Malignant mesotheliomas in a small village in the Anatolian region of Turkey: an epidemiologic study. An epidemiologic and cause-related study was done in Tuzköy, a small village in the city of Nevşehir, in the Anatolian region of Turkey. The neighboring village of Kizilböy was used as a control. People 25 years of age or older were studied: 312 persons (145 males and 167 females) from Tuzköy and 95 persons (45 males and 50 females) from the control village. The annual incidence of malignant pleural mesothelioma (MPM) was found to be 6.5 cases (22 cases per 10,000 people) in Tuzköy. Several other respiratory disorders were detected as well. Although no type of asbestos could be found in Tuzköy and its vicinity, the asbestiform mineral zeolite was found in soil samples from the roads and fields of Tuzköy, in its building stones, and in lung tissues of the villagers. Chest X-rays revealed no cases of MPM or other respiratory abnormalities in the control group. No zeolite could be found in the control village. Therefore, zeolite was thought to be the cause of MPM and the other respiratory disorders in Tuzköy.


Sequential histopathology and cell kinetic changes in rat pyloric mucosa during gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine. The effect of administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at a dose level of 83 mg/liter in the drinking water was followed in the pyloric mucosae of male noninbred Wistar rats. Autoradiographic studies were done on animals killed after 10, 15, 26, and 36 weeks of treatment. In the normal-appearing mucosae of the rats treated with MNNG for 10 weeks, the number of epithelial cells per pit column was significantly increased over that in control rats. Simultaneously, a shift in the major zone of epithelial cell proliferation was noted in the treated rats. Along with the formation of a longer pit in MNNG-treated rats, the greatest number of DNA-synthesizing cells was displaced from the middle third of the pit in a downward direction toward the muscularis mucosa. In addition, at this early experimental time period, pits lined with more immature, cuboidal, mucus-depleted cells were recognizable. These pits not only had higher labeling indices than normal-appearing pits of the same animals but also expressed a dual nature with increased proliferative activity extending either upward to the luminal surface or further in a downward direction. Focal areas of cellular atypism were present by week 10 of treatment with a threefold to sevenfold greater DNA synthesis activity than that found in the normal-appearing mucosa of the same animal. A wide range of values in proliferative activity was found not only among invasive pyloric tumors within the same animal but also within different areas of the same tumor. The mechanism for the formation of adenomas and invasive adenocarcinomas is believed to be related to the dual character of the hyperplastic pits described.


Carcinogenicity of N-nitrosobis(2-hydroxypropyl)amine and N-nitrosobis(2-oxopropyl)amine in MRC rats. Weekly sc injections of equitoxic doses of N-nitrosobis(2-hydroxypropyl)amine (BHP) and N-nitrosobis(2-oxopropyl)amine (BOP) to Wister-derived MRC rats induced tumors. The incidence, latency, multiplicity, morphologic type, and distribution of these tumors varied according to the compound given. The esophagus was the main target organ for BHP (100%), followed by the respiratory tract (87%), pharynx (80%), colon and liver (each 73%), kidneys (20%), thyroid gland (20%), and urinary bladder and urethra (each 7%). BOP was ineffective in the esophagus and pharynx but induced a higher incidence of tumors in the kidneys (27%), thyroid gland (60%), urinary bladder (33%), and urethra (73%) and fewer neoplasms in the respiratory tract (20%), colon (67%), and liver (53%). In addition, BOP caused a few, apparently primary, prostate squamous cell carcinomas. The results are compared with results of BHP treatment in Sprague-Dawley rats and with results of BHP and BOP treatment in Syrian golden hamsters.


Tumorigenicity of the diastereomeric benz[a]anthracene 3,4-diol-1,2-epoxides and the (+)- and (-)-enantiomers of benz[a]anthracene 3,4-dihydrodiol in newborn mice. The tumorigenic activity of benz[a]anthracene (BA), the (+)- and (-)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene (BA 3,4-dihydrodiol), and the racemic diastereomers of the BA 3,4-diol-1,2-epoxides [i.e., either or both of the diastereomeric 1,2-epoxides derived from BA 3,4-dihydrodiol in which the epoxide oxygen is cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 4-hydroxyl group) was examined in newborn Swiss-Webster mice. The mice were administered ip a total dose of 280 nmoles of compound in divided doses consisting of 40 nmoles within 24 hours of birth, 80 nmoles at 8 days of age, and 160 nmoles at 15 days of age. The experiment was terminated when the animals were 26 weeks of age. BA 3,4-diol-1,2-epoxide-2 was the most potent compound tested. All animals treated with BA 3,4-diol-1,2-epoxide-2 developed pulmonary tumors with an average of 13.3 tumors per mouse. BA 3,4-diol-1,2-epoxide-1 produced pulmonary tumors in 42% of the mice with an average of only 0.56 tumors per mouse. The (-)-enantiomer of BA 3,4-dihydrodiol with [3R,4R] absolute stereochemistry was the second most tumorigenic derivative of BA tested; it produced pulmonary tumors in 71% of the mice with an average of 1.88 tumors per mouse. BA and the (+)-enantiomer of BA 3,4-dihydrodiol had little or no tumorigenic activity at the dose tested. A comparison of the average number of pulmonary tumors per mouse revealed that BA 3,4-diol-1,2-epoxide-2 was about 30-fold more tumorigenic than was BA 3,4-diol-1,2-epoxide-1, 8-fold more tumorigenic than was (-)-BA 3,4-dihydrodiol, and greater than 85-fold more tumorigenic than was BA. These data indicate that in newborn mice BA 3,4-dihydrodiol and a BA 3,4-diol-1,2-epoxide are proximate and ultimate carcinogenic metabolites of BA, respectively.


Renal carcinogenic and nephrotoxic effects of the flame retardant tris(2,3-dibromopropyl) phosphate in F344 rats and (C57BL/6N X C3H/HeN)F1 mice. Tris(2,3-dibromopropyl) phosphate (TBP) was administered in the feed at one of two concentrations to groups of 55 male and 55 female inbred F344 rats and to 50 male and 50 female (C57BL/6N X C3H/HeN)F1 mice. The high and low dietary concentrations of TBP administered orally were 100 and 50 ppm for the rats, respectively, and 1,000 and 500 ppm for the mice, respectively. For each rodent type, 55 animals of each sex were used as contols. In both rodent types, renal epithelial tumors developed at incidences that were significant for male and female rats and mice that received the doses. These tumors included renal tubular cell adenomas and carcinomas that developed from the proximal convoluted tubular epithelium. Among female mice and rats, hyperplasia and/or dysplasia of the proximal convoluted tubular epithelium with or without cystic dilatation of the tubules and increase in the size of cell nuclei were dose dependent and recognized as preneoplastic and/or toxic lesions. The comparative histogenesis of renal tubular neoplasms was discussed.


Epidemiology of cancer of the cervix in Buffalo, New York. An investigation was done to determine whether patients with cancer of the cervix from Buffalo and Kenmore, New York, differed from a random sample of women from the same communities on a variety of parameters associated with marital and reproductive history. A comparison of the 285 patients and 1,620 controls showed that patients were more likely than controls to have been black and non-Jewish, to have married young, to have had more than one husband, and to have been young at the time of their first pregnancy. In addition, they were more likely to have frequently used the vaginal douche and to have used it over many years. As frequency of douche increased, so did risk of cervix cancer.


Inhibition of human leukocyte migration in agar by 3-M potassium chloride extracts of stomach, colon, and lung cancers. Inhibition of leukocyte migration in agarose-agar was used as a probe for tumor-associated antigen in 3-M KCl solubilized extracts of gastric, colon, and lung cancers from humans. Twelve of 40 (30%) leukocyte preparations from gastric cancer patients, 10 of 21 (48%) from colon cancer patients, and 7 of 14 (50%) from lung cancer patients were inhibited by their respective histologically homologus cancer extract. However, among 75 preparations from various cancer patients, leukocytes from only 2 gastric cancer patients were inhibited by paired normal gastric tissue extracts. Only 2 of 68 preparations from normal individuals and none of 67 preparations from patients with nonmalignant diseases, such as gastric peptic ulcer, gastritis, colon polyposis, colitis, pulmonary tuberculosis, chronic bronchitis, and sarcoidosis, were inhibited by cancer extracts. These findings suggest the presence in KCl extracts of gastric cancer of presumed tumor-associated antigen(s) that is antigenically distinct from that of either colon or lung cancer.


Inhibition of thymidine incorporation into epidermal and dermal DNA of HRS/J mice after a Colcemid pulse. The introduction of a Colcemid pulse, which is known to inhibit microtubulin assembly and thereby influence cellular events dependent on the assembly, delayed the normal passage of G1 cells to S-phase. Experimental support for this hypothesis was obtained by the pulsing of cultured inbred HRS/J mouse skin for 30 minutes with 20 micrograms Colcemid/ml and by measurement of thymidine (dThd) incorporation for a 4-hour incubation period at different intervals after the pulse. Incorporation of dThd into epidermal and dermal DNA was maximally inhibited 9.5 and 17.5 hours after the Colcemid pulse. Maximal inhibition and minimal inhibition of dThd incorporation were observed 17.5 hours after different concentrations of Colcemid (1--30 micrograms/ml) were pulsed. Maximal inhibition of dThd incorporation occurred after pulsing with 20 micrograms Colcemid/ml. The acceleration of G0 or G1 events to S-phase in proliferative cells caused by mild epidermal stripping was also inhibited after the addition of the Colcemid pulse. The data suggest a time relationship between the Colcemid pulse, the subsequent disruption of microtubulin structure, and the inhibition of dThd incorporation into epidermal and dermal DNA. The intact microtubulin structure is apparently essential for the normal intracellular dependency among the cell membrane, the cytoskeleton structure, and the cell nucleus and occurs when the cells transit from G1 to S-phase.


Effect of palytoxin and serotonin on murine tumor cells. In vitro and in vivo effects of two fractions of a crude extract from the marine invertebrate Palythoa were investigated in Novikoff hepatoma and Ehrlich ascites tumor cells. These fractions, previously identified as serotonin and a partially purified toxin, inhibited thymidine incorporation into acid-precipitable material by tumor cells. Results from respiration studies indicated that the inhibitory effect was not toxic. The data suggested that: a) The partially purified toxin exerted its inhibitory effect at the membrane level; b) this inhibitory effect might have been accomplished through the Na+K+ transport mechanism, this conclusion being supported by data on the uptake of labeled amino acids by Ehrlich ascites tumor cells; and c) in an unconfirmed experiment, the partially purified toxin increased the survival time of 50% of 6 palytoxin-treated mice at least three times as long as 6 untreated control mice. All 12 mice had received 1 X 10(6) viable Ehrlich ascites tumor cells.


Effect of inhibitory and stimulatory agents on protein synthesis in hepatomas and host livers of rats. Female inbred BUF rats bearing intrahepatically transplanted hepatomas (5123 or 19) were subjected to acute exposure to a variety of hepatotoxic agents (actinomycin D, aflatoxin B1, CCl4, dimethylnitrosamine, ethionine, puromycin, or sparsomycin) or of stimulatory agents (hydrocortisone, phenobarbital, or whole-body X-ray). The responses in terms of changes in polyribosomes and protein synthesis (in vitro and in vivo) of host liver and hepatoma were evaluated. The responses of the host livers and hepatomas to the different agents varied. In general, the host livers responded much more than did the hepatomas. Of the two hepatomas, hepatoma 19 responded less (particularly in terms of polyribosome changes) than did hepatoma 5123. In a few experiments, different doses of actinomycin D, ethionine, or sparsomycin were used and in all instances the host livers responded more than did the hepatomas.


Histopathology of liver carcinomas in (C57BL/6N X C3H/HeN)F1 mice ingesting chlordane. (C57BL/6N X C3H/HeN)F1 male mice that ingested 30 or 56 ppm chlordane and female mice that ingested 30 or 64 ppm chlordane in the diet had highly significant incidences of carcinomas of the liver. The carcinomas varied from well differentiated to poorly differentiated and undifferentiated and were capable of invasion and metastasis. They were more poorly differentiated in mice receiving chlordane than in controls.


[ALL therapy study 1971-1974 of the German working group for leukemia research and therapy in childhood: prognostic significance of initial features and different therapeutic modalities (author's transl)]. 495 children and adolescents with acute lymphoblastic leukemia entered a non-randomized therapy study between January 1st, 1971, and December 31st, 1974. They were treated at 40 pediatric clinics in the Federal Republic of Germany according to the protocol of the Deutsche Arbeitsgemeinschaft für Leukämieforschung und -Behandlung im Kindesalter, which was closely adapted to the Memphis protocol VII. These patients were analyzed retrospectively, data of evaluation was July 31st, 1978. 7 patients (1.4%) did not achieve remission, 14 patients (2.8%) died during the induction period, and another 37 patients (7.5%) died in continuous complete remission (CCR). For purposes of comparison only the remaining 437 patients were evaluated (remission group). The 7 1/2 years' probability of CCR is 0.33 +/- 0.02 for the total group (495 patients) and 0.37 +/- 0.03 for the remission group. The probability of survival for these groups is 0.40 +/- 0.03 and 0.45 +/- 0.03, respectively. After relapse the 5 years' probability of survival is 0.03 +/- 0.02. Patients with testicular relapse have a markedly better prognosis compared to those with bone-marrow or central nervous system relapses. The most important prognostic factor was found to be the white blood count at diagnosis. There is a negative correlation between the initial white blood count and the probability of CCR. Girls fared significantly better than boys (0.46 +/- 0.04 vs 0.31 +/- 0.03 in CCR, p less than 0.01). There is convincing evidence that the impaired prognosis in boys is at least partly due to the incidence of testicular relapses (36 of 268 relapses were found to be testicular, isolated in 20 patients and combined with bone-marrow relapse in 16 patients). Patients with thymic involvement and/or positive reaction of the acid phosphatase in their blast cell did significantly worse (0.12 +/- 0.05 in CCR). Irradiation of the skull was performed in two different doses (1800 and 2400 rads). Concerning the incidence of relapses primarily located in the central nervous system patients with low-dose irradiation had no worse prognosis than those with high-dose irradiation but the incidence of bone-marrow relapses was found to be higher in the 2400 rad group (50.5% vs. 41.7% in the 1800 rad group), possibly due to the higher initial white blood count (median 8900/mul vs. 6500/mul). The addition of vincristine/prednisone reinduction courses during the first year of continuation therapy showed no significant advantage.


[The prognostic value of measuring cell size in acute childhood leukemia (author's transl)]. Prior to treatment we studied leukemia cell diameters in bone marrows of 89 children with acute lymphoblastic leukemia (ALL) and 26 children with myeloblastic leukemia (AML). Patients were treated according to the West-Berlin protocol. The median cell diameter (+/- S) in ALL measures 11,3 (+/- 1,1)mu, in AML 15,3 (+/- 1,1)mu. Both forms of leukemia show some overlapping of cell diameters in the range of 13,8--14,3 mu. with ALL, no difference is shown in the cell diameters of those who remained in complete remission or went into relapse. Likewise, there is no significant difference in cell-diameter, when correlating them to the following parameters: initial peripheral leucocyte count, acid  phosphatase- and PAS-reaction, T-cellmarkers. The risk of relapse is the same in patients with more than 20% macrolymphoblasts (diameter greater than 12mu) and in those less than 20% macrolymphoblasts. In 11 patients with ALL cellmeasuring was performed during the first hematological relapse. The median cell-diameter in 10 of these cases is higher than in the initial bone marrow.


[Concordant leukemia in identical twins (author's transl)]. We report female monozygous twins who developed acute lymphoblastic leukemia at the age of 5 1/2 years. The diagnosis in the first twin was made after pallor, lethargy, and petechiae developed. The diagnosis in the second twin was made two days later when a whole blood count was taken. The lymphoblasts of both patients showed with the exception of the PAS-reaction identical morphological, cytochemical, and immunological results. The PAS-reaction was positive in 55% of the lymphoblasts in one twin, negativ in the lymphoblasts of the other twin. Both patients are in continuous complete remission 14 months after diagnosis. The risk of leukemia is high in the other monozygous twin when one of the twins has already developed leukemia. In the literature it is estimated to be 1 : 5. The diagnosis after the second year of life is rarely made at the same time. There is only one previous report of this occuring in a case of acute myeloblastic leukemia in 4 1/2 year old monozygous twins.


[Occurrence of thyroid carcinoma after treatment of acute lymphoblastic leukemia (author's transl)]. A 21 month old boy with acute lymphoblastic leukemia diagnosed on 17th September 1971, was treated according to the Memphis Study VII of Pinkel. Bone marrow examination, 39 days after treatment, showed complete remission. The patient has since then been in continuous complete remission. Therapy was suspended twice (29 days and 18 days) because of interstitial pneumonia. The treatment was terminated on 9th October 1974 nearly 3 years after onset of the disease. Because of papillary thyroid carcinoma with metastases in the regional lymph nodes, a total thyreoidectomy with removal of lymphnodes was performed on 30th April 1977. Because of a relapse in the neck lymphnodes, the patient was treated in December 1977 with radioiodine. The prognosis is however not discouraging.


[Clinical trials with mercapto polycytidylic acid in the treatment of acute childhood leukemia (author's transl)]. Mercapto Polycytidylic acid (MPC) is a potent inhibitor of oncornaviral reverse transcriptase. The fact that this enzyme has been found in human leukemic cells, has let to the clinical trials of MPC in the treatment of childhood acute leukemia. This report discribes the first pilot clinical trial on 23 patients. The results are encouraging, and have let to a second pilot study including other clinical centers on the clinical efficacy of MPC in the treatment of leukemia.


[Common ALL-associated antigen on cells and in the serum of common ALL patients: clinical relevance and biochemical characterization (author's transl)]. The clinical application of an antiserum recognizing common ALL associated antigen (cALL-AG) is very useful in classifying leukemias and diagnosing bone marrow relapse as well as CNS-leukemia. We could demonstrate that sera of common ALL (cALL) patients contain cALL-AG; its partial biochemical characterization is described. The anti cALL serum (cALL-AS) was raised in rabbits with cALL-cells precoated with rabbit antiserum against normal human lymphocytes. After appropriate absorbtion the cALL-AS was highly specific for cALL cells. The isolation of serum cALL-AG was performed by ammoniumsulfat precipitation, gel chromatography and affinity chromatography on agarose lens culinaris hemagglutinin A (lentil lectin). The apparent molecular-weight of the serum glycoprotein is 125 000. Two cALL-AG active structures could be solubilized from cALL cell membrane. The apparent molecular-weights were calculated to be 55 000 and 110 000.


[Prognosis improvements in children with acute myelocytic leucemia after more intensive induction therapy (author's transl)]. Between October 1974 and October 1978 23 children with acute myelocytic leucemia (AML) received intensive therapy in the Univ.-Kinderklinik Münster: 4 children were treated according to the ALGB-protocol consisting of 5-7 day courses of ARA-C-infusion and 3 DNR-injections. 19 patients received the West-Berlin-protocol: The first 7 the original ALL protocol, 11 the modified form of AML, which will be presented here as AML-therapy-study BFM 78. 4 of the 23 patients died with early acute cerebral bleeding. 2 patients were nonresponders. 17 children went into remission. One girl died in remission of septicemic aspergillosis. 4 children had a relapse. In November 1978 there were still 12 patients in continuous complete remission, 3 of them already without therapy. 13 of the 19 patients, who were treated with the West-Berlin-protocol went into remission. 1 had a relapse. At present there are 11 patients in continuous complete remission. The above results and those found in the literature could signify that the long term prognosis of children with AML will be improved. To coordinate efforts toward this goal a cooperative AML-therapy-study in the "Deutsche Arbeitsgemeinschaft für Leukämieforschung" (BFM-group) using the here presented therapy protocol was formed in November 1978.


[Bleeding complications in acute myeloblastic leukemia (author's transl)]. Bleeding is common in acute myeloblastic leukemia (AML). At the time of diagnosis, the danger of bleeding cannot be predicted by laboratory means. However, the following factors represent increased risks: Promyeloblastic leukemia, high blast count, low fibrinogen, low plasminogen. From coagulation studies performed at the time of bleeding complications, the pathomechanism leading to bleeding complications usually cannot be detected. The question whether impairment of production, consumption coagulopathy, or primary fibrinolysis causes the bleeding complications can only be answered by controlling frequently clinical and hemostatic criteria, which include the thrombocytic stystem as well as plasmatic coagulation and fibrinolysis. At the present time, the therapy of bleeding complications in AML is symptomatic. It consists of transfusion with thrombocytes or fresh whole blood, respectively. Coagulation factor concentrates should only be given in combination with Heparin to prevent the deterioration of consumption coagulopathy.


Nocturnal intragastric therapy in type I glycogen storage disease: effect on hormonal and amino acid metabolism. The acute hormonal and amino acid responses to differing food substrates were examined in type 1 glycogen storage disease. Ingestion of a glucose load or a glucose-plus-beef meal caused an acute fall in the initially elevated plasma glucagon, alanine, proline, and lactate. Ingestion of beef alone caused a sharp rise in these parameters. Long term nocturnal intragastric therapy of a high carbohydrate and moderate amino acid content resulted in a similar fall in these parameters as well as a fall in the elevated plasma glutamate, uric acid, triglycerides, and RBC-reduced glutathione. A remarkable clinical improvement and growth spurt accompanied the improvement in these biochemical values. The possible relation between the disturbed plasma hormonal and amino acid findings and growth failure and hyperuricemia is discussed.


N-independent leftward transcription in coliphage lambda: deletions, insertions and new promoters bypassing termination functions. Lambda mutants capable of N-independent red-gam gene expression were isolated by selecting Fec+ plaque-forming derivatives of lambda N+ nutL- (Fec-) strains. In addition to true nutL+ reversions, three classes of second-site mutations were identified: (1) ninL deletions that remove a region containing either tL1 or both tL1 and tL2 termination signals, or only a small region (defining the rut site) just upstream from tL1, (2) new constitutive promoters that map just upstream from the tL2 termination site and which are created either by point mutations (hip) or by short insertion sequences (isp), (3) small internal deletions in gene cro. The positions and individual effects of these mutations, some of which only partially abolish termination function, provide evidence for a complex multipartite structure of the termination signals.


Mitochondrial DNA from Podospora anserina. I. Isolation and characterization. Mitochondrial (Mt) DNA from Podospora anserina was isolated and characterized with respect to density in CsCl, contour length and endonuclease restriction enzymes. The density of Mt DNA for four races examined was 1.694 g/cm3, compared with 1.712 g/cm3 for nuclear DNA. Extraction in the presence of a nuclease inhibitor, aurintricarboxylic acid and isolation in DAPI CsCl gradients allowed us to isolate high molecular weight DNA. Mt DNA isolated by total DNA extraction contained ca. 1% of circular molecules, 31 micron in contour length; Mt DNA isolated from purified mitochondria contained 2--4% of these 31 micron circles. Analysis with Eco RI restriction endonuclease revealed that each of the four races examined, s, A, T and E had a characteristic fragment pattern. Races s and A Mt DNA differed by only one fragment after Eco RI enzymatic digestion; similarly, these two DNA differed by only one or two fragments after Hae III digestion.


Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures. Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 mu) were present; instead, smaller circles characteristic for each mutant studied were found. Eco R1 digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type. The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 mu. These circles ranged in size from 0.89 mu to greater than 20 mu; only one molecule out of some 200 molecules was thought to be of full length (31 mu). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm3) and a second at 1.699 g/cm3. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23 x 10(6) daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20 x 10(6) daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length. These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho- mutation in the yeast, S. cerevisiae.


The nucleotide sequence of the region surrounding the replication origin of an R100 resistance factor derivative. The replication origin of a group of small plasmids derived from R100 was previously determined by electron microscopy (Ohtsubo et al., 1977). This region was subjected to extensive restriction enzyme analysis and the nucleotide sequence of the region containing the replication origin was determined using the Maxam and Gilbert sequencing technique. Various characteristics of this sequence, including a very interesting secondary structure are described and discussed.


Bacterial DNA synthesized under phage control in a DNA-defective Salmonella-mutant and packaged into a special fraction of transducing particles of phage P22. Lysates of P22 contain a small fraction of transducing particles with bacterial DNA replicated semiconservatively after the time of infection. It was demonstrated that the presence and relative amount of this class of transducing particles was unchanged, if infection of Salmonella occured under a condition nonpermissive for bacterial DNA replication. Analysis of particles with DNA fragments derived from different regions of the Salmonella chromosome indicated that the replication of the bacterial DNA carried by these transducing particles was not initiated specifically at the normal origin for bacterial chromosome replication.


Polar uncoating of tobacco mosaic virus (TMV) with dimethylsulfoxide (DMSO) and subsequent reassembly of partially stripped TMV. Increasing concentrations of dimethylsulfoxide (DMSO) strip tobacco mosaic virus (TMV) stepwise from the 3'end. The RNA tail increases in length up to 2,000 nucleotides (nu) reaching a region of very strong protein-RNA affinity. Thereafter, uncoating occurs from the other end and produces a second RNA tail 500 nu long. Further stripping of TMV proceeds from both ends, the long tail increasing in length up to 4,000 nu and the short one increasing more moderately and remaining below 2,000 nu. The region of strongest protein-RNA affinity is located between 4,000 and 5,000 nu away from the 3' end. Using the same conditions as for in vitro TMV reassembly, it is possible to recoat the RNA tails with viral protein preferentially in the 5' direction. The advantages of DMSO in studies of TMV protein-RNA interactions are discussed.


The arrangement of nucleotides in the coding regions of natural templates. The relation of the nucleotide sequences in the coding regions of natural templates and of the short nucleotide sequence in 3' terminus of 16 S ribosomal RNA was found to differ from random pattern. The observation is interpreted in terms of both the ribosomal interactions and the molecular evolution.


Gene expression in Drosophila: post-translational modification of aldehyde oxidase and xanthine dehydrogenase. Maroon-like homozygotes are completely deficient for xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), however, ma-l is not a structural locus for either enzyme. Quantitative immunoelectrophoresis of ma-l and wild type extracts suggests that the ma-l function must be post-translational. To determine whether the ma-l function involves some direct physical changes in XDH and/or AO the enzymes were characterized with respect to temperature sensitivity and behavior in gel sieving electrophoresis. Since the XDH and AO from complementary ma-l heterozygotes is more thermolabile and different in shape from wild type XDH and AO, we conclude that ma-l is involved in a post-translational modification of these enzymes.


Acute nonlymphocytic leukemia: expression in cells restricted to granulocytic and monocytic differentiation. Two patients with acute nonlymphocytic leukemia who were heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase were studied to determine the number and type of cells in which the disease arises. Both type A and B isoenzymes were found in normal tissues, but the myeloblasts showed only one enzyme type, indicating that at the time of study, the disease had a clonal origin. The observation in one patient that erythroid cells did not arise  from this clone contrasts with conclusions reached in patients previously studied with chromosomal markers. The results suggest that in this patient, the leukemic clone suppressed expression of normal granulopoiesis but did not inhibit erythroid differentiation from normal progenitors. They suggest also that the disease is heterogeneous. In some patients, the disease is expressed in cells with differentiation restricted to the granulocyte-macrophage pathway; in others, it involves stem cells that also differentiate into erythrocytes. This heterogeneity may reflect differences in causation and could have prognostic importance.


[The fine structure of granulocytic sarcoma (author's transl)]. Granulocytic sarcoma, a rare manifestation of leukemia, can present as solid and invasive tumors in the central nervous system. We report electron microscopic findings in two such cases of granulocytic sarcoma in this communication. The first case is a granulocytic sarcoma involving mainly the left frontal dura with invasion of the underlying leptomeninges and brain. This was the initial presentation of acute myelogenous leukemia that became apparent seven months later. The second case is a granulocytic sarcoma involving the cervical spine and epidural soft tissue in a known case of chronic myelogenous leukemia. Electron microscopic studies confirm the presence of immatuure granulocytic cells with specific granules which distinguish these cases from other tumors such as a malignant lymphoma. Granulocytic sarcoma should be considered in the differential diagnosis of neurosurgical cases which present clinically as acute intracranial dural or spinal epidural tumors.


The correlation of the computer quantitated treadmill exercise electrocardiogram with cinearteriographic assessment of coronary artery disease. One hundred and twenty-five consecutive patients being submitted for coronary cinearteriography were subjected to preliminary graded exercise testing using a Quinton treadmill and ECG data computer. Patients with disease of the left main coronary artery are at particular risk during coronary arteriography and it was hoped that exercise testing might identify these patients. This hope was not realised. A satisfactory end point to the exercise test was either the attainment of 85% of the maximal heart rate assessed for age, or ST segment depression greater than 2 mm. Exercise induced angina also formed an end point to the test and was considered a positive result. Results were determined by McHenry's discriminant analysis. The sensitivity of the exercise test was 66, 88 and 92% for one, two and three vessel disease respectively. Specificity was at least 83%. Exercise induced ST segment elevation occurred in four patients all of whom had previous extensive anterior wall infarction. Exercise testing is without risk in patients with ischaemic heart disease provided well defined precautions are taken.


A surgical approach to primary hyperparathyroidism, 1971-1976. Forty-two patients with primary hyperparathyroidism were seen in Auckland Hospital between 1971-1976. These patients were reviewed as to age, sex, mode of presentation, pathology and location of lesions. All were treated surgically. There were two deaths due to severe hypercalcaemia. The value of parathyroid hormone assay and neck vein catheterisation is assessed. The practical aspects of neck exploration for hyperparathyroidism are outlined. The two problems of the treatment of chemical hyperparathyroidism and the recognition and treatment of parathyroid hyperplasia are discussed.


Upper limb replantation: a 3-year follow-up. A case report of an upper limb replantation is presented detailing the initial treatment and the functional results three years later. Although wasted in appearance, the limb is very useful and the patient has been able to successfully adapt to his resulting residual disability.


Congenital dislocation of the hip over a ten-year period. A Malmo type neonatal screening programme for congenital dislocation of the hip has been conducted in New Plymouth by orthopaedic surgeons from 1964 to 1975 including 10 103 births. Follow-up for the 11-year period appears complete. Treatment by a Von Rosen type splint for six weeks instituted within seven days of birth appears to prevent established late CDH. Subluxation at six months, however, occurred twice and a longer period of splintage might be advisable. Fourteen cases of "walking" CDH have been prevented, with a 92% success rate, with five and 10-year x-ray follow-up demonstrating good hip development in treated cases.


Bacterial contamination of radial artery catheters. A prospective survey of 113 radial artery catheters showed a total contamination rate using an enrichment media culturing technique of 39%. The yield from routine culturing alone was only 9%. In six cases pathogenic bacteria were isolated. In this study no correlation between contamination and aspects of catheter insertion and management was shown.


Planning services for the intellectually handicapped in North Canterbury. The development of a wider range of service alternatives for the care of the intellectually handicapped (IH) has led to the establishment of multiple agencies in the field of service provision. A multi-agency, multi-disciplinary working party has undertaken a survey of all IH persons in the area, including an assessment of problems in service provision. There were a total of 2330 IH persons in the area. The prevalence rate for North Canterbury is 3.94 per 1000 which is substantially higher than the New Zealand figure of 3.29 of a previous survey. The working party has sponsored communication and mutual understanding and support through workshops, the formation of a parents committee and an information exchange system.


Meniscectomy.

Meniscectomy. Meniscectomy will predispose to degenerative osteoarthritis. To reduce the incidence of long-term morbidity the removal of normal menisci must be kept ot a minimum. The value of arthrography and arthroscopy in establishing a diagnosis and reducing the incidence of arthrotomy, is well recognised. Arthrography and arthroscopy are complementary and in many diagnostic knee problem both are indicated.


Bereavement in the elderly.

Bereavement in the elderly. One hundred bereaved spouses aged 65 and over were interviewed six months after the death for symptoms of depression. Twenty-nine were still depressed. Depression was not commoner with advancing age but was more frequent in men. Unexpected death was associated with more depression than if it was expected. Many derived benefit from supporting services.


Hypertension in valvar aortic stenosis.

Hypertension in valvar aortic stenosis. The indirect arterial blood pressure in 50 cases of catheter-proven aortic stenosis without significant incompetence was examined. Defining the upper limit of normal blood pressure as 140/90mmHg, 14 percent had borderline, 18 percent had mild and 8 percent had severe hypertension. There was a significant positive correlation of systolic and diastolic blood pressure with age; ie pressures rose with age. The rate of increase was virtually identical with that of the normal population except for those over 70. Hypertension interferes with reliable clinical and haemodynamic quantitation of the valve lesion. The two lesions summate to increase the systolic burden on left ventricle.


Spitz Naevi.

Spitz Naevi. Twenty-four benign melanocytic tumours from children and adults (range 1-31 years of age) excised at Auckland Hospital between 1967-1977 are defined as Spitz naevi. The necessary to differentiate these tumours from malignant melanoma is stressed.


Pregancy complicated by acute myeloid leukaemia. Combination cytotoxic chemotherapy was used to treat a case of acute myeloid leukaemia presenting in the 25th week of pregnancy with a sustained complete remission of the leukaemia and the successful delivery of a normal infant. The management of leukaemia presenting in pregnancy is discussed.


Factors associated with ex-nuptial birth. The paper examines the background to 210 live exnuptial births studied in the first phase of the Christ-church Child Development Study. The results show: 1. That nearly half of the children had been conceived within cohabiting situations. 2. That nearly one in five ex-nuptial children was the result of a planned pregnancy. 3. Maternal reactions to the birth and pregnancy varied with the mother's situation: cohabiting mothers reported considerably less adverse reaction to the birth than did non-cohabiting mothers. 4. Overall, mothers of ex-nuptial children had a fairly sophisticated appreciation of contraceptive methods. 5. One quarter of unplanned ex-nuptial pregnancies were the result of contraceptive failure and three-quarters were the result of contraceptive non-usage. 6. In common with previous findings for nuptial pregnancies, about 35 percent of unplanned ex-nuptial pregnancies were ascribed to a breakdown in the mother's pattern of usage of the contraceptive pill.


Ultrasound diagnosis of pancreatic disease. The gray-scale ultrasound features of the normal and pathological pancreas are presented in 37 patients, 25 of whom presented with jaundice, and 12 of whom had abdominal pain suspected to be of pancreatic origin. The ultrasound criteria for the diagnosis of pancreatic carcinomata, acute pancreatitis and pancreatic pseudocysts are illustrated. The correlation with findings at laparotomy and post-mortem are shown, suggesting that ultrasound is the best method of pancreatic imaging available in New Zealand at this time.


Cerebral embolisation of a shotgun pellet: case report. An 18-year-old male sustained a shotgun injury to his chest with the entry of pellets into both the heart and lungs. Cerebral embolisation of a pellet caused a dense hemiplegia. The literature is briefly reviewed.


Overdosage of buprenorphine: case report. A recent addition to the list of analgesics, buprenorphine, is still under trial. This paper reports the ingestion of a large dose taken with suicidal intent. Symptoms were minimal and recovery complete.


Trichobezoar in an infant: case report.

Trichobezoar in an infant: case report. A case of a trichobezoar in an infant is reported. This was caused by nursing the child on a sheep skin rug.


Acebutolol in the treatment of patients with hypertension and renal functional impairment. Eleven patients with hypertension and varying degrees of stable renal functional impairment were treated with the beta adrenoreceptor blocking drug, acebutolol (Sectral). Parameters of renal, cardiovascular and respiratory function were measured immediately prior to treatment and again after four and 12 weeks. In five patients the blood pressure was well controlled throughout the 12-week period on 400mg of acebutolol each morning, in three the blood pressure was satisfactory after four weeks treatment with 400mg each morning but control had been lost by 12 weeks, while in the remaining three patients 800mg each morning was ineffective. There was no significant change in the mean glomerular filtration rate of the 11 patients but in two of these patients with severe, but stable, chronic renal failure the introduction of acebutolol was associated with a decline in renal function and the onset of uraemic symptoms. One of these patients showed an improvement when the acebutolol was discontinued but the other required regular dialysis treatment. Beta adrenoreceptor blockers should be used cautiously in severe renal failure.


The first year of metroprolol (Lopresor): an analysis of a newly released drug in general practice. In 95 hypertensive patients treated in general practice, metoprolol given once daily was as effective as other beta-blockers given in an equivalent daily dose two or three times daily (P less than 0.001) and was well tolerated. Follow-up a year later produced no evidence of unusual side effects but there was a reduction in the mean daily dosage without loss of efficacy. Problems associated with surveys carried out in general practice and with the retrieval of information on the effects of long term administration in an identifiable patient population are discussed.


Water intoxication after oxytocin-induced midtrimester abortion. Two cases of water intoxication assoicated with midtrimester induction of abortion with oxytocin are presented. Both patients recovered from this potentially fatal condition which is associated with acute hyponatraemia. Some of the pertinent literature is reviewed and the possible mechanisms of causation and measures for prevention and treatment are discussed.


Detecting areas of deficiency in factual knowledge of general practitioners. Approximately one-third of all general practitioners returned a multiple choice questionnaire covering general medical knowledge. The overall average score was 63.6 percent and a definite fall off was observed with advancing age. It was also found that those who worked in group practices did rather better than those in single-handed practice--similarly full-time general practitioners performed rather better than part-time general practitioners. The proportion of respondents who were members of a recognised college of general practitioners approximates to the proportion of all general practitioners in New Zealand who are college members. The average mark for college members was exactly the same as the average mark for the group as a whole, suggesting that college members represent a typical cross section of the general practitioner community. This survey could be a useful baseline for further studies in the future should continuing education become obligatory for maintaining college membership.


Osteosarcoma arising in osteogenesis imperfecta. We report a case of osteosarcoma arising in osteogenesis imperfecta. The characteristic radiographic and pathological findings are discussed and emphasized to warrant a biopsy.


[Therapeutics future of liposomes (author's transl)]. The possibility to use the liposomes as drug vehicles has been largely investigated in recent years. Most of the studies have been carried out in animals and are related to disease involving predominantly the reticulohistiocytic system. Attempts to use these liposomes orally (induced diabetes) or locally (induced arthritis) are also reported. Further trials are required to get a better knowledge of the mechanisms involved in the exchanges between the cell membrane and the liposome the latter being variable as regards its composition, size and permeability according to the various investigators; the adjustment to the liposome of a complex capable to electively direct it towards the target organ is studied.


Protein-lipid interactions in bilayer membranes: a lattice model. A lattice model has been developed to study the effects of intrinsic membrane proteins upon the thermodynamic properties of a lipid bilayer membrane. We assume that only nearest-neighbor van der Waals and steric interactions are important and that the polar group interactions can be represented by effective pressure-area terms. Phase diagrams, the temperature T(0), which locates the gel-fluid melting, the transition enthalpy, and correlations were calculated by mean field and cluster approximations. Average lipid chain areas and chain areas when the lipid is in a given protein environment were obtained. Proteins that have a "smooth" homogeneous surface ("cholesterol-like") and those that have inhomogeneous surfaces or that bind lipids specifically were considered. We find that T(0) can vary depending upon the interactions and that another peak can appear upon the shoulder of the main peak which reflects the melting of a eutectic mixture. The transition enthalpy decreases generally, as was found before, but when a second peak appears departures from this behavior reflect aspects of the eutectic mixture. We find that proteins have significant nonzero probabilities for being adjacent to one another so that no unbroken "annulus" of lipid necessarily exists around a protein. If T(0) does not increase much, or decreases, with increasing c, then lipids adjacent to a protein cannot all be all-trans on the time scale (10(-7) sec) of our system. Around a protein the lipid correlation depth is about one lipid layer, and this increases with c. Possible consequences of ignoring changes in polar group interactions due to clustering of proteins are discussed.


Regulation of protein synthesis in rat ventral prostate: cell-free translation of mRNA. Androgenic steroids regulate both a class of abundant poly(A)-containing RNA and four major proteins secreted by the rat ventral prostate. That this class of RNA was mRNA coding for the major proteins was demonstrated by translating total poly(A)-containing RNA in a wheat-germ cell-free system and using the technique of hybrid arrested translation. RNA alone resulted in the synthesis of four major translation products, whereas RNA hybridized with DNA complementary to the class of abundant poly(A)-containing RNA failed to direct their cell-free synthesis. The synthesis of the polypeptides was androgen dependent, but their relative mobilities on denaturing acrylamide gels were not identical to those of the polypeptides synthesized in whole cells. Because these polypeptides are secreted to form part of the prostatic fluid, the possibility that the immediate translation products are precursors to the polypeptides was investigated. Prostatic RNA, translated in an L-cell cell-free system supplemented with microsomal membranes isolated from dog pancreas and rat prostate, caused the appearance of four translation products with relative mobilities similar to those of the androgen-dependent polypeptides synthesized in whole cells. The relationship of one of the polypeptides synthesized in minced tissue with the immediate translation products and processed polypeptides was further investigated by tryptic peptide fingerprint analysis, which revealed identical peptide maps.


Regulation of protein synthesis in reticulocyte lysates: immune serum inhibits heme-regulated protein kinase activity and differentiates heme-regulated protein kinase from double-stranded RNA-induced protein kinase. A specific immune serum to the heme-regulated inhibitor (HRI) has been prepared by immunizing chickens with highly purified reversible HRI prepared from rabbit reticulocyte lysates. Studies with this immune serum demonstrate that the behavior of purified reversible HRI is similar to that of the inhibitor activated in rabbit reticulocyte lysates: the immune serum (i) inhibits the phosphorylation of the small subunit (38,000 daltons) of the eukaryotic initiation factor eIF-2 by both crude and purified inhibitor preparations; (ii) prevents the concomitant inhibition of protein synthesis by both crude and purified inhibitor preparations; and (iii) prevents the autophosphorylation of the 95,000-dalton polypeptide in purified and crude HRI preparations. The protein kinase and inhibitory activities of crude and partially purified preparations of the double-stranded RNA-induced inhibitor of protein synthesis are not affected by the immune serum prepared to reversible HRI. These results indicate that the inhibitor induced by double-stranded RNA is antigenically distinct from the reversible HRI.


Isolation of maxicircle component of kinetoplast DNA from hemoflagellate protozoa. Maxicircle DNA has been isolated from the kinetoplast DNA of Leishmania tarentolae culture forms by bouyant separation in CsCl in the presence of the A + T-binding dye Hoechst 33258, after liberation from the kinetoplast DNA network by cleavage with the single-hit restriction endonuclease EcoRI. The purified linearized maxicircle DNA has a density in CsCl of 1.681 g/cm3 (79% A + T) and a molecular weight of approximately 18-20 x 10(6). The maxicircle molecule exhibited intramolecular base composition heterogeneity ranging from 85% A + T to 74% A + T.


Protection and reactivation of oxidative phosphorylation in mitochondria by a stable free-radical prostaglandin polymer (PGBx). A stable free-radical polymeric derivative of prostaglandin B1 (PGBx) has been synthesized that exhibits regenerative effects on oxidative phosphorylation in aged mitochondria. The molecular weights of the most active preparations fall between 2000 and 2600. PGBx is characterized by a single-line electron spin resonance spectrum that is stable at room temperature. PGBx restores phosphorylating ability and net ATP synthesis in isolated mitochondria aged for 4 days at 0 degrees C and protects against further degradation of phosphorylating activity when such aged mitochondria are preincubated at 28 degrees C in the absence of adenine nucleotide phosphate acceptors. This compound has been reported to exert beneficial effects in vivo in experimental pathological conditions, such as regional ischemia, in which the mitochondria of the ischemic region may have been damaged.


13C NMR studies of gluconeogenesis in rat liver cells: utilization of labeled glycerol by cells from euthyroid and hyperthyroid rats. The gluconeogenic pathway from [2-13C]glycerol and [1,3-13C]glycerol has been followed in suspensions of isolated rat hepatocytes at 25 degrees C by 13C NMR at 90.5 MHz. The flow of label through the major pathway from glycerol to L-glycerol 3-phosphate and into glucose was followed in cells from control and triiodothyronine-treated rats. Treatment increased the rates of glucose formation and glycerol consumption 2-fold and decreased the alphaGP level to 40%. We calculate that approximately 60% of the flux is through the mitochondrial glycerol phosphate dehydrogenase in cells from triiodothyronine-treated rats, compared with approximately 15% in cells from the controls. Equal distribution of label between the trioses of glucose was obtained and, because the C3-C4 spin-spin coupling gives the distribution of labeled carbons in the same molecule, it was possible to measure the amount of triose from unlabeled fructose incorporated into the glucose labeled at carbons 1, 3, 4, and 6. About 10% of the hexoses had flowed through the pentose cycle and back into the hexose pathway in cells from fasted rats. From the distribution of label at glucose carbons not labeled via the major pathway and from the carbon spin-spin splitting patterns observed, we conclude that transketolase is reversible whereas transaldolase is essentially irreversible in the nonoxidative pentose branch.


The lambda repressor contains two domains. Papain digestion of the lambda phage repressor produces two fragments that are relatively resistant to further digestion. One includes the amino terminus (residues 1-92) and the other the carboxyl terminus (residues 132-236). Calorimetry shows that the amino-terminal fragment denatures near 50 degrees C and that the carboxyl-terminal fragment denatures near 70 degrees C. Intact repressor undergoes two denaturations, one near 50 degrees C and another near 70 degrees C. These and other data show that lambda repressor consists of two domains joined by a "connector" 40 amino acids long that is sensitive to proteases. The amino-terminal domain binds DNA, and the carboxyl-terminal domain oligomerizes.


Transfer of 5'-terminal cap of globin mRNA to influenza viral complementary RNA during transcription in vitro. We have recently demonstrated that globin mRNAs are effective primers for influenza viral RNA transcription in vitro catalyzed by the virion transcriptase [Bouloy, M., Plotch, S. J. & Krug, R. M. (1978) Proc. Natl. Acad. Sci. USA 75, 4886-4890]. Here, we present direct evidence that the 5'-terminal methylated cap of the globin mRNAs is transferred to viral complementary RNA (cRNA) during transcription. Chemical (beta-elimination) or enzymatic removal of the cap of globin mRNAs eliminated essentially all their priming activity. Much of this activity could be restored by recapping the beta-eliminated globin mRNAs with the vaccinia virus guanylyl and methyl transferases. Globin mRNAs containing (32)P label only in the cap (m(7)G(32)pppm(6)A(m)-) were prepared by recapping beta-eliminated globin mRNAs with the vaccinia virus enzymes, [alpha-(32)P]GTP, and unlabeled S-adenosylmethionine. By using this labeled globin mRNA as primer and unlabeled nucleoside triphosphates as precursors, the viral cRNA segments that were synthesized were shown to contain a (32)P-labeled 5'-terminal cap structure. Gel electrophoretic analysis indicated that the globin mRNA-primed cRNA segments were 10-15 nucleotides longer at their 5' end than ApG-primed cRNA segments, which initiate exactly at the 3' end of the virion RNA templates. This suggests that, in addition to the cap, about 10-15 other nucleotides are also transferred from the globin mRNA to viral cRNA. A mechanism for the priming of influenza viral cRNA synthesis by globin mRNA is proposed.


Regulation of DNA replication in S phase nuclei by ATP and ADP pools. Synchronized 3T6 (mouse fibroblast) ghost monolayers (isolated nuclei) were utilized to study the effects of ATP and ADP levels on DNA replication in vitro. A system yielding discontinuous semiconservative DNA replication (without any detectable repair) in synchronized S phase nuclei has been developed. Lack of initiation of new sites has been observed in isolated S phase 3T6 nuclei without the presence of cytoplasmic material; DNA synthesis is comprised only of elongation at sites where initiation had previously taken place. DNA synthesis in S phase nuclei proceeded optimally at an ATP concentration of 4-5 mM. High ATP levels as well as high ATP/ADP ratios (produced by an ATP-regenerating system at a variety of ATP concentrations) yielded marked inhibition of [(3)H]dTTP incorporation. The cellular and nuclear pools of ATP and ADP in intact synchronized 3T6 cells were accurately determined by high-pressure liquid chromatography. A good correlation with the studies on isolated nuclei has been observed. Whereas total cellular ATP pools increase during the progression of 3T6 cells from G(1) to S phase of the cell cycle, nuclear ATP pools do not increase, and the nuclear ATP/ADP ratios decrease once the cells enter the S phase of their cycle. These experiments suggest that nuclear ATP pools and ATP/ADP ratios act as S phase controls, regulating DNA elongation at sites where its synthesis has previously been initiated by cytoplasmic factors.


Complete amino acid sequence of a histidine-rich proteolytic fragment of human ceruloplasmin. The complete amino acid sequence has been determined for a fragment of human ceruloplasmin [ferroxidase; iron(II):oxygen oxidoreductase, EC 1.16.3.1]. The fragment (designated Cp F5) contains 159 amino acid residues and has a molecular weight of 18,650; it lacks carbohydrate, is rich in histidine, and contains one free cysteine that may be part of a copper-binding site. This fragment is present in most commercial preparations of ceruloplasmin, probably owing to proteolytic degradation, but can also be obtained by limited cleavage of single-chain ceruloplasmin with plasmin. Cp F5 probably is an intact domain attached to the COOH-terminal end of single-chain ceruloplasmin via a labile interdomain peptide bond. A model of the secondary structure predicted by empirical methods suggests that almost one-third of the amino acid residues are distributed in alpha helices, about a third in beta-sheet structure, and the remainder in beta turns and unidentified structures. Computer analysis of the amino acid sequence has not demonstrated a statistically significant relationship between this ceruloplasmin fragment and any other protein, but there is some evidence for an internal duplication.


Choleragen (cholera toxin): a bacterial lectin. Choleragen (cholera toxin) agglutinated erythrocytes and liposomes containing the toxin receptor, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (ganglioside GM1). Cells that had been exposed to GM1 were agglutinated, but agglutination was not observed when cells had been exposed to other gangliosides (GM2, GM3, GD1a, GD1b). Choleragen-dependent agglutination of liposomes was slightly less specific, because liposomes containing either GM1 or GD1b, but neither GM2, GD1a, nor GM3 were agglutinated. The oligosaccharide isolated from GM1 inhibited both the agglutination of cells and liposomes containing GM1 and the binding of choleragen to liposomes containing GM1. Galactose and sialic acid were less effective inhibitors of liposomal agglutination and did not inhibit cellular agglutination or binding of choleragen to liposomes. Liposomal agglutination was dependent on choleragen concentration and occurred with the B but not the A protomer of choleragen. These results suggest that choleragen, through its binding to the oligosaccharide portion of a glycolipid, exhibits lectinlike activity, which results in agglutination of liposomes and erythrocytes.


Limited action of micrococcal nuclease on trout testis nuclei generates two mononucleosome subsets enriched in transcribed DNA sequences. Hybridization experiments show that DNA extracted from two distinct subsets of mononucleosomes (MNI and MN2) generated by a limited action of micrococcal nuclease on trout testis nuclei is enriched approximately 7-fold in sequences that are transcribed into cytoplasmic polyadenylated RNA in trout testis cells. Both subsets of mononucleosomes contain eight core histones, but MNI also possesses one or two molecules of a small, basic, high-mobility-group (HMG) protein H6 [Levy W., B., Connor, W. & Dixon, G. H. (1979) J. Biol. Chem. 254, 609-620], bound to a DNA fragment of 140 base pairs. In contrast, MN2 contains 1 molecule of H1 but no H6, and its DNA length is somewhat longer at 140-190 base pairs. The preferential release of these two subsets of mononucleosomes is correlated with the presence of a second larger HMG protein, HMG-T, in the linker regions flanking both types of mononucleosomes. The HMG-T-containing linker regions appear to be considerably more susceptible to attack by micrococcal nuclease than H1-containing linkers. Cross-reassociation reactions between the DNA from MN1 and MN2 subsets indicate that they share a significant extent of sequence overlap but also that each subset contains specific sequences that are absent in the other subset.


Evidence for covalent attachment of fatty acids to Sindbis virus glycoproteins. Selective binding of lipid to glycoprotein was detected when [3H]palmitate-labeled Sindbis virus particles or viral-infected cells were disrupted by heating with sodium dodecyl sulfate, and glycoproteins were isolated by electrophoresis in sodium dodecyl sulfate/10% polyacrylamide gels. The smaller glycoprotein (E2) retained 2 to 3 times more labeled lipid than did the larger EI glycoprotein, and the cell-associated glycoprotein precursor (PE2) bound even less lipid. No lipid was associated with the nonglycosylated glycoproteins that accumulated in infected cells treated with tunicamycin. The labeled lipid remained bound to the glycoproteins after exhaustive extraction with chloroform/methanol of virus particles, infected-cell extracts, or isolated glycoproteins, but it could be extracted by chloroform/methanol after treating glycoproteins with mild alkali. Analysis by gas/liquid chromatography showed that 60% of the label was in palmitate and the balance of label was distributed between oleate and stearate. There were approximately 2 mol of fatty acid bound per mol of E1 glycoprotein. Proteolysis of the fatty acid-labeled glycoprotein with pepsin, thermolysin, and Pronase degraded the polypeptide to fragments that retained the fatty acids in an alkali-labile state. These data suggest that a covalent attachment of fatty acid may occur during maturation of the viral glycoproteins.


Biochemical studies of taste sensation: binding to taste tissue of 3H-labeled monellin, a sweet-tasting protein. Binding of 3H-labeled methylated monellin to taste receptor tissue was demonstrated in vitro. Preparation of bovine and human circumvallate (taste) papillae bound more of the ligand  than did lingual and nonlingual epithelial preparations devoid of taste buds. Binding to the taste preparations saturated at high ligand concentrations. Furthermore, sugars and other sweet-tasting molecules appeared to compete to some extent with this sweet-tasting protein for its binding sites. These binding measurements of the intensity sweet-tasting protein monellin to taste receptor preparations help to establish the binding interactions as an initial step taste sensation.


Regulation of the threonine operon: tandem threonine and isoleucine codons in the control region and translational control of transcription termination. The DNA sequence of 178 base pairs preceding the first structural gene of the threonine operon of Escherichia coli has been determined. A region of perfect 2-fold rotational symmetry, involving 28 base pairs, precedes the first structural gene. The structural similarity of this sequence to known RNA polymerase termination sites suggests that this region is the termination site of the threonine operon leader RNA. Moreover a mutation (thr 79-20), which confers a depressed, constitutive phenotype, was sequenced and found to be a G.C insertion in the putative terminator. A potential coding region for a 21-amino acid leader peptide ends approximately 18 base pairs before the terminator. This peptide contains eight threonine and four isoleucine codons. Eleven of these codons are in tandem. A model for threonine operon regulation, involving alternative secondary RNA structures and translation of leader RNA, is discussed.


Presence of chondroitin sulfate in the neuronal cytoplasm. The distribution of glycosaminoglycans and glycoproteins has been studied in cytoplasmic and particulate fractions of neurons isolated in bulk from rat cerebrum. Lysis of the neurons in 25 mM sodium phosphate buffer at pH 7.5 released 20% of the protein and over 90% of the lactate dehydrogenase in a soluble form. Eighty-two percent of the chondroitin sulfate was also released, together with 55% of the heparan sulfate and 24-25% of the hyaluronic acid and glycoproteins. The chondroitin sulfate remaining in the membranes was completely depolymerized to disaccharides after treatment with chondroitinase ABC, and treatment of the neuronal membranes with 0.1% trypsin removed 55-63% of the chondroitin sulfate and heparan sulfate but only 25% of the sulfated glycoproteins. The results reported here support our previous conclusion that the soluble chondroitin sulfate proteoglycan of brain is largely a cytoplasmic constitutent of neurons (and astrocytes) and is not primarily present in nervous tissue as an extracellular ground substance.


In vitro regulation of DNA-dependent synthesis of Escherichia coli ribosomal protein L12. The DNA of the transducing phage lambdarifd18 contains, among others, the genes for the ribosomal proteins L11, L1, L10, and L12 and the beta and beta' subunits of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). In a coupled in vitro protein-synthesis system, lambdarifd18 DNA directs the synthesis of about four to five molecules of L12 per molecule of L10. This is consistent with the finding that there are four copies of L12 per ribosome. The ratio of L12/L10 was also examined from an EcoRI fragment of lambdarifd18 that contains the L10 gene and about 50% of the L12 gene. A significantly lower ratio of truncated L12/L10 was observed compared to the intact phage. The binding of RNA polymerase to various lambdarifd18 DNA restriction fragments was used to locate possible promoter sites. These binding experiments suggest that the beta and beta' subunits of RNA polymerase are cotranscribed with at least ribosomal protein L12 and, also, that there may be an additional promoter site for the L12 gene within the structural gene for L10.


Interactions of a photosensitive analog of cholesterol with hydroxymethyglutaryl-CoA reductase (NADPH) and acyl-CoA:cholesterol acyltransferase. 25-Azido[25-(3)H]norcholesten-3beta-ol, a radioactive photosensitive analog of cholesterol, was synthesized as a probe to study lipid-protein interactions [Stoffel, W. & Klotzbücher, R. (1978) Hoppe-Seyler's Z. Physiol Chem. 359, 199-209]. Upon UV irradiation a reactive nitrene is generated which can insert into carbon-hydrogen bonds of proteins to form covalent linkages. The compound has properties similar to those of cholesterol. When administered intravenously to rats it was transported to the liver, and within 4 hr it was found in all of the subcellular fractions, accounting for 8% of the microsomal sterol content. Concomitantly, it altered the activities of two membrane-bound enzymes. 3-Hydroxy-3-methylglutaryl (HMG)-CoA (NADPH) reductase [mevalonate:NADP(+) oxidoreductase (CoA-acylating), EC 1.1.1.34] activity was reduced to 20% of control values, and acyl-CoA:cholesterol acyltransferase (ACAT; acyl-CoA:cholesterol O-acyltransferase, EC 2.3.1.26) was stimulated at least 2-fold. Esterified products of the analog were found in both blood and liver, and no evidence was obtained for production of any hydroxylated or polar derivatives. UV irradiation of microsomes increased the radioactivity bound to the protein fraction and decreased it in the total lipid extract; in parallel a sharp decline in ACAT activity but unaltered HMG-CoA reductase activity was observed. These results are interpreted as a possible consequence of crosslinking of the sterol to the enzyme proteins and discussed in view of the evidence that HMG-CoA reductase is an extrinsic and ACAT an intrinsic membrane protein.


Release of in vitro-synthesized poly(A)-containing RNA from isolated rat liver nuclei: characterization of the ribonucleoprotein particles involved. Nuclei isolated from rat liver were incubated under conditions in which RNA continued to be labeled with precursors for long periods. After 1 hr, during which the rate of RNA synthesis was constant, 25-30% of the newly synthesized RNA was recovered in the postnuclear supernatant. About 3-5% of this fraction was characterized as poly(A)-containing ribonucleoproteins by the following criteria: (i) characteristic elution profile in oligo(dT)-cellulose chromatography; (ii) size distribution of the molecules and their deproteinized RNAs; (iii) buoyant densities in CsCl gradients; (iv) presence of RNaseresistant fragments resembling poly(A)-protein complexes; and (v) identification of the protein components by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The sensitivity of labeling of the RNA synthesized and released from the nuclei to low doses of alpha-amanitin suggests the presence of polymerase II products in the particles. Comparison of the sizes of proteins in these particles with those of free and polysomal messenger ribonucleoproteins as well as with heterogenous nuclear ribonucleoproteins indicates that the released particles contain a protein of 78,000 daltons, which is also present in the other three classes of ribonucleoproteins, presumably at the 3'-poly(A) segments. In addition, a few other proteins, similar in size to those found in the cytoplasmic ribonucleoproteins, were also present in the released particles. It is suggested that proteins associated with heterogenous nuclear RNA are mostly exchanged before or at the time of release of mRNA from the nucleus; the remaining mRNA-associated proteins are added in the cytoplasm, possibly in relation to cytoskeleton attachment, followed by the removal of most of these proteins during polysome formation.


Prostaglandin D2 formation by malignant melanoma cells correlates inversely with cellular metastatic potential. B16 malignant melanoma cell lines transform arachidonic acid and its transient metabolite, prostaglandin endoperoxide H2, into prostaglandin D2. The highly metastatic line, B16 F10, forms less prostaglandin D2 compared to the moderately metastatic parent line, B16 F1. Since platelet aggregation may be one factor involved in B16 metastasis and since prostaglandin D2 inhibits platelet aggregation, this prostaglandin could affect the outcome of platelet-tumour interactions, which may contribute ultimately to metastasis. Arachidonic acid metabolism may be another one of the intrinsic biochemical properties of tumor cells that affects their metastasis. Our results suggest that quantitative release of unusual prostaglandins must be considered in this context.


Conformation of an oligopeptide in phospholipid vesicles. To demonstrate a method by which the conformation of membrane proteins may be determined spectroscopically in model membranes, we determined the structure of a hydrophobic oligopeptide, t-butyloxycarbonylprolylleucylvalylmethyl ester, in phospholipid vesicles by nuclear magnetic resonance, circular dichroism, and infrared spectroscopy. 13C nuclear magnetic resonance and circular dichroism techniques demonstrated that the conformation of this peptide in linear hydrocarbon solutions was essentially identical to its conformation in lipid vesicles. 1H nuclear magnetic resonance and infrared spectroscopy of the peptide in hydrocarbon solution then provided additional high-resolution information concerning the structure of the peptide as found in the hydrophobic portion of the lipid bilayer. The conformation of this peptide in hydrophobic media a differs from its structure in hydrophilic solvents, not only in bond angles and the proportion of cis/trans isomers about the X-proline bond, but also in its intermolecular associations.


Ultrasonic absorption evidence of structural fluctuations in viral capsids. When the coat protein of the small icosahedral virus, brome mosaic virus, reassembles into capsids, the ultrasonic absorption of the solution greatly increases. Submitting the solution to an ultrasonic field thus appears to reveal spontaneous molecular motions within a protein assembly. Confirmatory evidence of a dynamics of a protein shell comes from measurements on brome mosaic virus at various degrees of swelling and on tomato bushy stunt virus treated with the crosslinking agent glutaraldehyde. The detected fluctuations may be related either with cooperative deformational motion in the capsid or with more localized structural changes. Such structural changes may help liberate the RNA at an early stage of viral infection.


Direct measurement of changes in sodium pump current in canine cardiac Purkinje fibers. Purkinje fibers from dog hearts may have either a "high" resting potential of about -90 mV or a "low" resting potential of about -40 mV when immersed in low-Cl(-) solution containing 4 mM K(+). Brief exposure of Purkinje fibers at the low level of resting potential to K(+)-free fluid causes further depolarization, and return to K(+)-containing solution elicits a transient hyperpolarization which reaches a peak within a few seconds and then declines within a few minutes. Repeating these changes in K(+) concentration after clamping the membrane potential at its steady resting level in K(+)-containing fluid allows the changes in net membrane current presumably underlying the depolarization and transient hyperpolarization to be measured. Net inward current is recorded when the fiber is exposed to K(+)-free solution, and a transient net outward current arises when it is returned to K(+)-containing solution. The transient net outward current reflects a temporary increase in the rate of electrogenic Na(+) extrusion caused by the rise in intracellular Na(+) concentration that occurs while the sodium pump is slowed in K(+)-free fluid. Sodium extrusion remains enhanced, presumably until the internal Na(+) concentration has been brought back to its resting level. The transient outward current is completely abolished by the cardiac steroid acetylstrophanthidin, and its amplitude is increased as the prior exposure to K(+)-free fluid is prolonged. The decay of the transient outward current and the decline in intracellular Na(+) concentration both appear to follow first-order kinetics.


Autotrophic growth of H2-uptake-positive strains of Rhizobium japonicum in an atmosphere supplied with hydrogen gas. Previous research from this laboratory has demonstrated CO(2)-fixing and H(2)-uptake capacities of certain strains of Rhizobium japonicum. In this report we have shown that SR, a H(2)-uptake-positive (Hup(+)) strain of R. japonicum, is capable of autotrophic growth with H(2) as the energy source. Growth occurred on mineral salts/vitamins/Noble agar, mineral salts/vitamins liquid medium (0.27 mug of C as vitamins per ml), and in mineral salts liquid medium with no added vitamins when cultures were provided with NH(4)Cl and incubated in an atmosphere containing H(2), CO(2), O(2), and N(2). Little or no growth occurred when either H(2) or CO(2) was omitted from the atmosphere or when the culture was inoculated with SR3, a Hup(-) mutant of SR. Growth was measured by protein synthesis, fixed organic carbon, and increase in cell number in liquid cultures. The organism that grew autotrophically was verified as R. japonicum by (i) apparent purity on streak plates; (ii) retention of the double antibiotic resistance markers; and (iii) its capability to nodulate soybeans. H(2)- and CO(2)-supported growth was demonstrated for three additional Hup(+) wild-type R. japonicum strains (USDA 136, 3I1b 6, and 3I1b 143), while three Hup(-) wild-type strains (USDA 120, 3I1b 144, and USDA 117) were incapable of growth on the Noble agar medium containing mineral salts/vitamins in the H(2)/CO(2)/O(2)/N(2) atmosphere. This demonstrated capability of Hup(+)R. japonicum strains to grow autotrophically requires revision of current concepts regarding conditions for survival and competition of these bacteria in the soil and their relationships to other microorganisms.


Induction of rat sarcomas in rats treated with antithymocyte sera after transplantation of human cancer cells. Human cancer cells that had had high (greater than 160) tissue culture passages, when transplanted into antithymocyte-treated F344 newborn rats, caused induction of rat sarcomas in the rats within 2 or 3 subcultures, whereas human cancer cells with low (5-33) passages in vitro did not cause overt induction of rat sarcomas until after 5-10 subtransplantations. Because oncornavirus activity was not detected in either rat or human tumors, it is suggested that transforming sequences located on the human tumor cells may have been transferred to supporting rat reticulum cells in close contact with the human cancer cells.


Conditions for inhibiting and enhancing effects of the protease inhibitor antipain on x-ray-induced neoplastic transformation in hamster and mouse cells. Using cultured normal hamster embryo cells and the heterploid mouse C3H cell line 10T1/2, clone 8, we have studied the effect of the protease inhibitor antipain on x-ray-induced neoplastic transformation. We found in both cell systems that, while there was no effect on cell survival as compared to irradiated controls, the addition of antipain at a concentration of 6 microgram/ml to the cultures 24 hr prior to irradiation resulted in enhanced transformation as compared to the frequency in cultures exposed to radiation alone. Yet the addition of antipain to cultures 10 min after irradiation resulted in a decreased transformation rate. This decrease was not found when antipain was added to the mouse cells 24 hr after irradiation or to the hamster cells 48 hr after irradiation. These results suggest that the protease inhibitor antipain has more than one mechanism of action in modulating the fixation and expression of transformation by x-irradiation, possibly by the modification of DNA repair.


Purification of human platelet-derived growth factor. Human platelets contain a polypeptide growth factor that stimulates the proliferation of connective tissue cells. Purification of this platelet-derived growth factor (PDGF) was accomplished by heat (100 degrees C) treatment of washed platelets and subsequent ion-exchange chromatography, gel filtration in 1 M acetic acid, isoelectric focusing, and preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis. PDGF has an isoelectric point of 9.8 and a molecular weight ranging from 13,000 to 16,000 as judged by gel filtration in 1 M acetic acid or analytical sodium dodecyl sulfate gel electrophoresis under reducing conditions. The specific activity of the purified PDGF is 20 million times greater than that found in unfractionated human serum. Purified PDGF stimulates replicative DNA synthesis and cell proliferation in quiescent density-arrested cultures of BALB/c 3T3 cells at concentrations of 1 ng/ml (0.1 nM).


Fibroblast interferon induces synthesis of four proteins in human fibroblast cells. Treatment of human diploid fibroblasts with fibroblast interferon for 8 hr inhibited replication of vesicular stomatitis virus. When the total cell protein of cells treated with interferon for 8 hr was compared to the total cell protein of untreated cells by two-dimensional gel electrophoresis, the interferon-treated cells were found to contain four proteins not found in untreated cells. Addition of actinomycin D to the cells concurrently with interferon inhibited the synthesis of the four proteins. We conclude that these proteins are induced by interferon and that they may be involved in the inhibition of virus replication.


Stimulation of Ca2+-dependent neurotransmitter release and presynaptic nerve terminal protein phosphorylation by calmodulin and a calmodulin-like protein isolated from synaptic vesicles. Synaptic vesicles have a Ca(2+)-dependent protein kinase system that may play a role in mediating Ca(2+)-stimulated neurotransmitter release and vesicle function. Calcium's ability to initiate norepinephrine release and protein phosphorylation in synaptic vesicle preparations was shown to be stimulated by the presence of an endogenous heat-stable vesicle protein fraction. The heat stability and characteristics of this endogenous vesicle fraction were similar to those of calmodulin (Ca(2+)-dependent regular protein) isolated from rat and bovine brain. Calmodulin, like endogenous heat-stable vesicle factor, restored calcium's ability to stimulate vesicle neurotransmitter release and protein kinase activity. Calmodulin-like vesicle protein and purified calmodulin were also equally effective in stimulating cyclic nucleotide-dependent phosphodiesterase, further indicating that these two proteins are functionally equivalent. Depolarization-dependent Ca(2+) uptake in intact synaptosomes simultaneously stimulated release of neurotransmitter and phosphorylation of particular synaptic vesicle proteins that were shown in the isolated vesicle preparation to be dependent on Ca(2+) and calmodulin. The results suggest that calcium's effects on neurotransmitter release and presynaptic nerve terminal protein phosphorylation may be mediated by endogenous calmodulin-like proteins.


Migratory patterns of cloned neural crest melanocytes injected into host chicken embryos. Cloned quail melanocytes grown in tissue culture for 8 days or more were injected into 2 1/2-day-old chicken embryos. The pigment cells were placed directly into the somitic lumen by means of an injection micropipette. This technique for introducing marked neural crest cells into host embryos causes far less damage than previous methods which require extirpation and replacement of the neural tube. In addition, small numbers of homogeneous cells can be implanted by this procedure. When injected into one of the posterior somites, cultured pigment cells migrated along the ventral neural crest pathway. Three days after injection the melanocytes had migrated ventral to the dorsal root ganglia and prevertebral and primary sympathetic chain ganglia and were seen associated with the adrenal gland and aortic plexi. Melanocytes were frequently found in or adjacent to the gonads and often had migrated as far as the gut.


Most myosin heavy chain mRNA in L6E9 rat myotubes has a short poly(A) tail. The mRNA for rat muscle myosin heavy chain (MHC) was isolated from L6E9 myotubes by two rounds of sucrose density gradient centrifugation followed by fractionation on an agarose/polyacrylamide gel. The purity of the mRNA isolated was determined by translation in vitro, peptide analysis of the in vitro product and comparison with authentic MHC, analysis of the kinetics of hybridization with cDNA prepared with this RNA, and titration analysis of total cytoplasmic RNA from muscle and nonmuscle sources. By using the MHC cDNA as probe of myogenic differentiation, it was observed that the level of cytoplasmic MHC mRNA increased approximately 200-fold as the dividing myoblast differentiated into the fused myotube. Titration analysis of RNAs fractionated by oligo(dT)-cellulose chromatography indicated that the majority of the increase occurred in that RNA population that failed to bind to an oligo(dT)-cellulose column.


Growth and morphology of colonies of Chinese hamster ovary cells growing on agar is affected by insulin. As a model for the effect of hormones and growth factors on three-dimensional growth of mammalian cells, we have analyzed the effect of insulin on the three-dimensional growth and morphology of Chinese hamster ovary (CHO) colonies grown on the surface of agar. Sequential photographs in dark-field illumination of growing colonies have been analyzed with computer-assisted techniques. In this analysis the entire shape of each colony in a sizeable population (up to 10(5) colonies per experiment) can be measured and distributions of parameters derived from these measurements can be studied. In fetal calf serum (FCS), insulin has a dose-related stimulatory effect on cell growth that is most pronounced when growth has slowed down. In 10% FCS, insulin has a similar but diminished effect. When CHO cells are grown conventionally on plastic substrata or in suspension, insulin has little effect on cell growth at 4% serum concentration. Computer analysis of changes in the distribution of colony morphology proved to be a sensitive, dose-dependent, and reproducible assay of a hormonal effect. As little as 5 ng of insulin per ml added to 10% FCS causes a shift in the distribution of colony morphologies. In 4% FCS, 50 ng of insulin per ml is required to produce a detectable change in the colony morphology distribution. Computer analysis of cells grown three-dimensionally on agar provides a powerful approach to studying the effects of hormones and provides observations not available when cells are grown on plastic substrata.


Catabolic control of the enhanced alanine-preferring system for amino acid transport in glucose-starved hamster cells requires protein synthesis. In cultured hamster cells starved for glucose for 24 hr there is an enhancement of the rate of alpha-aminoisobutyric acid transport ("shiftup"). When the starved cells are re-fed with glucose, the rate of transport shifts back down to the low, "regulated" rate typical of cells continuously fed with medium containing glucose ("shiftdown"). The high, deregulated rate of transport is maintained, however, when cycloheximide is present for 24 hr during the re-feeding with glucose. Maintenance of the high transport rate is evident only when the cells are incubated in amino acid-free medium just prior to the transport assay or when the assays are conducted with isolated membrane vesicles. A premature, pseudoshiftdown was observed in intact cells within as little as 2 hr after re-feeding when care was not taken to deplete the amino acid pool prior to the transport assay. In addition, a cycloheximide-insensitive increase in transport was observed when cultures were re-fed for 2 hr with amino acid-free medium containing fresh serum. These results emphasize the often overlooked precautions that should be taken to guard against artifacts that could mislead interpretations of amino acid transport data. More important, however, is the finding that Na(+)-dependent amino acid transport in cultured animal cells is regulated in part by a factor (or factors) that becomes inactivated when the cells are maintained under nonglycolytic culture conditions. In order to reactivate the control mechanism, starved cells that have been re-fed with glucose must resynthesize the regulatory factor(s). Thus, in at least cultured hamster cells, Na(+)-dependent amino acid transport regulation is much like the hexose transport regulation in that catabolic control (shiftdown) requires protein synthesis.


Tumor promoter-mediated inhibition of cell differentiation: suppression of the expression of erythroid functions in murine erythroleukemia cells. Previous studies demonstrated that 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a tumor promoter, is a potent inhibitor of inducer-mediated differentiation of murine erythroleukemia cells. Inhibition of cell differentiation was associated with inhibition of cell growth. The present studies, employing a cell line adapted for growth in TPA, demonstrate that inhibition of differentiation is not dependent upon inhibition of cell growth or a change in the cell division cycle; neither is inhibition of differentiation accompanied by detectable effect on cell uptake of [3H]hexamethylene bisacetamide, the inducer used in these studies. TPA causes an inhibition of expression of all hexamethylene bisacetamide-inducible erythroid characteristics measured, including commitment to terminal cell division, accumulation of globin mRNA, and synthesis of globins, spectrin, heme synthetic enzymes (delta-aminolevulinic acid dehydratase and uroporphyrinogen-I synthase) and heme. A hypothetical model for the inhibitory action of tumor promoters on terminal cell differentiation is discussed.


Isolation of lysosomal alpha-mannosidase mutants of Chinese hamster ovary cells. A method is presented for detection of lysosomal hydrolase activity in situ in colonies of Chinese hamster ovary cells. This method was used to screen for mutants deficient in lysosomal alpha-mannosidase. Mutagenized cells were replicated onto filter papers [Esko, J. D. & Raetz, C. R. H. (1978) Proc. Natl. Acad. Sci. USA 75, 1190-1193] and allowed to divide for 11-14 days; cells on the filter replicas were lysed, and the filters were incubated with 4-methylumbelliferyl-alpha-D-mannopyranoside at pH 4.4 and then photographed under UV light. Colonies for which the replicas exhibited little or no fluorescence were picked from the master plates and purified; mutants with decreased alpha-mannosidase activity were obtained at a frequency of 1 per 1500 mutagenized cells. Analysis of one of these mutants showed that lysosomal alpha-mannosidase activity was 18% of that from wild-type cells, whereas the activities of alpha-mannosidases not of lysosomal origin were similar in the wild type and mutant (these included both Golgi-associated and cytosolic enzymes as well as a novel acid alpha-mannosidase seen only in cells from confluent culture). The mutant contained normal levels of other lysosomal hydrolases. Both wild-type and mutant cells secreted alpha-mannosidase into the medium at levels proportional to those found inside the cells. Incubation of mutant cells with secretions from the wild type resulted in partial restoration of enzyme activity. Added enzyme was localized in the lysosomal fractions; uptake of added enzyme was inhibited by mannose 6-phosphate and fructose 1-phosphate, which are known to inhibit uptake of lysosomal enzymes into human diploid fibroblasts.


Biochemical and electron microscopic evidence that cell nucleus negatively controls mitochondrial genomic activity in early sea urchin development. Enucleated halves of sea urchin eggs obtained by centrifugation contain almost all the mitochondrial population of the egg. Removal of the nucleus followed by parthenogenetic activation stimulates the incorporation of [3H]thymidine into the mitochondrial DNA, whereas no such incorportion is observed in activated whole eggs. The block is not the result of a modification in the permeability of the mitochondrial membrane. Electron microscopic observations demonstrated duplication of mitochondrial DNA molecules in activated enucleated halves. No duplication was found in the mitochondrial DNA from activated whole eggs or from nonactivated enucleated halves. We conclude that the cell nucleus exerts a negative control on the activity of the mitochondrial genome through some short-lived nuclear substance(s).


Contrasted modes of evolution in the same genome: allozymes and adaptive change in Heliconius. Butterflies in the South American genus Heliconius have undergone a spectacular adaptive radiation (with convergent evolution between some lines) in their color patterns; this has been produced by natural selection for muellerian mimicry. The genetic basis of this radiation, shown by crossing highly differentiated races within two of the species, is homozygosity for alternative alleles at some half dozen loci. In complete contrast, allozyme loci in these butterflies are strongly heterozygous and show only frequency differences (never amounting to homozygosity of alternative alleles) between races; the amount of allozyme divergence is the same between races of H. erato and H. sara, although in color pattern the first forms marked races and the other does not. For the allozymes, there is a strong correlation over loci for rate of divergence between species and average heterozygosity. This is not true of the genes controlling color pattern. Heterozygosity of the enzymes is correlated with subunit molecular weight. Thus, different parts of the genome can evolve in different ways simultaneously; genes controlling color pattern in the "classical" mode, and allozymes in a different mode in which the rate of evolution is related to their heterozygosity (a "balance" or "neutral" mode).


Molecular model for the transposition and replication of bacteriophage Mu and other transposable elements. A series of molecular events will explain how genetic elements can transpose from one DNA site to another, generate a short oligonucleotide duplication at both ends of the new insertion site, and replicate in the transposition process. These events include the formation of recombinant molecules which have been postulated to be intermediates in the transposition process. The model explains how the replication of bacteriophage Mu is obligatorily associated with movement to new genetic sites. It postulates that all transposable elements replicate in the transposition process so that they remain at their original site while moving to new sites. According to this model, the mechanism of transposition is very different from the insertion and excision of bacteriophage lambda.


Germ line integration of Moloney leukemia virus: identification of the chromosomal integration site. The chromosomal integration site of the structural gene of Moloney murine leukemia virus (M-MuLV) in the genome of BALB/Mo mice was mapped genetically. These mice transmit the exogenous M-MuLV as an endogenous virus at a single Mendelian locus. Two independent experimental approaches were used: (i) Non-virus-producing fibroblasts prepared from homozygous BALB/Mo embryos were fused to Chinese hamster Wg3-h-o cells. In an analysis of 30 independent mouse-Chinese hamster cell hybrid clones, the segregation of the viral genome measured by molecular hybridization and enzymes assigned to 16 different mouse chromosomes were compared. We found a highly concordant segregation of M-MuLV sequences and the mouse enzyme triosephosphate isomerase (TPI, EC 5.3.1.1), whose gene has been assigned to chromosome 6. A further karyotype analysis of 9 clones, in which the chromosomes were identified cytochemically, supported this result. (ii) The segregation of the viral genome was studied in backcrosses of BALB/Mo with ABP/J mice. In the backcross ABP/Jx(ABP/JxBALB/Mo) a linkage of the M-MuLV genome to the morphological marker wa-1 on mouse chromosome 6 was found. This confirmed the conclusion that the M-MuLV genome is integrated in mouse chromosome 6. These experiments define the genetic locus Mov-1, denoting the genetically transmitted structural gene of M-MuLV in BALB/Mo mice.


Effect of tumor promoters, protease inhibitors, and repair processes on x-ray-induced sister chromatid exchanges in mouse cells. The induction of sister chromatid exchanges (SCE) in the second postirradiation mitosis was studied in mouse 10T1/2 cells irradiated with 400 rads (4 grays) and maintained in stationary growth for several hours after x-ray exposure (similar to liquid holding recovery experiments in bacterial cells). X-irradiation with no recovery period induced few SCE. With short recovery intervals, however, the SCE frequency rose in parallel with the increase in survival, reaching a maximum increase of 2-fold after 4 hr; SCE declined with longer recovery intervals. The influence of postirradiation incubation with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and with the protease inhibitors antipain and leupeptin was studied on spontaneous, x-ray-induced (no recovery), and recovery-induced (4 hr) SCE. TPA (0.1 microgram/ml and 1.0 microgram/ml) increased the frequency of both spontaneous and direct x-ray-induced SCE, but not of recovery-induced SCE. Incubation with the protease inhibitors suppressed both TPA- and recovery-induced SCE, but had no effect on direct x-ray-induced SCE. These results are discussed in relation to the hypothesis that promotional events in carcinogenesis may involve the expression of mutational damage in cells by mitotic segregation.