responses. No clinical symptoms were observed in 2 steers inoculated with WD-42. Each had a 4-fold or greater antibody response to the virus. Results indicate that the winter dysentery syndrome observed in New York is produced by an agent other than WD-42 virus and that it is produced by an agent other than an enterovirus.


Increased serum calcitonin levels in bronchogenic cancer. A study of 26 men with bronchogenic cancer demonstrated high serum calcitonin levels in 62 percent (16). Levels were particularly high in patients with small-cell cancer and adenocarcinoma. Two varieties of hypercalcitonemia have been encountered: (1) ectopic hypercalcitonemia, in which the hormone is secreted by the tumor, and (2) thyroidal hypercalcitonemia, in which the high values emanate from the thyroid gland. In several patients, serum calcitonin levels decreased following therapy for the cancer. Further studies are needed to evaluate the diagnostic value and clnical utility of serum calcitonin levels as a marker substance in bronchogenic cancer.


Mechanisms of functional loss and recovery in spinal cord damage. Two main classes of morphological change follow trauma to central nerve fibres: (1) axonal disruption leads to total disintegration of the fibre distal (with respect to the cell body) to the lesion; (2) less severe trauma produces focal demyelination with preservation of axonal continuity. Large experimental demyelinating lesions produce complete conduction block. The histologically normal portions of the fibres, proximal and distal to the lesion, retain the ability to transmit impulses. Smaller lesions allow conduction to continue, but at a reduced velocity, and the ability of the fibres to carry long trains of impulses faithfully is impaired. All three defects of conduction contribute to functional loss. After acute transient compression of the spinal cord of the cat, demyelination increases during the first week. Evidence of remyelination appears in the third week. Inappropriately thin myelin is seen surrounding histologically normal axons. By one month, 90% of the fibres in the lesion have acquired new sheaths. Studies on single fibres have shown that the myelin is organized into segments bounded by nodes. The segments are abnormally thin and short. The myelin increases in thickness with time but thin segments are still present at 18 months. Electron microscopy shows that many of the known ultrastructural prerequisites for conduction are present in the new segments. It is not yet known, however, whether the chains of very short internodes which occur on some fibres allow conduction to be restored.


[Ultrastructure of the lymphocytic nucleus under the effect of phytohemagglutinin stimulation]. The author presents a review of the literature, in which he describes the blast transformation of lymphocytes, induced by lectines, as a proper model for examining the metabolic and connected with them morphological changes in cellular activation. The literature connected with the basic achievements in examining the ultrastructure of the interphase nucleus is also described. The author presents his studies on the dynamic changes, occuring in the nucleus in the course of the blast transformation, induced by phytohemagglutinin. The author found early changes in the nuclear chromatin, discovered within the first hour, using together with the ordinary double staining and the method for visualization of RNA structures (EDTA method of W. Bernar) and the method of G. Moyne for establishing DNA. It was established by means of nuclear morphometry that within the first hour the reduction of dense chromatin on account of the dispersed chromatin was 7,7%, the 24th hour was 26,0% and on the 48th hour was 26,3%. On the basis of the morphometric examination the author found also that the largest was the number of perichromatin granules in nontransformed lymphocytes of PHA-cultures. The supposition that these were B-lymphocytes was supported by analogous morphometric examination on lymphocytes obtained from the thymus and bursa Fabricius of newly shelled chickens and it was established that this number in the bursa was more than twice larger. There were three types of arrangement of the interchromatin granules: in groups, diffuse and in chains and it was found that the last type was connected with the largest number of perichromatin granules. The author discusses the possibility for a transfer of whole perichromatin granules through the nuclear pores in cases under strong stimulation such as that of phytohemagglutinin.


[Mechanism of the antispasmogenic action of some substances that affect the 3', 5'-AMP system]. The authors carried out studies on isolated segmets of ileum with a length of 3 sm. obtained from both male and female, mature and immature guinea pigs and adult male and female rats as well as on isolated vasa deferntia, obtained from guinea pigs and rats. They examined the influence of a group of substances, known as stimulators and inhibitors of adenyl-cyclase and phosphodiesterase (isoprenaline, lithium sulfate, imidazol, theophiline and eupaverine) on the spasmogenic effects of acethylcholine (on ileum) and of noradrenaline (on vas deferens). These substances were used in doses, which were subthreshold in respect to their effect on the biogenesis and exzymic inactivation of the cyclic AMP. The authors found certain species, adult and sex differences in the spasmogenic effects of acethylcholine and noradrenaline. The examined substances lowered the spasmogenic effects of acethylcholine and noradrenaline in most of the experiments. Since the examined substance-classical means for altering the endogenous production of the cyclic AMP-show antispasmogenic effect in concentrations subthreshold for their influence on the level of the cyclic AMP, an inference is made that their inherent antispasmogenic effect is due also to a mechanism, which does not require the inclusion of the system of the cyclic AMP.


Basal and dibutyryl cyclic AMP-stimulated release of newly synthesized and stored growth hormone from perifused rat pituitaries. The basal rate of pre-labeled stored 14C-labeled rat growth hormone ([14C]rGH) release from perifused rat pituitary explants is a constant fraction of pituitary GH content, suggesting random release from the storage pool. Basal release of newly synthesized [3H]rGH occurs in two phases: 1) immediate and associated with [3H]rGH synthesis, and 2) late (delayed by 60 min) and independent of concurrent [3H]rGH synthesis. Dibutyryl cyclic AMP (10(-2)M)-stimulated release of stored [14C]rGH is characterized by an initial acute rise followed by a second phase of continuous rapid release. Immediate and late release of new [3H]rGH is increased by dibutyryl cyclic AMP, and the late phase of [3H]rGH is less delayed. Simultaneous exposure of pituitary explants to [3H]alanine and [14C]leucine resulted in the release of immunoprecipitable rGH whose ratio of incorporated 3H and 14C varied with time. The observed changes suggest that after it is synthesized, a GH molecule may either be released directly or be processed into the somatotroph's storage compartment. In addition, stored GH is composed of two pools, one of which is immediately releasable. The differential incorporation of [3H]alanine and [14C]leucine into "big" and "small" rGH, together with the ability to differentially displace 3H-labeled "big" and 14C-labeled "small" rGH from the GH antibody suggest that "big" rGH is a heterogenous molecule including "small" rGH and another peptide rather than simply a dimer of "small" rGH.


Characterization and modulation of growth hormone and prolactin binding in mouse liver. The specific binding of 125I-labeled insulin, human hormone ([125I]hGH), bovine growth hormone ([125I]bGH), and ovine prolactin ([125I]oPRL) was studied in mouse liver membranes. [125I]hGH and [125I]oPRL bound to adult liver membranes. Pregnancy increased the specific binding of [125I]hGH but not that of [125I]oPRL. [125I]hGH was displaced from membranes of pregnant mice by hGH, oPRL, and bGH, but only by hGH and oPRL from liver membranes of nonpregnant mice. Significant specific binding of [125I]bGH was seen only in pregnancy. The binding of [125I]bGH to pregnant mouse liver membranes increased with increasing concentration of either membrane protein or [125I]bGH. Both the specific binding and dissociation of [125I]bGH were greatly influenced by the time and temperature of incubation. Binding of [125I]bGH was inhibited by growth hormones, including hGH and rat GH, and not by lactogenic hormones (various prolactins and human placental lactogen), ACTH, glucagon, or insulin. The inhibition of [125I]hGH binding by hGH and bGH, in the presence of excess (2 mug/ml) of PRL, was very similar to that seen with [125I]bGH. Scatchard plots of displacement dose-response curves obtained under steady state conditions of 4C were nonlinear and very similar with either [125I]bGH or [125I]hGH. This contrasted with the linear Scatchard plots obtained from displacement dose-response curves of either [125I]oPRL or [125I]hGH in the presence of excess (2 mug/ml) bGH. Termination of pregnancy, either naturally or by hysterectomy, reduced [125I]bGH specific binding to nonpregnant levels by 24 to 36 h. Estrogen administration did not increase [125I]bGH binding in hepatic membranes. Nonpregnant mice possess hepatic lactogen binding sites which are uninfluenced by pregnancy. GH specific binding sites are markedly augmented during pregnancy. The close correlation between the level of these sites and pregnancy suggests that they are regulated by a product of the fetoplacental unit.


Capacity of immunologically purified FSH to stimulate cyclic AMP accumulation and steroidogenesis in Graafian follicles and to induce ovum maturation and ovulation in the rat. Reference preparations of ovine follicle-stimulating hormone (NIH-FSH-S8 and S9; 10-50 mug/ml) induced ovum maturation and stimulated cyclic AMP formation, as well as progesterone and 17beta-estradiol secretion, by rat Graafian follicles in vitro. These actions of NIH-FSH were retained after immunoabsorption of any contaminating luteinizing hormone (LH) present in the preparations, by treatment with an antiserum to the beta-subunit of purified ovine LH (anti-betaLH). In contrast, the corresponding biological actions of NIH-LH-S18 (0.5-10 mug/ml) were abolished by treatment with this anti-betaLH serum. A highly purified FSH preparation (64-96 CD, 0.25 mug/ml) also triggered oocytic meiosis and increased follicular progesterone secretion in vitro. Intraperitoneal (ip) administration of anti-betaLH-treated NIH-FSH-S9 (50 mug/rat at 1430 h) consistently induced ovulation in proestrous rats in which the endogenous gonadotropin surge had been blocked by ip injection of either Nembutal (1345 h) or antiserum to the LH-releasing hormone (1200 h). Injection (ip) of anti-betaLH serum on its own into proestrous rats at 1200 h prevented ovum maturation and follicular rupture. We conclude that currently available reference preparations of ovine FSH possess the capacity to stimulate follicular adenylate cyclase, steroidogenesis, and ovum maturation in vitro, as well as ovulation in vivo, in the rat, and that this capacity cannot be attributed to contamination with material immunochemically identical with LH. However, it is inferred that the physiological triggering of ovulation and related events in this species depends principally on LH.


Comparison of the metabolism and distribution of L-triiodothyronine and triiodothyroacetic acid in the rat: a possible explanation of differential hormonal potency. Previous studies from this laboratory have shown that nuclear displacement of triiodothyronine (T3) is more rapidly dissipated after iv injection of triiodothyroacetic acid (triac) than after equimolar doses of T3. This suggested that the discrepancy between the strong nuclear binding of triac and its relatively weak thyromimetic effect could be explained by a more rapid fractional rate of triac metabolism. To test this hypothesis, tracer studies with radioactively labeled T3 and triac were carried out in normal male Sprague-Dawley rats. Noncompartmental analysis showed that the average residence time of tracer triac in the exchangeable compartment was 5.5 h compared with 10.9 h for T3. The metabolic clearance rate of triac was 14.4 ml/h/100 g BW and of T3 17.6 ml/h/100 g. The average distribution space of triac was 78.2 ml/100 g and of T3 190.7 ml/100 g. The fraction of isotope excreted via the fecal route was 0.46 for triac and 0.41 for T3. Triac was approximately 16 times as firmly bound to plasma proteins as was T3. Isotopic studies suggested that a rapid exchange of tracer triac occurred between plasma, cytosol, and nuclei, similar to previously observed relationships for T3. Based on the daily dose of T3 and triac required to maintain a normal concentration of alpha-glycero-phosphate dehydrogenase in thyroidectomized animals, the T3:triac potency ratio was estimated to be 3.0. Our observations support the concept that a shorter duration of nuclear occupancy observed in previous experiments can be attributed to a more rapid fractional metabolism of triac and may explain the lesser hormonal effect of this compound. Since labeled triac could not be identified in nuclear extracts after the injection of isotopically labeled T3, it appears unlikely that the initiation of hormonal effects by T3 at the nuclear level is dependent on its prior conversion to triac.


Corticoid receptors in rat brain: evidence for an aldosterone receptor. The existence of high and low affinity mineralocorticoid-binding macromolecules (receptors) has been demonstrated in vitro in cytosols derived from the adrenalectomized rat brain by the specific binding of [3H]aldosterone (3H-A). The high-affinity aldosterone sites can be distinguished from those sites which have a higher affinity for either [3H]dexamethasone (3H-DM) or [3H]corticosterone (3H-B) on the basis of selectivity for spirolactone SC-9420 or non-radioactive A, DM, and B. The binding of 3H-A to the receptors was maximal after 2 hours of incubation of 0-4C. No significant binding of 3H-A to the receptors could be demonstrated when incubations of the radioactive ligand were performed at either 20 or 37 C, indicating that the receptor is heat-liabile. Scatchard analysis of the 3H-A binding data over a 200-fold concentration range of 3H-A indicated that there are two binding sites for aldosterone, a high affinity component (a1) with a Kd approximately equal to 1.5 X 10(-9)M and a low-affinity component (a2) with a Kd approximately equal to 6.3 X 10(-8)M. A similar study using 3H-DM as the radioactive ligand demonstrated only one site for the 3H-DM binding with a Kd = 6.2 X 10(-9)M. The presence of specific aldosterone receptors in the brain with high affinity, limited capacity, and selectivity for aldosterone suggests a possible extra-renal mechanism of action of the hormone in or mediated through the CNS.


Androgen-uterine interaction: nuclear translocation of the estrogen receptor and induction of the synthesis of the uterine-induced protein (IP) by high concentrations of androgens in vitro but not in vivo. High concentrations of androgens in vitro [10(-6) and 10(-7)M 5alpha-dihydrotestosterone (DHT) and testosterone (T)] translocate the estrogen receptor from cytoplasmic to the nuclear fraction of the immature rat uterus, and the androgen translocated sites are capable of eliciting the synthesis of the specific uterine "induced protein" (IP), formerly attributed to estrogenic compounds only. The magnitude of receptor translocation and IP synthesis induction is related to the concentration of androgen, and, at equal concentrations, DHT is more effective than T. Competitive protein-binding asssays with cell-free uterine cytosol indicate that DHT and T bind with barely detectable affinity to the cytosol estrogen receptor detectable affinity to the cytosol estrogen receptor [relative binding ability ca. 0.001% that of estradiol (E2)], and radioactive E2 uptake into whole uterus after pretreatment with androgens indicates that the androgen-translocated nuclear sites are readily filled by E2. DHT and T translocate the estrogen receptor to the nucleus in vitro, and the salt-extracted nuclear receptor is present as a 5 S ("transformed") receptor; however, concentrations of DHT and T that effect translocation are unable to elicit the heat-activated transformation of the estrogen receptor in cell free cytosol under conditions in which low concentrations of E2 fully transform the receptor. Under in vivo conditions, high levels of androgens (5--2,000 mug DHT or T) do not evoke any detectable translocation of the estrogen receptor and do not elicit any IP synthesis induction although uterine weight is increased (greater than 200 mug androgen) in long-term (2--3 day) assays. The analysis of androgen uptake and metabolism indicates that 500 mug or higher doses of DHT or T in vivo result in uterine concentrations of unmetabolized DHT or T equal to those seen after exposure to 5 X 10(-7) - 1 X 10(-6)M DHT or T in vitro. Hence, under conditions where in vivo and in vitro tissue uptake of androgen is equivalent, in vivo androgens are unable to affect the estrogen receptor system as is seen in vitro. These studies indicate that the in vitro and in vivo effects of androgens on the uterus are clearly different and suggest that the actions of androgens on the uterus in vivo are probably not directly mediated through the estrogen receptor system.


Studies on ACTH dynamics in cultured adenohypophyseal cells: effect of adrenalectomy or dexamethasone in vitro. Basal or hypothalamic extract-induced ACTH or TSH secretory capacity as well as intracellular ACTH content were assessed in cultured rat adenohypophyseal cells after various pretreatments of the donor animals. Medium and intracellular ACTH or TSH were measured by radioimmunoassay. Ten-day to 2-month adrenalectomy enhanced, whereas dexamethasone pretreatment suppressed, both synthesis and secretion of ACTH in the adenohypophyseal cells. Dexamethasone may also impair the replication of the adenohypophyseal cells in vivo, as indicated by the smaller number of cells obtained per animal in the dexamethasone-treated group. No significant increase in TSH secretion was observed when adenohypophyseal cells were derived from rats fed propyl-thiouracil (PTU) for 3 weeks instead of from intact rats.


Regulation of estrogen receptors in ovarian-dependent rat mammary tumors. I. Effects of castration and prolactin. The "ovarian-dependent" rat mammary tumors, induced by 9,10 dimethyl-1,2-benzanthracene, were assayed for their estrogen receptor content by dextran-coated charcoal adsorption and sucrose gradient ultra-centrifugation. The estradiol receptors bound estrogens with a high affinity (KD approximately 0.25 nM), limited capacity and high specificity, and sediment at 8 S in a sucrose gradient. The cytosol receptors were transferred to the nucleus after binding to estrogens either in vivo or in vitro. The tumor area regressed by 70% during the first 10 days of castration while the concentration of estradiol cytosol receptors decreased from 225 to 16 fmoles/mg of protein. Three to five days after in vivo administration of estradiol (2 mug daily) or prolactin (1 mg daily) the concentration of estrogen receptors was increased in spayed rats. In biopsy experiments, prolactin, but not estradiol, was shown to increase the estrogen receptor concentration when endogenous prolactin release was blocked by CB 154. Prolactin did not modify the intracellular distribution of the estrogen receptor or its binding affinity for estrogen. The uterine estrogen receptor sites were not modified by prolactin under the same conditions. We, therefore, suggest that in mammary tumors prolactin sensitizes the action of estrogens at the target level by increasing the concentration of their available receptor sites.


Effect of feeding and fasting on the early steps of glucagon action in isolated rat liver cells. The effect of the nutritional state (fasted or fed) on the binding of glucagon and on the glucagon-stimulated cyclic AMP accumulation in the presence of theophylline was studied in isolated rat liver cells. The binding of glucagon was higher in cells from fed than in those from fasted rats at each concentration of glucagon tested between 0.1 and 36 nM. The specific binding of the hormone was about 2-fold higher in cells from fed than from fasted rats. At concentrations of glucagon between 0.1 and 2.2 nM, the accumulation of cyclic AMP in the presence of 1 mM theophylline was higher in the cells from fed rats. Furthermore, 4 times as much glucagon was required to elicit half-maximal cyclic AMP accumulation in the cells of fasted (1.47 nM) than in the cells of fed (0.35 nM) rats. These data suggest that, in isolated rat liver cells, both glucagon binding to receptor sites and glucagon-stimulated cyclic AMP levels in the presence of theophylline can be affected by the nutritional status of the animal.


Binding of cytosol receptor-glucocorticoid complexes by isolated nuclei of glucocorticoid-responsive and nonresponsive cultured cells. Nuclear binding of the AtT-20 cytosol receptor-glucocorticoid complex was studied in a cell-free system using nuclei from steroid-responsive (AtT-20) and nonresponsive (EPO-G1) cell lines, both of which synthesize ACTH. The AtT-20 cell line was derived from a mouse pituitary adenocarcinoma,  while the EPO cell line was established from a human malignant melanoma. The nonresponsive EPO cells lacked a cytosol receptor for glucocorticoids, and, when whole cells were incubated with labeled glucocorticoid, they were unable to concentrate the steroid in their nuclei. A cell-free system using AtT-20 cytosol preincubated with labeled glucocorticoid was used to study binding by isolated nuclei. Binding to isolated nuclei from both cell lines was indistinguishable, in terms of temperature sensitivity, binding capacity, and saturability. Sucrose density gradient analyses of KCl extracts of nuclei labeled under these cell-free conditions showed 3.2-3.6 S peaks. In contrast, a 4.0 S peak was observed consistently when unreacted cytosol was analyzed on high-salt gradients, suggesting that interaction with nuclei from both cell lines caused the receptor to alter its sedimentation characteristics. These findings suggest either that all cells contain nuclear acceptor sites and that target cell responsiveness is conferred solely by the presence or absence of the cytosol receptor, or that binding sites detected in isolated nuclei may be different from those observed in intact cells and may, in fact, obscure them.


A possible role of cyclic AMP in mediating the effects of thyrotropin-releasing hormone on prolactin release and on prolactin and growth hormone synthesis in pituitary cells in culture. Thyrotropin-releasing hormone (TRH) has 3 effects on clonal strains of rat pituitary cells in culture (GH-cells). Two long-term effects of TRH on GH-cells, which are measurable after 3 h or longer, have been previously reported; these are an increase in prolactin synthesis and a decrease in growth hormone production. We report here that TRH also stimulates the rapid release of stored intracellular prolactin. We have investigated the role of cyclic AMP as a possible mediator of the effects of TRH on GH-cells. Cyclic AMP concentrations are higher in cells treated with TRH compared with paired controls; a maximum difference of greater than 150% of control values is detected at 15 min if the incubation is performed in serum-free medium in the presence of 1 mM theophylline. The concentration of TRH required to give half-maximum increases in both prolactin release and cyclic AMP accumulation is 0.3 nM; half-maximal increases in prolactin synthesis occur at 3 nM TRH. Exogenous cyclic AMP (1 mM) causes only a slight increase in prolactin release; 8-bromo-cyclic AMP and 8-methylthio-cyclic AMP (1 mM) do not cause significant release. Phosphodiesterase inhibitors (0.3 mM theophylline, 0.03 mM isobutyl-methylxanthine) increase prolactin release but their effects on hormone synthesis are more complicated. Isobutylmethylxanthine, 8-bromo-cyclic AMP and 8-methylthio-cyclic AMP (0.4 MM) increase prolactin synthesis, but do not significantly affect growth hormone synthesis. Theophylline increases the synthesis of both hormones. Dibutyryl cyclic AMP (0.5 mM or more) increases prolactin release and both growth hormone and prolactin synthesis, but equivalent amounts of sodium butyrate have the same effects. We conclude that in GH-cells under carefully defined experimental conditions: 1) TRH causes an increase in intracellular cyclic AMP concentrations; 2) the increase in endogenous cyclic AMP and the effects of phosphodiesterase inhibitors are consistent with a model with cyclic AMP as a mediator of the effects of TRH on prolactin release; however, they do not prove this model, because the interpretation of these results depends on assumptions which may not all be valid; and 3) none of the analogs of cyclic AMP or the phosphodiesterase inhibitors tested mimic the decrease in growth hormone production caused by TRH.


Dog thyroid cells in monolayer tissue culture: adenosine 3', 5'-cyclic monophosphate response to thyrotropic hormone. A simple, rapid, and efficient method is described for establishing dog thyroid cells in tissue culture. Thyroid cell yield from a small amount of tissue (1 g) is high and viability is excellent. The adenosine 3',5-cyclic monophosphate (cAMP) response to thyrotropin (TSH) was investigated in these thyroid cells. Peak cAMP values were achieved after 10-15 min of TSH stimulation (in the presence of 0.5 mM 3-isobutyl-1-methyl-xanthine, MIX), with a subsequent decline to about half the maximal value after approximately 4 hours. This decline in cAMP concentration was associated with the development of refractoriness to TSH stimulation. Half-maximal stimulation of thyroid cell cAMP content was observed at a TSH concentration of between 1 and 2 mU/ml. Maximal cAMP values achieved were approximately 30-fold greater than basal values in the presence of MIX. The threshold of sensitivity of the cAMP response to TSH was very low, with significant stimulation being observed at a TSH concentration of 5-10 muU/ml. As determined by double reciprocal plots, the net cAMP response to TSH appeared to represent a single function over the entire TSH concentration range tested.


A receptor-like testosterone-binding protein in ovaries from estrogen-stimulated hypophysectomized immature female rats. A soluble thermolabile protein with many characteristics of an androgen receptor has been demonstrated for the first time in the cytosol (100,000 X g supernatant) of estrogen-stimulated ovaries from hypophysectomized immature female rats (HIFR) treated with diethylstilbestrol in Silastic capsules (DESC). This binding protein is organ-specific and androgen-specific, has a high affinity with a Kd of 2.4 X 10(-9)M for testosterone, and is saturable with 2.1 X 10(-13) moles of binding sites per mg cytosol protein. The number of binding sites is linear with cytosol protein concentration and the binding protein sediments at 7-8 S on sucrose gradients. Estradiol is an effective inhibitor of testosterone binding. A role for this testosterone-binding protein as an effector of ovarian morphologic change is hypothesized.


Hormonal control of renal phosphoenolpyruvate carboxykinase activity during development of the rat. The effect of injections of hormones in utero on fetal rat kidney and liver extramitochondrial phosphoenolpyruvate carboxykinase activity has been studied. Glucagon and thyroxine induced the liber enzyme but none of the hormones tested affected the renal enzyme. In the postnatal rat, the hepatic phosphoenolpyruvate barboxykinase activity is increased after triamcinolone or thyroxine injection but only triamcinolone injection increases the activity of the kidney enzyme. It is suggested that the rise in renal phosphoenolpyruvate carboxykinase activity at about 10 days of age is due to the increase in blood corticosterone content occurring at the same age.


Intramicrosomal location in intestine of enzymes which synthesize complex lipid. Smooth and rough microsomal fractions were prepared from hamster intestinal mucosa and assayed for RNA, diglyceride acyltransferase, cholinephosphotransferase and lysolecithin acyltransferase. The specific activity of cholinephosphotransferase was 4-fold more in the rough than in the smooth microsomal fractions, whereas diglyceride and lysolecithin acyltransferases were respectively 26 and 57% more active in the rough microsomal fraction. The two acyltransferases were similarly located in the microsomes and more closely corresponded to the location of dietary lipid than cholinephosphotransferase.


Undifferentiated patterns of key carbohydrate-metabolizing enzymes in injured livers. I. Acute carbon-tetrachloride intoxication of rat. In acute CCL4 intoxication of rats significantly increased activities of hepatic low-Km hexokinases, glucose-6-phosphate dehydrogenase, phosphofructokinase, aldolase A and pyruvate kinase M2 with concurrently decreased activities of glucokinase, glucose-6-phosphatase, fructose-1,6-diphosphatase, aldolase B and pyruvate kinase L were observed. The resulting enzyme pattern was apparently different from that in dietary induction. Principal component analysis revealed that the degree of enzyme deviation in the injured liver was much greater than that in the regenerating liver after partial hepatectomy and was closer to that in fetal liver or hepatoma tissue.


A reassessment of the phospholipid dependence of membrane-bound enzymes, with special reference to glucose-6-phosphatase and Na+, K+-dependent adenosine triphosphatase. It is suggested that the specific involvement of phospholipid in either the expression or constraint of glucose-6-phosphatase activity is not conclusively established by the existing experimental evidence. The physiological significance of an apparent requirement for phosphatidylserine for Na+, K+-dependent adenosine triphosphatase activity is also questioned. The need for a critical reassessment of past conclusions in the light of present knowledge is emphasized.


Serological detection of equid herpesvirus 1 infections of the respiratory tract. An investigation was made of 3 serological tests (virus neutralization, complement fixation and indirect immunofluorescence), which are applicable to epidemiological studies of infections by Equid herpesvirus 1 (EHV-1). Sera from gnotobiotic foals inoculated intranasally with various strains of EHV-1 were unable in some cases to neutralize heterologous strains and these results were not consistent with the existence of clearly-defined subtypes of EHV-1, as previously proposed. The cross-reactions in complement-fixation tests paralleled those with neutralization but immunofluorescence tests were found to be both more sensitive and more broadly reactive than the other two. Complement-fixing antibodies declined more rapidly following experimental infection than did those measured by neutralization or immunofluorescence. The results are discussed in relation to the diagnosis of EHV-1 infection and the significance they may have for the epidemiology of this disease.


A possible correlation between lipid hydration and lipid activation of the C55-isoprenoid alcohol phosphokinase apoprotein. 1. A direct method for determining the binding of triated water to lipids is described. The experimental conditions were practically identical to those previously employed (1974) in the determination of the cofactor activities of a series of oleyl-lipids in reactivation of the C55-isoprenoid alcohol phosphokinase apoprotein. 2. Active cofactor lipids (dioleyl lecithin, sodium oleate, 1-monoolein, 1-monomyristin)bound between 2.3 and 5.3 nmol 3H2O per nmol lipid, whereas less than 0.14 nmol 3H2O were bound per nmol of the inactive lipids (1,2- and 1,3-diolein, triolein, oleyl alcohol, methyl oleate, cholesteryl oleate). 3. When exposed to 3H2O vapour, the active lipids adsorbed between 1 and 2 nmol 3H2O per nmol lipid, whereas the inactive lipids adsorbed less than 0.1 nmol 3H2O per nmol lipid. 4. The active lipid cofactor, egg lecithin, bound more than twice as much 3H2O as egg phosphatidylethanolamine which was devoid of cofactor activity in the absence of detergent. 5. Appropriately hydrated lipid polar groups are concluded to be required for an alignment with polar amino acid side chains of the enzyme apoprotein in the formation of a mixed micellar lipoprotein complex. The enzyme reaction might occur at the resulting lipoprotein/water interface.


Homoserine kinase from Escherichia coli K12. Homoserine kinase was purified to apparent homogeneity from a derepressed strain of Escherichia coli K12, using standard fractionation techniques. It is a dimer (Mr = 60000) composed of apparently identical polypeptide chains (Mr = 29000). Its amino acid composition and N-terminal sequence have been determined. L-Threonine is a competitive inhibitor of the substrate L-homoserine; this inhibition is straighforward and shows no sign of co-operativity. Evidence is presented that homoserine and threonine bind to the same site of this non-allosteric enzyme. The binding of homoserine and threonine can also be studied by difference spectroscopy; the latter studies reveal an unexpected effect of magnesium ions, which might be the basis for the unusual high Mg2+ requirement for optimal enzyme reaction.


Phosphorylation of microtubule-associated proteins. 1. Tubulin is not an adenosine-3':5'-monophosphate-dependent (cyclic-AMP-dependent) protein kinase. Both entities have been clearly separated by sucrose gradient ultracentrifugation. With a tubulin preparation obtained by the polymerization-depolymerization technique protein kinase had a sedimentation coefficient of 8.7 S whereas tubulin sedimented with 6.4 S. After preincubation with both cyclic AMP and histone the kinase dissociated into its catalytic subunit with a sedimentation coefficient of 3.4 S. 2. Tubulin prepared by the polymerization-depolymerization technique was neither phosphorylated in vivo nor in vitro. On the contrary if this preparation was further purified by the Weisenberg's procedure (DEAE-Sephadex batch absorption) before incubation with [gamma-32 P]ATP, phosphorylation occurred. Thus, phosphorylation depended on the method used to purify tubulin i.e. was likely to an an artefact.


Conformational changes associated with transient activation of phosphorylase in glycogen particles. Studies using activity, electron-spin-resonance and phosphorus-nuclear-magnetic-resonance measurements. 1. Calcium-dependent transient phosphorylation of phorphorylase b has been monitored in a rabbit muscle glycogen particle fraction. Using a phosphorus nuclear magnetic resonance assay, the changes in concentrations of small phosphate-containing metabolites associated with this event have been measured. In addition, the conformation of phosphorylase has been monitored during transient activation by observing changes in the electron spin resonance signal from added spin-labelled phosphorylase. 2. The transient activation was associated with a loss of glucose-6-phosphate from phosphorylase b; newly formed phosphorylase a binds the nucleotides ADP, AMP, or IMP. Because of the fast interconversion of these nucleotides the species bound to phosphorylase a change throughout the process. 3. Lowering the [Mg2+] : [Ca2+] ratio during transient activation causes accumulation of ADP. Electron spin resonance data from spin-labelled phosphorylase shows that, under these conditions, ADP binding to phosphorylase a is potentiated. 4. Calcium-dependent activation in the glycogen particle fraction is compared to the activation of phosphorylase in vivo.


Spin-labelled AMP - an activator of phosphorylase. 1. A spin-labelled AMP derivative and its diamagnetic analogue activate phosphorylase b in the same way, but do not activate phosphorylase a. 2. The electron-spin-resonance spectra of the spin-labelled AMP derivative bound to phosphorylase b and a have "powderlike" characteristics indicating that the spin label is immobilised on the protein. From changes in the electron-spin-resonance spectrum of spin-labelled AMP as phosphorylase b or a is added, the dissociation constants were calculated. 3. The interactions of spin-labelled AMP and the diamagnetic analogue with phosphorylase b and a have been monitored by observing changes in the spectral properties of fluorescent and spin-label probes covalently attached to the enzyme. 4. The dissociation constants of spin-labelled AMP and phosphorylase b or a are 175 +/- 25 muM and 15 +/- 5 muM respectively. Similar dissociation constants are obtained for the diamagnetic analogue. The effect of these AMP derivatives on the covalently attached probe groups and on phosphorylase activity is compared to the effect of AMP and IMP.


Gangliosides of hepatoma 27, normal and regenerating rat liver. The highly malignant rat hepatoma 27 was found to have increased amounts of lipid-bound sialic acid as compared with normal liver whereas in regenerating liver the lipid-bound sialic acid level was reduced. In contrast to the liver the hepatoma contained higher amounts of disialogangliosides and no trisialogangliosides, which are abundant in the liver. The main disialoganglioside of the hepatoma had no analogue among the liver gangliosides and was identified as Gal-GalNAc(AcNeu-AcNeu)-Glc-Cer (GD1b), which in other tissues is known to be a precursor of trisialogangliosides. These findings may be explained by a reduced activity of glycosyltransferases in the hepatoma and apparently do not simply reflect differences in growth rate since the ganglioside pattern of regenerating rat liver was not altered significantly in comparison with the liver. Liver and hepatoma microsomes were found to be enriched in gangliosides as compared with whole cells, liver mitochondria were slightly poorer, while the ganglioside level of hepatoma mitochondria was much higher than that of the hepatoma cells. It thus appears that the existing image of the plasma membranes as the only sites of high ganglioside concentration may not hold true for weakly differentiated hepatomas of high malignancy.


Horse-liver alcohol dehydrogenase and Pseudomonas testosteroni 3(17)beta-hydroxysteroid dehydrogenase transfer epimeric hydrogens from NADH to 17beta-hydroxy-5alpha-androstan-3-one. An exception to one of the Alworth-Bentley rules. In the reduction of 17beta-hydroxy-5alpha-androstan-3-one to the 3beta-alcohol, horse liver alcohol dehydrogenase utilizes the 4-pro-R hydrogen of NADH whereas the 3(17)beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni utulized the 4-pro-S hydrogen. These observations provide an exception to the rule proposed by Alworth and Bentley that with regard to the paired methylene hydrogens at C-4 of NADH and NADPH "the stereospecificity of a particular reaction is fixed and does not vary with the source of the enzyme preparation". It is also apparent that for these two enzymes, the selection of the side of NADH from which hydride is transferred to substrate cannot in both cases be dictated by the "best fit" of substrate and cofactor.


Urinary excretion of adenosine 3'5' monophosphate in vitamin D deficiency. Urinary cyclic adenosine 3'5' monophosphate (AMP) excretion has been determined by radioimmunoassay in children with rickets and in control children. Cyclic AMP was greatly increased in children with rickets. The excretion of cyclic AMP correlated significantly with parathyroid hormone levels (PTH) and alkaline phosphatase, but not with age, calcaemia and serum inorganic phosphate. Calcium infusion led to a decrease in the excretion of cyclic AMP. The data are consistent with following hypothesis. During vitamin D deficiency, high PTH levels can increase the renal excretion of cyclic AMP. The effects of PTH on bone resorption fail to maintain the levels of serum calcium due to the lack of vitamin D. The mechanism by which the secondary hyperparathyroidism develops during vitamin D deficiency remains to be investigated.


Effects of dopamine on kidney function and on the adenyl cyclase phosphodiesterase system in man. Clearance studies were made to determine the influence of intravenous infusions of dopamine (between 2.5 and 3.5 mug.kg.-1min.-1) on renal function and on the adenyl cyclase phosphodiesterase system in eleven patients with chronic renal disease. Glomerular filtration rate (+ 19%), effective renal plasma flow )+ 29%), sodium (+ 199%) and potassium (+ 40%) clearances were significantly increased. These effects were associated with a stimulation of the adenyl cyclase phosphodiesterase system demonstrated by an increase of cyclic adenosine 3'5'-monophosphate in plasma and urine. The results suggest that dopamine probably affects renal function by activating the adenyl cyclase phosphodiesterase system.


The metabolism of low density lipoprotein in endogenous hypertriglyceridaemia. The metabolism of low density lipoprotein (LDL) was studied in eighteen hypertriglyceridaemic patients by injecting autologous radioiodinated LDL. Over 95% of the label was bound to the protein moiety of LDL and therefore the metabolic data reflect the fate and distribution of LDL apoprotein (apo B). The hypertriglyceridaemic subjects included ten with Type V, five with Type IV, two with Type III and one with Type IIb hyperlipoproteinaemia. For comparison identical studies were carried out in seven normal subjects and five patients with heterozygous familial hyperbetalipoproteinaemia (Type IIa). The groups differed considerably in mean LDL-cholesterol concentration. The patients with Type V lipoprotein pattern had significantly lower LDL-cholesterol concentration (mean 0.754 g/1) than the normal group (mean 1.237 g/1). Raised LDL-cholesterol levels were observed in all patients with heterozygous familial hyperbetalipoproteinaemia. The synthetic rate of LDL-apoprotein was found to be similar in all three groups (hypertriglyceridaemic, normal and hypercholesterolaemic). The highest synthetic rate was observed in the patient with Type IIb pattern. However, the fractional catabolic rate (FCR) of LDL-apoprotein differed significantly. The highest mean FCR was found in the Type V group (0.65 +/- 0.17 day-1) compared with 0.41 +/- 0.09 day-1 in the normal group and 0.185 +/- 0.05 day-1 in the Type IIa group. A strong inverse correlation was found between FCR and LDL apoprotein concentration in the whole series (r = -0.90, p less than 0.001) as well as within the Type V group (r = -0.87, p less than 0.01). These data indicate that the low plasma levels of LDL frequently observed in patients with very high plasma triglyceride levels are due to a high removal rate of LDL in these patients rather than to abnormal LDL synthesis.


Lipids and lipoproteins in chronic uraemia. A study of the influence of regular haemodialysis. Plasma lipids and lipoproteins were studied in a group of chronic uraemia patients some of whom were maintained by regular haemodialysis. Compared with healthy individuals, there was a significant increase in plasma triglycerides and in the prebeta-1- and prebeta-2-lipoprotein plasma concentrations. There was no difference between dialyzed and undialyzed patients. Carbohydrate intake was normal, basal plasma insulin and free fatty acid levels were within the normal range. There was no correlation between plasma triglyceride levels and the degree of hypoalbuminaemia, the latter being marked in 30% of the patient. Basal plasma glucagon levels were very high in nearly all dialyzed patients and post-heparin lipoprotein-lipase activity was very low in dialyzed patients. In our experience, regular haemodialysis for 32 weeks did not improve hypertriglyceridaemia.


Metabolism of very low density lipoproteins in hyperlipidaemia: studies of apolipoprotein B kinetics in man. The metabolism of very low density lipoprotein-B (VLDL-B) peptide was studied in nineteen subjects with endogenous hypertriglyceridaemia (Types V, IV and IIb), three patients with heterozygous familial hyperbetalipoproteinaemia (Type IIa) and eight healthy subjects, by reinjecting autologous radioiodinated VLDL. The kinetics of VLDL-B peptide were followed. The mean turnover rate of VLDL-B peptide was significantly higher in the hypertriglyceridaemic group than in the control group but a considerable overlap in turnover rate was found between these groups. The patients with heterozygous familial hyperbetalipoproteinaemia had a normal turnover rate of VLDL-B peptide. A significant positive correlation was found between the turnover rate of VLDL-B peptide and VLDL-triglyceride concentration in the whole series. It is concluded that the underlying defect in endogenous hypertriglyceridaemia is heterogeneous. Overproduction of VLDL is a major determining factor in some patients whereas a reduced clearance is the determining factor in others.


Central dopaminergic neurons: effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid. Stimulation of the nigro-neostriatal or mesolimbic dopamine pathway results in a stimulus dependent increase in the accumulation of dihydroxyphenylacetic acid (DOPAC) in the neostriatum and olfactory tubercles, respectively. A block of impulse flow induced pharamacologically by administration of gamma-butyrolactone or by placement of a lesion in the dopamine pathway results in a decrease in the steady state levels of DOPAC. Drugs which have previously been shown to alter impulse flow in central dopaminergic neurons also produce a predictable change in the brain levels of DOPAC. Drugs which increase impulse flow in nigro-neostriatal or mesolimbic dopamine neurons increase DOPAC levels in the striatum and olfactory tubercles and drugs which reduce impulse flow cause a reduction in DOPAC. Pargyline, a monoamine oxidase inhibitor, causes a rapid depletion of striatal DOPAC suggesting that this metabolite is rapidly cleared from the brain. Administration of benztropine, a potent inhibitor of dopamine reuptake, causes a significant decrease in striatal DOPAC and partially prevents the stimulus-induced increase in the accumulation of DOPAC. These observations together with the finding that about 85% of the DOPAC in the striatum disappears when the dopamine neurons in the nigro-neostriatal pathway are destroyed suggests that the majority of striatal DOPAC is formed within the dopaminergic neurons and may reflect the metabolism of dopamine which has been released and recaptured. We conclude that short-term changes in brain levels of DOPAC appear to provide a useful index of alterations in the functional activity of central dopaminergic neurons.


Automatic registration of behaviour related to dopamine and noradrenaline transmission. In a search for behavioural tests where certain behaviours related to dopamine and noradrenaline transmission can be recorded automatically we have developed and tested an automatic version of the hole board. The test measures two behaviour variables: the open field variable defined as the number of interruptions of photocell beams symmetrically covering an open field area and the 'hole' variable, defined as the number of head-dips into holes recorded by photocell beams positioned underneath the floor of the cage. The method was evaluated by observations of the rats concomitant with the automatic registrations. The animals were tested on dopamine agonistic drugs, which were found to decrease the 'hole' counts and cause an increase in the open field counts as compared to saline injected controls. d-Amphetamine which is known to increase the release of dopamine as well as of noradrenaline caused an increase in both the open field counts and the 'hole' counts. The increase in 'hole' counts caused by d-amphetamine was reduced when the animals were pretreated with a dopamine-beta-hydroxylase inhibitor (FLA 63) or a noradrenaline receptor blocker (phenoxybenzamine). These results suggests that the increase in the 'hole' variable was related to an increased noradrenaline transmission while the increase in the open field variable was related to an increased dopamine transmission. The lowest dose of apomorphine caused a behavioural inhibition which may be explained by a preferential stimulation of dopamine autoreceptors.