Hormonal influences on motivation, learning, and memory processes. Experiments have shown that the hypothalamic, pituitary, and adrenocortical hormones play an important role in the motivational value of environmental cues, in the acquisition of knowledge, and in the long-term memory of learned behavior. In human volunteers, a synthetic fraction of ACTH seems to improve visual memory, enhance alertness, and increase motivation--all without the hormone's usual endocrine effects.


Options for care of the aged sick.

Options for care of the aged sick. Hospital workups should be done only when they will produce information not otherwise obtainable. Even when patients are acutely ill, home care may be preferable; after care and day care are often best for long-term treatment.


Diagnosis and management of congenital adrenal hyperplasia. After a generation of appropriately treated patients, it is now clear that most of those affected with congenital adrenal hyperplasia can lead virtually normal lives, in terms of ultimate height and sexual development. But the key is early diagnosis. The underlying biochemical mechanisms in the various forms of the disease are discussed, together with the principles and specifics of adrenal hormone replacement therapy.


Latent genomes of Epstein-Barr virus.

Latent genomes of Epstein-Barr virus. Of the EBV DNAs in Raji cells that contained 50 to 60 viral genomes per cell, 90% were separated from high molecular weight cellular DNA by centrifugation in a neutral glycerol gradient after lysis of the cells by treatment with pronase and Sarkosyl, indicating that the major portion of the EBV DNA in Raji cells was not integrated linearly into cellular DNA.


Structure and function of herpesvirus saimira DNA. Two types of genomes are present in HVS stocks: (1) the defective H-genome consisting of highly reiterated heavy (71% C+G) sequences; and (2) the M-genome, which carries the whole genetic information of HVS and is infectious in tissue culture. The sequences in the M-genome are made up of 70% unique L-DNA (with 36% C + G) and 30% H-DNA, which is highly repetitive. H-sequences in the M-genome are clustered at the ends of the molecule.


Nucleic acid biosynthesis in nuclei of cell infected with herpesviruses (HSV and EBV). Analysis of nuclei isolated from herpes simplex virus (HSV)-infected cells by electrophoresis in polyacrylamide gels showed that only virion-associated DNA molecules migrated into the gels. The viral DNA molecules, which do not migrate in the gels, are the precursors for the mature viral DNA. Centrifugation of deoxycholate-treated infected nuclei in sucrose gradients revealed that most of the viral DNA co-sedimented with the cellular DNA. The DNA-dependent RNA polymerase activity also co-sedimented with the viral and cellular DNA. Incubation of nuclei isolated from HSV-infected cells, under in vitro conditions that support DNA synthesis, resulted in the synthesis of viral molecules of low molecular weight. Under the same conditions, nuclei from Burkitt lymphoblasts did not synthesize DNA. The nature of the association between cellular DNA and EBV DNA was studied by hybridization with EBV cRNA. EBV-specific sequences were found in the lymphoblast DNA; they banded at a density of 1.707 g/cm3. Some of the viral mRNA transcribed in HSV-infected nuclei is symmetrically transcribed from HSV DNA. The symmetrical portions of the RNA are removed, since poly (A)-containing mRNA molecules lack homologous sequences. Most of the RNA synthesized in HSV-infected nuclei can be released after incubation of the nuclei in vitro in the presence of ATP. The released RNA consists of poly(A)plus and poly (A)-minus molecules. The mechanism of RNA transport from the nuclei still remains to be studied.


Partial purification and biochemical characterization of herpesvirus saimiri. HVS propagated in Vero cells was labelled with 3H-leucine and purified by isopycnic flotation in sucrose. Electron microscopic examination revealed both complete and incomplete HVS. No significant contamination  with cellular debris was noted. Polyacrylamide gel electrophoresis analysis of solubilized virus revealed at least 12 proteins varying in molecular weight from 8 000 to 200 000 daltons.


Immunological studies with hamster cells transformed by cytomegalovirus and herpes simplex virus type 1. Evidence is presented that cells transformed by CMV and HSV-1 express virus-associated antigens on the cell surface. The finding that hyper-immune antivirus sera block cellular immunity in vitro may explain why virus-immunized hosts have thus far not been found to reject isografts of these cells.


Differences between laboratory strains of Epstein-Barr virus based on immortalization, abortive infection and interference. Biological activities of extracellular Epstein-Barr virus (EBV) from two laboratory strains, namely P3J-HR-1 (P-H) from Burkitt's lymphoma and B95-8 (B95) from infectious mononucleosis, were compared. Virus stocks from both sources contained approximately the same number of virions. Virus from the P-H line induced early antigen in six non-producer EBV-genome carrier cell lines; virus from B95 did not induce early antigen. Extracellular virus from B95 regularly caused lymphocytes from human umbilical cords to form continuous lines (immortalization); P-H virus did not cause primary cultures of human lymphocytes to grow continuously. B95 virus stimulated DNA synthesis, as determined by the rate of incorporation of 3H-thymidine into acid-insoluble material; P-H virus did not stimulate DNA synthesis. Pretreatment of lymphocytes with undiluted P-H virus inhibited immortalization and stimulation of DNA  synthesis by B95 virus. The inhibitory properties of the P-H virus were sedimented at 100 000 g and inactivated by heat and UV irradiation; interference by the P-H virus was neutralized by human serum with antibody to EBV and not by antibody-negative human serum. The hypothesis most consistent with these results is that the P-H virus is defective in gene(s) needed for initiation of immortalization. We speculate that the absence of this gene allows early antigen to be expressed upon superinfection of non-producer cell lines. The availability of two laboratory strains of two laboratory strains of EBV that differ in biological behaviour provides starting material for analysis of the mechanism of lymphocyte immortalization by EBV and of virus structural differences that affect immortalization.


Appearance of Epstein-Barr virus nuclear antigen in human cordblood lymphocytes. Transformation and induction of EBV-specified EBNA was studied in human umbilical cord-blood lymphocytes infected with EBV, HVS or HVA. Transformation of human umbilical umbilical cord-blood lymphocytes into continuous lymphoblastoid cell lines was successful with EBV derived from five of seven human or simian cell lines but not with HVS or HVA derived from simian lymphoblastoid cell lines. EBNA induction was studied using B95-8 virus for infection of lymphocytes; as early as 24 hours after infection, a few EBNA-positive cells were identified in 10 of 12 samples studied. Thereafter, the number of EBNA-positive cells increased progressively and by day 12, 50% of cells contained EBNA; after 18-20 days, approximately 85-90% of cells were EBNA-positive.


Presence of a herpes simplex virus type 1 genome fragment in HSV-transformed cells. LTK-cells infected with UV-irradiated HSV produce transformants that contain a thymidine kinase  (TK) activity not found in the parental LTK-line (Munyon et al., 1971). One of these (TK+) transformants (clone 139) has been analysed for the presence of the HSV genome. Reassociation kinetics studies with iodinated HSV DNA of specific activity of about 9 x 107 cpm/mug have established that there are approximately six copies of a fragment comprising about 15% of the HSV genome in HSV-transformed clone 139. Neither the parental LTK-nor a "revertant" cell line (clone 139 BUDR) obtained from clone 139 showed any detectable HSV-specific sequences. Analysis of data on RNA-125I-HSV DNA reassociation kinetics indicates that perhaps 5% of the HSV genome is transcribed in HSV-transformed clone 139. These results indicate that transformation is probably maintained by the presence of only a fraction of the HSV genome in the TK+ clones.


Some biological properties of herpesvirus saimiri from chronically infected monolayer and suspension cultures. HVS-MEST cells, which are adherent, produced more infectious HVS at 37 degress C than at 33 degrees C. This was paralleled by the appearance of EA and LA in rounded cells and CF antigen in the cell cultures. In contrast, MLC-1 cells, which grow in suspension, produced hardly any infectious virus, but did produce more HVS S antigen than did the HVS-MEST cells. Production of S antigen in MLC-1 cells was augmented by treatment of the cells with BUDR. HVS-MEST cells could be superinfected with HVS, but MLC-1 cells were resistant to super-infection. The unpurified S antigens from both cell lines gave almost identical spectra when tested in CF against a battery of 10 owl and squirrel monkey sera. Thus, two cell lines have been described, one that produces infectious virus and S antigen, and another that produces more S antigen, but in the absence of any great amounts of infectious virus.


Source, alterations, characteristics and use of a new dog cell line (Cf2Th). A cell line derived from normal fetal canine thymus (Cf2Th) has been in culture since 1967. During cultivation the cells have changed morphologically from a fibroblast-like to flat, fusiform appearance and karyologically from diploid (2n=78) with 76 telocentric autosomes to hypodiploid with newly formed atelocentric chromosomes. The cells retain canine characteristic enzyme activity (G6PD and LDH) as well as cell membrane fluorescence and are free of mycoplasma. High passage cells produce tumors in ATST mice. No endogenous viruses have been detected in these cells. No original publication exists, to date, on the origin of this line, but seed stocks thereof have been distributed to many laboratories and the cells have served as experimental substrates in a number of published works on oncology albeit under different designations. The present information is offered in order to establish the provenance of this valuable cell line and to list characteristics which may serve to monitor for its purity and to distinguish it from other existing cell lines of dog origin also in common use.


Persistent infection with bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) in cultured hamster cells. Bovine herpesvirus-1 infection in hamster embryo cells was found to be dependent upon input multiplicity; productive infection was achieved at input multiplicities greater than one, while persistent infection was established when input multiplicities were about 0.5. This persistence was characterized by a noncyclic, minimal degree of cytopathic effect with a low level of released virus. Maintenance of the persistently infected cultures did not require external supportive measures. Subcultivation of the persistently infected cultures led to virus replication followed by CPE and then cell regrowth. With 3 to 4 weeks after subcultivation a persistent infection was re-established. The possible mechanism for the bovine herpesvirus persistence in hamster cells is discussed.


[Comparison of value of four immunological methods (AST, ADNase B, AHy, ANADase) by A-streptococcal infections and their significance in the routine diagnostic (author's transl)]. In the sera of approximately 600 patients of all age groups, who were on clinical suspicion of an A-streptococcal infection, AST, ADNase B, AHy and ANADase were determined. The ANADase alone detects 90% of the acute A-streptococcal infections, the AHy and the ADNase B 80% and the ASO 50%. In skin infections superinfected by A-streptococci, the AHy is positive in 85%, the ANADase in 80%, the ADNase B in 75% and the AST in 40% of the cases. The most frequent A-streptococcal infection, Angina tonsillaris, is determined in the best manner possible through the combination ADNase B + ANADase + AHy. According to the statistical evaluation, the ADNase B and the ANADase are the most reliable tests. The age group of the 4 to 15 year-olds reaches for ASO, ADNase B and AHy a percentage of highly increased titers twice as high in comparison with the adult group. In no case does one test determine 100% of the collective; therefore, a combination of several antibody reactions is desirable for the diagnosis. Increased titers should be checked every four weeks, also normal titers with patients, who continue on the clinical suspicion of having an A-streptococcal infection, because their blood sample may have been taken before the immune reaction occurred.


Effects of in vivo administered B. pertussis and other adjuvants on the mitotic responses of lymphocytes in vitro. Intraperitoneal treatment of mice with adjuvants affects the in vitro response of their lymphocytes toward class-specific mitogen. Spleen cells from animals injected with Corynebacterium parvum organisms showed in some cases an increase in their response to all mitogens, while in other experiments, a moderate decrease in the reaction to T-specific mitogens (concanavalin A and phytohemagglutinin) was found. Injection of lipopolysaccharide (LPS) and in particular Bordetella pertussis bacteria, brought about a marked reduction in the response of spleen cells to B mitogens (LPS and PPD) but had little or no effect on the reaction to the T mitogens. Intraperitoneal administration of B. pertussis caused a marked depletion of lymph nodes and a high level of lymphocytosis. Blood cells of the treated mice showed an increased response to T mitogens, whereas mesenterial lymph node cultures reacted higher than the controls to LPS and without stimulation. No change was noted in the responses of cells from the axillary lymph nodes of these pertussis-treated mice.


Role of cyclic nucleotides in human lymphocyte-mediated antibody-dependent cytotoxicity. Experiments were carried out in order to throw light on the role of cyclic AMP and cyclic GMP in the modulation of the expression of antibody-dependent cytotoxicity mediated by human peripheral blood lymphocytes in vitro. Chicken erythrocytes were used as target cells. Support for an inhibitory role of cyclic AMP was derived from the marked suppression of cytotoxicity by dibutyryl cyclic AMP. Cyclic AMP itself had little effect. Isoproterenol, prostaglandin E1 and prostaglandin E2 increased cyclic AMP concentrations and strongly inhibited the cytotoxic effect of the lymphocytes. Histamine showed very slight elevation of the cyclic AMP level and suppression of the cytotoxicity. Theophylline did not increase the cyclic AMP concentration, but potentiated the effects of isoproterenol and prostaglandin E1 on the cyclic AMP levels, and significantly inhibited cytotoxicity. No clear effects were seen when cyclic GMP, dibutyryl GMP, acetylcholine or carbacholine were added to the cells. The results indicate that in this system cyclic AMP inhibits cytotoxicity, while no evidence for a role of cyclic GMP has been obtained.


Detection of Epstein-Barr virus early antigen-D and its antibodies by passive hemagglutination. These data describe the use of a passive hemagglutination test (PHAT) for the detection of antibodies to the Epstein-Barr virus (EBV) associated antigen, early antigen-D (EA-D). EA-D was obtained from EBV-superinfected RPMI 64-10 cell preparations which showed by immunofluorescent staining (IF) that 25-35% of the cells contained EA-D. Solubilization of EA-D was achieved by sonic oscillation, treatment of the pellet-associated material with triton X 100, ammonium sulfate precipitation, and chromatography on diethylaminoethyl celluiose. Successful use of the PHAT for detection of antibodies to EA-D was dependent on the degree of purity of the partially purified antigen. Throughout these studies preparations from uninfected 64-10 cells were used as control reagents. More than 100 human antisera from patients with EBV-associated diseases, which had been previously tested by standard IF procedures were assayed by PHAT. A good correlation was found between IF and PHAT titers for EA-D and, in addition, the PHAT was 50-100 times more sensitive. Most human sera gave no agglutination of red cells coated with control preparations. An inhibition (blocking) of PHAT was developed which was useful in the purification and characterization of EA-D. These data indicate that the PHAT may provide a rapid, reliable, objective and sensitive method for studying EA-D and possibly other virus-associated antigens.


Herpesvirus infection and cervical anaplasia: a seroepidemiological study. In a cohort of 23,146 pregnant females followed for 37-56 months subsequent to collection of prenatal sera, 57 cases of carcinoma of the cervix were encountered. Three controls were selected for each case from the residual cohort matched to cases for age, residence, number of prior cytology smears taken, and the data of entry to the study. The antibody activity to herpesvirus type 1 and type 2 was examined by the indirect hemagglutination test. The proportion of cases positive for HSV-2 infection was greater than that of the controls; however, the difference was not significant at the 5% level. The relative risk value for the association between HSV-2 infection and carcinoma of the cervix was found to be 2.33. The geometric mean titers of IHAT revealed that the cases of cervical cancer had a higher titer than the controls with respect to HSV-1 as well as HSV-2 antibodies. A sufficiently longer follow-up, yielding a larger sample size, is desired to assess the specific role of HSV-2 infection in development of cervical anaplasia.


Polyoma genome transcription in transformed mouse cells growing in culture and as tumors in syngeneic mice. The strands of the polyoma genome coding for early and late viral RNA were separated by means of asymmetric cRNA synthesized under high salt conditions by Escherichia coli RNA polymerase. Each strand was then employed in RNA-DNA hybridization experiments to determine the degree to which it is transcribed in transformed cells under several conditions. Except for a concanavalin-A-selected revertant, approximately one-quarter of the early strand was expressed in all of the situations investigated. In contrast, while no significant late strand transcription was detected in transformed cells in culture, the tumors induced by these cells contained transcripts complementary to about 12% of this strand. The results are discussed in terms of current knowledge of the amount of the virus genome required to transform cells.


Effects of ethylnitrosourea administration during pregnancy on three subsequent generations of BDVI Rats. A single dose of 40 mg/kg of N-nitrosoethylurea (ENU) was administered to BDVI rats on the 16th day of pregnancy. The first generation descendants (F1) were mated on a brother-to-sister system to produce a second generation (F2) which was then mated to produce a third generation. A high incidence of nervous tissue tumors and a few kidney tumours were observed in F1 descendants. A few nervous tissue tumours were observed in F3 but not in F2 descendants. These results partially confirm previous observations obtained with 7,12-dimethylbenz(a)anthracene in mice and nitrosomethylurea in rats and indicate that prenatal exposure to a chemical carcinogen may result in an increased cancer risk which can persist for more than one generation.


Detergent solubilization and partial purification of tumor specific surface and transplantation antigens from SV40-virus-transformed mouse cells. A solubilization technique employing 0.5% Triton X-100 was developed to obtain both SV40 virus (SV40)-induced tumor-specific surface antigen(s) (TSSA) from SV40-transformed mouse cells, as determined by a serum-mediated microcytolytic assay, and tumor-specific transplantation antigen(s) (TSTA), as determined by in invivo experiments. High yields (approximately 50%) of TSSA were obtained in whole-cell extracts and also after ammonium sulfate fractionation. Additional fractionation of a 30-50% ammonium sulfate fraction by gel exclusion chromatography on Sephadex G-150 resulted in two pooled fractions which contained TSSA activity. The first eluted close to the void volume, and the second in the 45,000 molecular weight region. The various TSSA active fractions were also active in vivo TSTA tests. Detergent solubilization provides a suitable technique to recover the SV40-induced antigens in good yield, and apparently in intact form.


Follow-up of a token economy applied to civilly committed narcotic addicts. Thirty-one civil-commitment, male, heroin addicts participated in an intramural token economy designed to reinforce the rehabilitative process by programming the incentive of length of residential stay. The project evaluated participation in rehabilitative activities during intramural stay, personality change as measured by the MMPI, and follow-up of the participants' community aftercare retention. The token economy had a substantial impact on the participants' institutional adjustment but had little influence on the evaluative dimensions of personality change and rate of aftercare retention.


Effects of gaba, gabob and some anticonvulsant drugs on dibutyryl cyclic AMP evoked seizures. The present experiments were undertaken to study the possible effects of Gaba, Gabob, phenobarbitone, diphenylhydantoin, and diazepam given systemically on convulsant activity evoked in chicks by intrahypothalamic injection of dibutyryl cyclic AMP (cAMP). Diphenylhydantoin and phenobarbitone antagonized the effect of dibutyryl cAMP but unfortunately the effects of Gaba and Gabob were complicated by osmotic effects. Diazepam, an anticonvulsant benzodiazepine, did not affect dibutyryl cAMP-induced electrocortical spiking. The dibutyryl cAMP-induced syndrome seems to be of limited value as a model for assessing the activity of anticonvulsant drugs are present.


Purification of protected syntheic peptides by preparative high performance liquid chromatography on silica gel 60. A simple preparative system is described for rapid and efficient purification of protected synthetic peptides on a gram scale by high performance liquid chromatography on prepacked silica gel 60 columns. A variety of protected peptides up to tetradecapeptides have been chromatographed at pressures of 50 to 150 psi and obtained in analytically pure from within 2 to 4 h. With such commonly used protecting groups as N-benzyloxycarbonyl (Z), N-2-(p-biphenylyl)-2-propyloxycarbonyl (Bpoc), N-t-butyloxycarbonyl (Boc), O- and S-t-butyl (But), and S-acetamidomethyl (Acm), compounds were sufficiently soluble in chloroform, alcohols, acetic acid, or mixtures of these solvents for column loading. Dimethylformamide was also used as a solvent for loading. Solvent systems for column elution in isocratic, stepwise, or gradient modes were composed of chloroform, isopropanol, ethanol, or methanol and acetic acid in ratios that differed for each protected peptide depending on Rf values on t.l.c. plates. A simple chromatography is described which was self-assembled using standard instruments commonly in use in most laboratories. A shut-off valve was designed to prevent loss of material between fractions.


Synthesis and transport of herpes simplex virus proteins in arginine-deprived BSC-1 cells. The synthesis and transport of [35S]methionine-labeled peptides from the cytoplasm to the nucleus of herpes simplex virus-infected cells was studied by polyacrylamide gel electrophoresis, using autoradiography of the labeled peptides, In complete medium the transport of proteins from the cytoplasm to the nucleus is a selective process and different peptides are transported at different rates. In arginine-deficient medium, the synthesis of viral peptides in the cytoplasm is reduced and the labeled proteins are transported slowly to the nucleus. These viral peptides gradually disappear from the cytoplasm, suggesting degradation of the synthesized proteins.


[Report of a case of Goltz-Gorlin syndrome]. A case of Goltz-Gorlin-syndrome (congenital ectodermal and mesodermal dysplasia) in a 8 1/2 year old girl with focal dermal aplasia after birth, poikilodermia, anomalies of nails, hair, teeth, eyes and bones is presented.


Cytophotometry of post mortem glycogenolysis in different histochemical types of muscle fibres of the pig. Serial transverse sections of porcine longissimus dorsi muscle (18 pigs, 50 to 178 kg live weight) were reacted for NAD tetrazolium reductase and ATPase at pH 9.4, and for glycogen with the periodic acid-Schiff (PAS) reaction. Three histochemical types of muscle fibre were identified; (1) strong ATPase and weak NADH oxidative activity; (2) strong ATPase and intermediate NADH oxidative activity; and (3) weak ATPase and strong NADH oxidative activity. Immediate post mortem samples from one side of each animal were compared with a later post mortem sample from the other side by measuring the absorbance of PAS-stained glycogen at 570 nm with a microscope photometer. Later post mortem absorbance was expressed as a percentage of immediate post mortem absorbance in each category of muscle fibre in order to compensate for distributional error and different starting levels of glycogen. Muscle fibres with weak ATPase and strong NADH oxidative activity showed a progressive decrease in absorbance of PAS-stained glycogen post mortem. In some animals, fibres with strong ATPase and intermediate or weak NADH oxidative activity showed an initial post mortem increase in absorbance of PAS-stained glycogen which was then followed by a progressive decrease. The maximum rates of decrease in absorbance in the three fibre types did not differ to any great extent.


[Applied phoniatry. IV. Rhinophony (author's transl)]. Rhinophony, which is a change of vocal sound, can appear as a major symptom in many peripheral or central disorders of childhood, adolescence and adulthood. Its investigation, aetiology and management from various points of view are discussed. These are considered broadly as rhinophony aperta (palatal paralysis, functional rhinophony aperta, congenitally short palate); as rhinophony clausa (organic in origin, functional nasal speech); and mixed types of rhinophony. Typical phoniatric problems related to cleft lip and palate are also presented.


Fatal generalized feline viral rhinotracheitis in a young adult cat. A 7-month-old cat died following a 17-day illness. Necropsy findings included sharply demarcated lingual ulcers and multifocal necrotic hepatitis. Intranuclear inclusions were found in the lesions, and a herpesvirus was recovered from specimens of liver and tongue. The virus was identified by neutralization with specific antiserum as feline rhinotracheitis virus.


A comparison of age estimation using discriminant function analysis with some other age estimations of unknown skulls. Available methods of estimating the ages of unknown skulls are notoriously inaccurate. Making use of a unique opportunity to view several hundreds of remains from two nineteenth century cemeteries, various methods of estimating age were tested. A statistical method which gives relative weighting to each age indicator before reducing the data to a single pooled age estimate was found to be the most reliable. The greater the number of criteria which were utilized for age estimation, the more highly correlated was the estimated age of remains with the actual age at death. The degree of accuracy in estimating the ages of skulls of under 20 years was relatively high, when only the stage of development of dental tissues was examined. With increasing age over 20 the accuracy decreased, particularly at ages over 45. The range of age distribution of skulls estimated from published data on suture closure tended to be more restricted than that which may acutally occur.


Regeneration of parietal and visceral peritoneum: an enzyme histochemical study. The healing of parietal and visceral peritoneum has been studied by the techniques of enzyme histochemistry in an attempt to define more precisely the type of cell responsibile for forming the new mesothelium. The changes occurring in the distribution of the following enzymes throughout the course of healing have been investigated: acid phosphatase, alkaline phosphatase, ATPase and non-specific esterase. Regenerating mesothelial cells have been found to have several enzyme histochemical properties in common with subperitoneal connective tissue cells. It has not been possible to distinguish between primitive mesenchymal cells and subperitoneal fibroblasts by the histochemical techniques used in this study and therefore the study has not been fruitful in determining whether the new mesothelium arises from primitive mesenchymal cells or subperitoneal fibroblasts. The present study does, however, lend weight to the view that the new mesothelium is not derived from peritoneal macrophages.


The distribution and relative sizes of three histochemical fibre types in the rat tibialis anterior muscle. A quantitative study of the tibialis anterior muscle of the adult rat showed that the proportions of each 'histochemical' fibre type varied between adjacent regions along the deep to superficial and medial to lateral axes of the muscle. The distribution of fibre types classified with oxidative enzymes was similar to that using phosphorylase, but differed from that obtained with sections stained for myosin ATPase. This apparent discrepancy in classifation was confirmed by comparison of the ranges of fibre cross sectional areas for each fibre type classified on the basis of their oxidative enzme and ATPase activites, and by an independent analysis of individual fibres in serial sections stained for succinic dehydrogenase and ATPase.


The postnatal development of the alimentary canal in the opossum. III. Small intestine and colon. The duodenum of the newborn opossum exhibits a patent lumen containing scattered elongate villi, whereas the distal segments of the small intestine are smaller in diameter and are filled with short immature villi. The muscularis externa through the small intestine consists of a single layer of myoblasts. Interposed between the intestinal lining epithelium and the muscularis externa is an extensive capillary bed that occupies a considerable proportion of the intestinal wall. Additional villi appear to form during the postnatal period as a result of evaginations of the epithelium, together with underlying connective tissue and vasculature, into the intestinal lumen. Intestinal glands are not observed until 8.5cm, and are shallow in depth even in the adult. The epithelium of the entire small intestine is modified for absorption until just prior to weaning. The principal intestinal lining cells show an extensive apical endocytic complex, large supranuclear vacuoles and numerous cytoplasmic inclusions. Intestinal epithelial cells of the colon also appear to be modified for absorption during the first two weeks after birth. Although goblet cells and Paneth cells are present during the suckling period, they do not comprise a significant population in the intestinal epithelium until after weaning. In contrast to the small intestine, goblet cells are numerous in the colon by the ninth postnatal day. The significance of macromolecular absorption and the possibility of passive immunity being transmitted in the opossum during suckling are discussed and related to similar events that occur in the slckling young of several eutherian species. The possible functional significance of two large membranes that develop in the lamina propria of the intestines after weaning also is discussed.


Histochemical characteristics and contractile properties of the spinotrapezius muscle in the rat and the mouse. In the spinotrapezius muscle of the rat and the mouse approximately equal proportions of A,B and C fibres are present. The spinotrapezius therefore contains a lower proportion of B fibres than are known to be present in soleus, but a higher proportion than in tibialis anterior. These results are consistent with the functional properties of the muscles, for the values for isometric twitch contraction time, the half relaxation time, and the twitch summation frequency of the spinotrapezius are also intermediate between those of soleus and tibialis anterior. The present experiments support the view that the proportion of B fibres to A and C fibres govern the overall functional characteristics of mammalian muscles.


Lack of electrical interaction between proximal bundle branches and subjacent muscle. Microelectrode techniques were used to assess the importance of subthreshold electrotonic interactions between the canine proximal bundle branches and adjacent septal myocardium, and vice versa. Bundle branch action potential duration, maximal rising velocity of phase O, current threshold requirements for all-or-none depolarization, transmembrane voltage, and spontaneous frequency were not altered by adjacent septal muscle activation. Activation of the proximal bundle branches did not change the transmembrane voltage of immediately subjacent muscle cells; likewise, all-or-none activation of ventricular septal muscle did not effect a voltage change in the overlying proximal bundle branches. We conclude that a high ohmic resistance barrier between proximal bundle branch and subjacent muscle precludes significant electrotonic interactions between these neighboring structures.


cAMP in temperature- and ADH-regulating centers after thermal stress. cAMP concentrations in temperature-regulating sites of the brain and plasma osmolality were measured after exposure of male rats to 36 degrees C and 37-42% rh. for 10, 20, or 30 min. Plasma osmolality was affected by none of the heat exposures. In both the preoptic area and the posterior medial hypothalamus, cAMP concentrations were increased compared to controls, after 10, 20 and 30 min of heat exposure. In the supraoptic-paraventricular nuclei, neither 10 nor 20 min of exposure resulted in augmented cAMP concentrations; but after 30 min of heat exposure, cAMP levels in these nuclei were significantly greater than in controls. Neurohypophysial cAMP concentrations were increased after both 10 and 30 min of exposure. Cerebral cortical cAMP concentrations were not affected by thermal stress. It is concluded that cAMP is involved in the neural mechanisms which are brought into play to regulate body temperature during acute heat exposure. The significance of this involvement and its relation to the overall temperature-regulating mechanisms of the body are discussed.


Neonatal chest wall afferents and regulation of respiration. We have studied two groups of eight preterm infants, relating chest wall afferent information to respiratory timing. Rib cage and abdominal motion were monitored by magnetometers and flow and tidal volume via a face mask. In the first group, studies were done in REM sleep when spontaneously occurring distortion of the rib cage occurred and a significant linear relationship between the rate of distortion of the chest wall and shortening of the inspiratory time (Ti) was found in all infants. Reduction in this distortion by the use of continuous positive airway pressure (CPAP) or continuous negative pressure at the body surface (CNeg) was associated with a significant (P less than 0.01) lengthening of Ti. Absence of changes in Ti when pressure was applied in quiet sleep suggested that lung volume or chemical changes were not involved. In the second group of infants we artificially generated the afferent inflow by using vibratory stimuli applied in one intercostal interspace and produced a significant (P less than 0.05) shortening in Ti. We suggest that the distortion of the rib cage in REM sleep generates afferent information from intercostal muscle spindles that is related to the rate of distortion and this, via a supraspinal reflex, inhibits phrenic motoneuron discharge. It may then be of importance in the etiology of apneic episodes in these infants. Applied pressure may be of benefit because it reduces an inhibitory afferent inflow.


Galactose transport in Salmonella typhimurium. We have studied the various systems by which galactose can be transported in Salmonella typhimurium, in particular the specific galactose permease (GP). Mutants that contain GP as the sole galactose transport system have been isolated, and starting from these mutants we have been able to select point mutants that lack GP. The galP mutation maps close to another mutation, which results in the constitutive synthesis of GP, but is not linked to galR. Growth of wild-type strains on glaactose induces GP but not the beta-methylgalactoside permease (MGP). Strains lacking GP are able to grow slowly on galactose, and MGP is induced; however, D-fucose is a much better inducer of MGP. Induction of GP or MGP is not prevented by a pts mutation, although this mutation changes the apparent Km of MGP for galactose. pts mutations have no effect on GP. GP has a rather broad specificity: galactose, glucose, mannose, fucose, 2-deoxygalactose, and 2-deoxyglucose are substrates, but only galactose and fucose can induce this transport system.


Reduction of iron and synthesis of protoheme by Spirillum itersonii and other organisms. Membranes from Spirillum itersonii reduce ferric iron to ferrous iron with reduced nicotinamide adenine dinucleotide or succinate as a source of reductant. Iron reduction was measured spectrophotometrically at 562 nm using ferrozine, which chelates ferrous iron specifically. Reduced nicotinamide adenine dinucleotide or succinate was also effective as a source of iron. The effects of respiratory inhibitors suggested that reduction of iron occurs at one or more sites on the respiratory chain before cytochrome c. Reduction of iron and synthesis of protoheme with the physiological reductants were also observed with crude extracts of other bacteria, including Rhodopseudomonas spheroides, Rhodopseudomonas capsulata, Paracoccus denitrificans, and Escherichia coli. The effect of oxygen upon reduction of iron and formation of protoheme was examined with membranes from S. itersonii, using succinate as a source of reductant. Both systems were inhibited by oxygen, but this effect was completely reversed by addition of antimycin A. We conclude that reduced components of the respiratory chain serve as reductants for ferric iron, but with oxygen present they are oxidized preferentially by the successive members of the chain. This could be a mechanism for regulating synthesis of heme and cytochrome by oxygen.


Evidence for stable messenger ribonucleic acid during sporulation and enterotoxin synthesis by Clostridium perfringens type A. Stable messenger ribonucleic acid (mRNA) was shown to be involved in both enterotoxin synthesis and synthesis of other spore coat proteins in Clostridium perfringens. When used at a concentration that inhibited [14C]uracil incorporation, rifampin, a specific inhibitor of deoxyribonucleic acid-dependent RNA polymerase, prevented incorporation of a mixture of labeled amnoo acids by 3-h sporulating cells. At that time, enterotoxin protein was first detectable and cells were primarily at stage II or III of sporulation. When rifampin or streptolydigin was added to 5-h sporulating cells, which were primarily at stage IV or V and had significant toxin levels, incorporation of labeled amino acids continued through 30 min despite its presence. Rifampin also failed to prevent the specific synthesis of enterotoxin, a structural protein of the spore coat. The half-life of enterotoxin RNA was estimated to be at least 58 min. When cell extracts from 5-h sporulating cells that had been exposed to 3H-labeled amino acids for 10 min were subjected to electrophoresis on polyacrylamide gels and the gels were subsequently analyzed for radioactivity, two major peaks of radioactivity were obtained. The two peaks corresponded to enterotoxin and another spore coat protein(s). Similar results were obtained when the cells had been preincubated for 60 min with rifampin before label addition, indicating the functioning of stable mRNA.


Control of morphogenesis in Arthrobacter crystallopoiets: effect of cyclic adenosine 3',5'-monophosphate. The intracellular levels of cyclic adenosine 3',5'-monophosphate (cyclic AMP) were measured at various intervals during growth and morphogenesis in Arthrobacter crystallopoietes. Cyclic AMP levels remained relatively constant throughout growth in spherical cells grown in glucose-based media. Immediately after inoculation of spheres from glucose- to succinate-containing media, a 30-fold increase in intracellular cyclic AMP was detected. This dramatic rise in cyclic AMP preceded the observed change in cellular morphology from spheres to rods. The cyclic AMP level in rod-shaped cells rapidly dropped to a relatively stable concentration during the exponential growth phase. At the onset of stationary phase and rod-to-sphere morphological transition, a second peak of cyclic AMP was observed. Neither of these two peaks was detectable in a morphogenetic mutant that grew only as spheres. The intracellular levels of cyclic AMP in this mutant remained constant throughout exponential growth and decreased slightly during stationary phase. Effects of exogenously added cyclic nucleotides and their derivatives to both parent and mutant cultures were investigated. The data presented indicate that dramatic changes in intracellular cyclic AMP levels occur just before the morphological transitions characteristic of the morphogenetic cycle in A. crystallopoietes. It is suggested that cyclic AMP is a contributing factor in the regulatory phenomenon associated with morphogenesis in this bacterium.


Constitutive mutations in the controlling site region of the araBAD operon of Escherichia coli B/r that decrease sensitivity to catabolite repression. Strains of Escherichia coli B/r containing a deletion of the regulatory gene araC are Ara-. Slow-growing revertants of these strains were isolated and designated aralc because they contain a second mutation in a controlling site, aral, that allows for a low level of constitutive expression of the araBAD operon (Englesbert et al., 1969). We mutagenized aralc delta C strains and selected mutants that grow faster in mineral L-arabinose medium. The new mutations, called araXc, map very close to the original aralc mutations and are in the controlling site region between araB and araC. The aralcXc delta C strains have a higher constitutive level of expression of the araBAD operon than the aralc delta C parents. The araXc mutations are cis acting and decrease the araBAD operon's sensitivity to catabolite repression. The araBAD operon is expressed equally well in ara delta C and ara C cya crp backgrounds. The repressor form of ara C protein is able to repress the constitutive synthesis due to the ara Xc allele.


Functional mosaicism of membrane proteins in vesicles of Escherichia coli. Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.


Gluconeogenesis in Saccharomyces cerevisiae: determination of fructose-1,6-bisphosphatase activity in cells grown in the presence of glycolytic carbon sources. The activity of fructose-1,6-bisphosphatase (FBP), a gluconeogenic enzyme, was determined in wild-type Saccharomyces cerevisiae X2180 grown in the presence of the glycolytic carbon sources, glucose, fructose, and galactose. The activities of phosphofructokinase (PFK), a glycolytic enzyme, and phosphoglucose isomerase (PGI), an enzyme functioning both in glycolysis and gluconeogenesis, were determined for purposes of comparison. A measurable amount of FBP activity was present in 20-h-old cells grown with moderate shaking in 1% glucose-nutrient or minimal medium. This activity increased significantly in 40 and 60-h-old cells. Similar levels of FBP activity were also present in 20-, 40-, and 60-h-old cells grown in 1% fructose-nutrient medium. A higher level of FBP activity was present in 20-h-old cells grown in 1% galactose-nutrient medium than in 20-h-old cells grown in 1% glucose- or fructose-nutrient medium. The FBP activity in glucose- or fructose-grown cells was higher than the corresponding activity in cells grown under similar conditions for 40 and 60 h in the presence of ethanol, a gluconeogenic carbon source. The PFK activity was significantly less in galactose- and ethanol-grown cells. The PGI activity was relatively constant in 20-, 40-, and 60-h-old cells grown in the presence of glucose, fructose, and galactose, but this activity was reduced approximately 50% in ethanol-grown cells. It is concluded from these results that, depending upon the concentration of carbon source and the time of incubation, FBP, a strictly gloconeogenic enzyme, is synthesized by S. cerevisiae grown in the presence of glycolytic carbon sources.


Isolation of the major glycoprotein from plasma membranes of an ascites hepatoma AH 66. Plasma membranes were isolated from AH 66 cells, some of which had been labeled with [14C]glucosamine, by the following procedure: homogenization of cells which had been hardened by treatment with Zn ions, fractionation of the homogenate by sucrose density gradient centrifugation and purification of the membranes by partition in an aqueous two-phase polymer system. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) of the plasma membranes and subsequent staining of the gel for protein and carbohydrate, and determination of radioactivity on the gel eluates indicated the presence of at least 10 bands of glycoproteins. The major band contained 27% of the total radioactivity incorporated into the plasma membranes and was most heavily stained with the periodate-Schiff reagent. To isolate the major glycoprotein, the membranes were solubilized with 0.6 M lithium diiodosalicylate containing 0.5% Triton X-100, then the solution was treated with phenol. The major glycoprotein, obtained in the aqueous phase, was further purified mainly by repeated chromatographies on Sepharose 6B. The purified preparation was practically homogeneous on SDS-polyacrylamide gel electrophoresis, as judged by radioactivity determination and by carbohydrate staining, but contained small amounts of carbohydrate-free proteins. The major glycoprotein had an apparent molecular weight of 160,000, as determined by SDS-polyacrylamide gel electrophoresis. The final preparation contained about 44% carbohydrate on a weight basis, and the carbohydrate moiety was composed of glucosamine, galactosamine, galactose, mannose, fucose, and sialic acid. This composition indicates that the major glycoprotein contains both N- and O-glycosidically linked oligosaccharide moieties.


Chemical characterization of the serum very low density and high density lipoproteins from bullfrog, Rana catesbeiana. The chemical properties of very low density and high density lipoproteins of adult bullfrog serum were determined. This serum contained extremely low levels of both very low density lipoprotein (10-30 mg/100 ml) and high density lipoprotein (5-10 mg/100 ml). The constituents of very low density lipoprotein, on a weight percentage basis, were found to be 48.1% triglyceride, 17.3% cholesterol ester, 8.8% cholesterol, 11.6% phospholipid, and 12% protein. These constituents were also present in high density lipoprotein with weight percentage values of 3.7%, 19.3%, 11.9%, 25.2%, and 36.8%, respectively. The fatty acid compositions of the triglycerides, cholesterol esters, and phosphatidylcholine were quite similar in the very low density lipoprotein and high density lipoprotein. However, shingomyelin fatty acid composition was appreciably different in the two lipoproteins. Disc gel electrophoresis in sodium dodecyl sulfate-polyacrylamide gels produced patterns with one major (approximate molecular weight, 7,000) and several minor bands for the apoprotein of very low density lipoprotein and one major (approximate molecular weight, 28,000) and several minor bands for that of high density lipoprotein.


Cardiac adenosine 3':5'-monophosphate. Free and bound forms in the isolated rat atrium. Adenosine 3':5'-monophosphate (cyclic AMP), a mediator of hormone action in a variety of tissues, has been measured in its free and bound forms in intact cardiac tissue. We have used a rapid high dilution technique which involves tissue homogenization, subcellular fractionation, and separation of bound from free cyclic AMP by Millopore filtration. The precision of this method is dependent upon minimization of binding and dissociation of cyclic AMP that occur during the preparation and handling of tissue homogenates. In each experiment, a tracer of cyclic [3H]AMP prebound to isolated cardiac binding protein was freed of unbound cyclic [3H]AMP by Sephadex gel filtration and added to the tissue just prior to homogenization in cold EDTA buffer. This tracer was therefore treated identically to the sample through all subsequent dilution, fractionation, and filtration procedures, and provided an acurate internal monitor for total cyclic AMP dissociation during the course of the free-bound determination. Each tissue sample was then individually corrected for dissociation. Rapid dilution to produce a 1:1000 homogenate was found to lower endogenous cyclic AMP levels sufficiently to make binding (or rebinding) during the procedure negligible (less than 5%). Spontaneously beating rat right atria (controls) contained 5.96 +/- 0.28 pmol of cyclic AMP/mg of protein (n = 19) of which 41 and 14% were bound to soluble and particulate proteins, respectively. The remaining cyclic AMP was free. Pretreatment of the tissue with 1 muM isoproterenol (30 s at 30 degrees) increased both the bound and free forms of cyclic AMP (n = 8). While free cyclic AMP increased 420% with the catecholamine, the bound forms increased 240% (soluble) and 60% (particulate). Similar results were obtained when atria (n = 6) were treated with the phosphodiesterase inhibitor, methylisobutylxanthine (0.5 mM, 10 min at 30 degrees). When both agents were used together, cyclic AMP bound to soluble proteins was elevated 4-fold over control while free cyclic AMP increased 27-fold (n = 7), indicating saturation of the soluble sites. It could be calculated that less than one-third of these sites are occupied in the unstimulated cell. These sites may represent the R subunit of cyclic AMP-dependent protein kinase. The data suggest that half-maximal binding in vivo occurs at an intracellular free cyclic AMP concentration of about 1 muM.


Metabolism of 5-fluorouracil in sensitive and resistant Novikoff hepatoma cells. N1-S1/FdUrd Novikoff hepatoma cells, which lack thymidine kinase activity, are resistant to 5-fluorouracil (FUra) as well as 5-fluorodeoxyuridine (FdUrd), suggesting that the pathway, FUra leads to FdUrd leads to FdUMP, is utilized for the conversion of FUra to FdUMP. However, the inhibition of thymidylate synthetase activity, the presumed target of FdUMP, by 1 X 10(-4) M FUra in intact N1-S1 Novikoff hepatoma cells, which have significant levels of thymidine kinase activity, is completely eliminated by 5 X 10(-4) M hydroxyurea, which is a potent inhibitor of ribonucleotide reductase. These results imply that the formation of ribonucleotides and does not involve thymidine kinase. This apparent dichotomy can be explained by the fact that, in addition to the well known lack of thymidine kinase activity, [14C]FUra conversion to ribonucleotides is greatly depressed in the N1-S1/FdUrd cells. Hence, the formation of FdUMP from FUra in Novikoff hepatoma cells apparently proceeds primarily via the intermediate formation of ribonucleotides. The decreased conversion of FUra to ribonucleotides in N1-S1/FdUrd cells decreases not only the ability of the analog to inhibit DNA synthesis, but also its effect on RNA metabolism. FUra, at a concentration of 1 X 10(-5) M, inhibits rRNA maturation in N1-S1 cells, but not in N1-S1/FdUrd cells. Since N1-S1/FdUrd cells are completely resistant to 1 X 10(-5) M FUra, whereas N1-S1 cells are completely inhibited by 1 X 10(-5) M FUra, even in the presence of 1 X 10(-4) M thymidine, the effects of FUra on RNA metabolism appear to contribute significantly to the cytotoxicity of the analog at higher drug concentrations.


Portein kinases in brown adipose tissue of developing rats. State of activation of protein kinase during development and cold exposure and its relationship to adenosine 3':5'-monophosphate, lipolysis, and heat production. The addition of norepinephrine to brown fat in vitro produced a dose-dependent increase in the protein kinase activity ratio (the ratio of activity assayed without cAMP to that assayed with cAMP) in extracts subsequently prepared in the presence of 0.5 M NaCl. The ratio was slightly increased by insulin. The effects of norepinephrine were potentiated by theophylline and reduced by propranolol. There was a significant linear regression between protein kinase activity ratio and the rate of glycerol release for ratios between 0.32 and 0.52. Higher activity ratios were associated with a slight but nonsignificant increase in glycerol release. The relationship between the protein kinase activity ratio and the concentration of cAMP in brown fat could be expressed by simple saturation kinetics. There was a significant linear regression between the reciprocal of the concentration of cAMP in the tissue and the reciprocal of the activity ratio over the whole range of observed values. Exposure of 1-month-old rats to cold increased the protein kinase activity ratio in their brown fat. This confirms that activation of protein kinase is involved in the physiological response of a tissue to a specific environmental stimulus. As the rat became fully adapted to the cold, the activity ratio declined. The protein kinase activity ratio in brown fat was low in late fetuses but greatly increased immediately after birth and remained high for the next 2 weeks. During this period the ratio was not further increased by the injection of norepinephrine but was reduced after chemical sympathectomy. The activity ratio in brown fat fell during the 3rd and 4th weeks after birth. At this time injection of norepinephrine increased the ratio whereas chemical sympathectomy had little effect. These observations confirm that the stimulation of the tissue by the sympathetic nerves results in an activation of protein kinase and reflect the reduced requirement for heat production in brown fat as the animals grow.


Transmission of the cytochrome c structural gene in horse-donkey crosses. Donkey cytochrome c was shown to differ from horse cytochrome c by having a serine in position 47 rather than a threonine. The rest of the amino acid sequences are identical. Mules and hinnies, both males and females, carry equal amounts of horse and donkey cytochromes c. The same ratio is found in hinnies in preparations from heart tissue and from skeletal muscle. These results demonstrate that cytochrome c is transmitted in horse-donkey crosses as a simple Mendelian character which is neither sex-linked nor shows dominance. The cytochrome c gene is therefore located in the nuclear genome, as earlier shown to be the case for Saccharomyces iso-1-cytochrome c.


Characterization and regulation of heart adenosine 3':5'-monophosphate-dependent protein kinase isozymes. There is broad species variation in the type of cAMP-dependent protein kinase isozyme present in supernatant fractions of heart homogenates as determined by DEAE-cellulose chromatography, Isozyme I, which elutes at less than 0.1 M NaCl, is predominant in mouse and rat hearts; while isozyme II, which elutes at greater than 0.1 M NaCl, is the predominant type in beef and guinea pig. Human and rabbit hearts contain about equal amounts of the two types. The type I heart kinases are more easily dissociated into free regulatory and catalytic subunits by incubation with histone than are the type II kinases, and the separated regulatory and catalytic subunits of isozyme II of rat heart reassociate more rapidly than the subunits of isozyme I under the conditions used. The data from several experiments using rat heart indicate that the basal activity ratio of the protein kinase in crude extracts (approximately 0.15) is due mainly to basal endogenous cAMP and that cAMP elevation accounts entirely for the epinephrine effect on the enzyme. Addition of epinephrine and 1-methyl-3-isobutylxanthine to the perfusate causes a rapid (1 min) increase in cAMP, active supernatant protein kinase, and active phosphorylase in perfused hearts of both rat (mainly isozyme I) and guinea pig (mainly isozyme II). The elevation percentage in cAMP is about the same in the two species, but the increase in active protein kinase is greater in rat heart. If hearts from either animal are perfused continually (10 min) with epinephrine (0.8 muM) and 1-methyl-3-isobutylxanthine (10 muM), the cAMP level, active protein kinase, and active phosphorylase remain elevated. Likewise, all parameters return rapidly to the basal levels when epinephrine and 1-methyl-3-isobutylxanthin are removed. Most of the epinephrine effect on the rat heart supernatant kinase is retained at 0 degrees if cAMP is removed by Sephadex G-25 chromatography, although this procedure completely reverses the epinephrine effect in the guinea pig heart. The epinephrine effect on the rabbit heart kinase (approximately equal amounts of isozymes I and II) is partially reversed by Sephadex G-25. These species differences can be accounted for by differences in association-dissociation behavior of the isozymes in vitro. The data suggest that epinephrine causes activation of both isozymes. The activity present in the particulate fraction comprises nearly half of the total cAMP-dependent protein kinase activity in homogenates of rabbit heart. Triton X-100 extracts of low speed particulate fractions from hearts of each species tested, including rat heart, contain predominantly or entirely the type II isozyme, suggesting differences in intracellular distribution of the isozymes. The binding of the protein kinase to the particulate fraction is apparently due to the properties of the regulatory subunit component. Differences in topographical distribution of the isozymes could provide for differences in either physiological regulation or substrate specificity.


Regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase in minimal deviation hepatoma 7288C. Immunological measurements in hepatoma tissue culture cells. The mechanism of action of serum lipoproteins and 25-hydroxycholesterol on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells was investigated using antiserum against purified rat liver HMG-CoA reductase (Heller, R. A., and Shrewsbury, M. A. (1976)J. Biol. Chem. 251, 3815-3822). This antiserum cross-reacted with solubilized and membrane-bound HMG-CoA reductase from HTC cells. The enzymes from rat liver and HTC cells appeared antigenically identical. The increase in HMG-CoA reductase activity of HTC cells grown in medium which lacked serum lipoproteins was shown to be due to an increase in immunoprecipitable enzyme. In contrast, the 25-hydroxycholesterol suppression of reductase activity leads to a reduction in the antigenicity of the enzyme rather than a decrease in its number of molecules.


Relative importance of 7-methylguanosine in ribosome binding and translation of vesicular stomatitis virus mRNA in wheat germ and reticulocyte cell-free systems. Vesicular stomatitis virus mRNAs with these four types of 5'-termini, (a) m7G5'ppp5'(m)Am, (b) ppp5'(m)Am, (c) m7G5'-ppp5' Am, and (d) G5'ppp5'A, were prepared and their translation and ribosome binding analyzed in wheat germ and reticulocyte cell-free protein synthesis systems. The relative efficiencies of translation of individual vesicular stomatitis virus (VSV) mRNAs having type 2 termini ranged from 23 to 29% of the control (type 1) RNA in the reticulocyte system and 6 to 7% of control RNA in the wheat germ system. A similar difference between the two systems was seen in ribosome-binding experiments in which type 2 RNA formed an 80 S initiation complex with high efficiency (70% of control type 1 RNA) in the reticulocyte system, but with low efficiency (17% of control RNA) in the wheat germ system. Similar differences in the importance of m7G in translation in the two systems were seen when VSV mRNAs synthesized in vitro with type 3 and type 4 termini were analyzed. However, the analysis of type 4 RNA (which was synthesized in vitro in the presence of S-adenosylhomocysteine) was complicated by the presence of abnormally large poly(A) at its 3'-end. Another series of experiments showed that compounds such as 5'pm7G and m7G5'ppp5'Np are potent and specific inhibitors of translation of all types of VSV mRNAs in the wheat germ system (greater than 98% inhibition) but cause less than 20% inhibition of translation in the reticulocyte system. Taken together, all of the results indicate that a 5'-terminal m7G is far more important in translation of VSV mRNAs in the heterologous plant cell-free system than in the reticulocyte lysate system.


Role of apolipoprotein A-I in the structure of human serum high density lipoproteins. Reconstitution studies. For a better definition of the role of human serum apolipoprotein A-I (apo A-I) in high density lipoprotein structure, a systematic investigation was carried out on factors influencing the in vitro association of this apoprotein with lipids obtained from the parent high density lipoprotein (HDL); these lipids include phospholipids, free cholesterol, cholesteryl esters, and triglycerides. Following equilibration, mixtures of apo A-I and lipids in varying stoichiometric amounts were fractionated by sequential flotation, CsCl density gradient ultracentrifugation, or gel-permeation chromatography, and the isolated complexes were characterized by physicochemical means. As defined by operational criteria (flotation at density 1,063 to 1.21 g/ml), only two types of HDL complexes were reassembled; one, reconstituted HDLS, small with a radius of 31 A, and the other, reconstituted HDLL, large with a radius of 39 A. The two types incorporated all of the lipid constituents of native HDL and contained 2 and 3 mol of apo A-I, respectively. A maximal yield of reconstituted HDL (R-HDL) was observed at an initial protein concentration of 0.1 muM, where apo A-I is predominantly monomeric. At increasing protein concentrations, the amount of apo A-I recovered in R-HDL was found to be proportional to the initial concentration of monomer and dimer in solution. The composition and yield of the complexes were independent of ionic strength and pH within the ranges studied. Both simple incubation and cosonication of apo A-I with HDL phospholipids produced complexes of identical composition, although the yeild of complexes was higher with co-sonication. When the comparison of the same methods was extended to mixtures of apo A-I and whole HDL lipids, the results confirmed previous observations that co-sonication is essential for the incorporation of the neutral lipid into the R-HDL complexes. The results indicate that (a) in vitro complexation of apo A-I with lipids is under kinetic control; (b) apo A-I can generate a lipid-protein complex with properties similar to those of the parent lipoprotein; (c) the process requires well defined experimental conditions and, most importantly, the presence in solution of monomers and dimers of apo A-I; (d) the number of apo A-I molecules incorporated into R-HDL determines the size and structure of the reassembled particle. All of these observations strongly support the essential role of apo A-I in the structure of human HDL.


Adrenal glucocorticoids, adenine nucleotide translocation, and mitochondrial calcium accumulation. The administration of dexamethasone to rats markedly diminished the initial rate and maximal extent of substrate-dependent calcium uptake in subsequently isolated liver mitochondria, and enhanced the release of calcium. The apparent Km for calcium transport was not altered by dexamethasone treatment and it ranged from 50 to 80 muM when an EDTA/Ca buffer system was used in the presence of magnesium, and 20 muM when an NTA/Ca buffer system without magnesium was employed. In contrast, when ATP was employed as the energy source, there was no significant difference in initial rate, Km, or the extent of calcium accumulation between mitochondria from control and dexamethasone-treated animals. Although mitochondria from dexamethasone-treated animal showed 15% less cytochrome c oxidase activity/mg of protein, overall respiratory capacity and ATP production from ADP were the same as in control mitochondria. However, mitochondria from dexamethasone-treated animals translocated ATP from inside to outside faster than those from control animals. When the ATP in the medium was depleted by glucose and hexokinase, both types of mitochondria retained essentially all the preloaded calcium until total ATP reached a critical level (7 approximately 5 mumol of ATP/mg of protein). When ATP content fell below this critical level, mitochondria released all the calcium quickly. Dexamethasone treatment increased the susceptibility of mitochondria to the depletion of ATP. These data indicate that the dexamethasone-induced decrease in maximal calcium transport and in calcium retention carrier system per se, but o an altered ability of the mitochondria to regulate intramitochondrial ATP content.


Direct generation of superoxide anions by flash photolysis of human oxyhemoglobin. The results presented in this report suggest that human oxyhemoglobin can directly form methemoglobin and superoxide anion when flashed with low intensity (38 joules) white light. The effect only occurred in quartz but not glass (cut off lambda approximately equal to 300 nm) cuvettes. The formation of O2 was established by observing the reduction of oxidized cytochrome c concomitant with MetHb formation at pH 9, and by showing that superoxide dismultase and catalse inhibit cytochrome c reduction at that pH. The inhibition of cytochrome c reduction by catalase led us to explore the possibility that H2O2 might reduce oxidized cytochrome c at pH 9. We show that H2O2 does reduce oxidized cytochrome c at that pH but not at pH 7. Furthermore, catalase but not superoxide dismutase, almost completely inhibited this reduction process. These experiments serve to confirm our interpretation of the effect of catalase on the reduction of oxidized cytochrome c in the photolytic experiments, thus establishing that H2O2 was also formed. In addition, we were able to identify the production of O2 and H2O2 due to the photolysis of water in agreement with the results of McCord and Fridovich ((1973) Photochem. Photobiol. 17, 115-121). Production of O2 from this source was considerably less than that observed when HbO2 was present. Addition of MetHb to aerated solutions of oxidized cytochrome c did not cause additional reduction, unlike addition of HbO2. The production of MetHb was found to have at least two components. One component was the primary photolytic process, and the second was a strongly pH-dependent reattack of HbO2 by O2. Addition of superoxide dismutase inhibited this second component, but did not significantly effect the primary photolytic process.


Total conversion of glycogen synthase from the I- to the D-form by a cyclic AMP-independent protein kinase from rabbit skeletal muscle. A newly discovered cyclic AMP-independent protein kinase, which catalyzes the total conversion of glycogen synthase from the I- to the D-form, has been isolated from rabbit skeletal muscle. This enzyme, designated glycogen synthase kinase, is separable from cyclic AMP-dependent protein kinase by column chromatography on phosphocellulose. Synthase kinase and cyclic AMP-dependent protein kinase are distinct in their specificity for protein substrates, the effects of cyclic AMP and the inhibitor of cyclic AMP-dependent protein kinase on their activities, and the extent to which they phosphorylate I-form glycogen synthase. The phosphorylation of I-form enzyme by synthase kinase results in the incorporation of 4 mol of phosphate/85,000 subunit; however only two of the phosphate sites seem predominantly to determine glucose-6-P dependence. The resulting multiply phosphorylated enzyme, which is highly dependent on glucose-6 P for activity, has a phosphate content comparable to the D-form enzyme isolated from rabbit muscle.


Expression of genes for metabolism of cyclic adenosine 3':5'-monophosphate in somatic cells. beta-Adrenergic and PGE1 receptors in parental and hybrid cells. Using the ligands [125I]iodohydroxybenzylpindolol and [3H]prostaglandin E1 ([3H]PGE1), we have studied the relationship of receptors for beta-adrenergic agents and for PGE1 to adenylate cyclase in membranes of parental, hybrid, and variant mammalian cell lines. Fusion of parental clones responsive to beta-adrenergic agonists (beta+) with unresponsive clones (beta-) produced hybrid clones with a greatly diminished beta-adrenergic response; beta+ X beta leads to beta-. Binding studies with [125I]iodohydroxybenzylpindolol showed a decreased concentration of beta receptors in six such hybrid clones. Thus, paucity of beta-adrenergic receptors is probably a sufficient, albeit not necessarily complete, explanation for the decreased beta-adrenergic responsiveness of the hybrid clones. When a clone with beta receptor but without apparent adenylate cyclase activity (HC-1) was hybridized with a beta- clone that has adenylate cyclase (B82), a responsive hybrid clone was obtained. In nine cell hybrids produced by the fusion of clones responsive (PGE1+) and unresponsive (PGE1-) to PGE1, high affinity binding sites for [3H]PGE1 were expressed in the same manner as was PGE1-sensitive adenylate cyclase: PGE1+ X PGE1 leads to PGE1+. The chemical specificities and affinities of the parental receptors and responsive adenylate cyclases were faithfully reproduced in the hybrid clones. Activation by PGE1 was proportional to the occupation of the high affinity receptors. In a wild type lymphoma clone (24.3.2), the concentration dependences for binding of [3H]PGE1 and for activation of adenyalte cyclase by PGE1 were identical. In a variant lymphoma clone (94.15.1) lacking adenylate cyclase activity, no high affinity receptors for PGE1 were detected, whereas beta-adrenergic receptors have been demonstrated in this variant clone (Insel, P.A., Maguire, M.E., Gilman, A.G., Coffino, P., Bourne, H., and Melmon, K. (1976) Mol. Pharmacol. 12, 1062-1069). Hybrid cells formed by the fusion of 94.15.1 with cell line RAG (PGE1-) responded to PGE1. Clone 94.15.1 may have receptors for PGE1 of reduced affinity or in low concentration. Alternatively, RAG and 94.15.1 may have complementary genetic defects such that the RAG X 94.15.1 hybrid cells express a hormonally responsive receptor-adenylate cyclase system.


Serum angiotensin-converting enzyme. Isolation and relationship to the pulmonary enzyme. Angiotensin-converting enzyme from rabbit serum was purified almost 60,000-fold to apparent homogeneity by a procedure exploiting its affinity for antibodies prepared against the enzyme from lung. The pure serum and pulmonary enzymes exhibited identical behavior during gel filtration, sucrose gradient centrifugation, and disc gel electrophoresis in the reduced, denatured state. Their catalytic properties with hippurylhistidylleucine, angiotensin I, and bradykinin as substrates were similar and their reactivity with antilung enzyme antibody was indistinguishable as examined by immunodiffusion, inhibition dose-response curves, and radioimmunoassay. Their content of fucose, mannose, galactose, and N-acetylglucosamine was also comparable; however, N-acetylneuraminic acid was much more abundant in the serum glycoprotein. This difference may reflect selective removal of sialic acid-deficient enzyme molecules from the circulation by the hepatic lectin which has been postulated to initiate the catabolic phase for plasma glycoproteins (Ashwell, G., and Morell, A.G. (1974) Adv. Enzymol. Relat. Areas Mol. Biol. 41, 91-128).


Electron paramagnetic resonance study of carp methemoglobin. The g anisotropy of the EPR spectra of carp azidomethemoglobin is found to be pH-dependent, whereas, the spectra of human azidomethemoglobin are not. The two hemoglobins have the same g values at alkaline pH values. Crystal field analysis yielded values of 2.25 and 3.31, respectively, for the rhombic distortion, V/lambda, and the tetragonal distortion, delta/lambda. The spin orbit coupling constant is lambda. At pH 4.0 the values of V/lambda and delta/lambda for carp azidomethemoglobin became 1.95 and 4.76, respectively, whereas those for the human hemoglobin are virtually unchanged. The results are interpreted to mean an increase of out-ofplane displacement of the iron atom and stabilization of the T form of carp azidomethemoglobin by high proton concentration. At pH 6.0 and lower, the EPR spectra of carp azidomethemoglobin showed the presence of about 1.5% of high spin species, the amount is not affected by excess of either inositol hexaphosphate or sodium azide. The EPR spectra of aquo- and fluoroderivatives of carp methemoglobin were not affected by pH changes.


Nonequivalence of subunits in [15N]nitrosylhemoglobin Kansas. A single crystal electron paramagnetic resonance investigation. EPR spectra of Hb15NO crystals of mutant Kansas (Asn G4(102) beta leads to Thr) have been recorded at every 5' intervals and in three orthogonal planes. The nitrosylhemes are nonequivalent for the alpha and beta subunits, their assignments are made possible by comparison with the powder EPR specrtra of Hb15NO of mutant Iwate (His F8(87)alpha leads to Tyr) (Trittelvitz, E., Gersonde, K., and Winterhalter, K.H. (1975) Eur. J. Biochem. 51, 33-42). The EPR parameters for the beta-nitrosylhemes of Hb Kansas are: gxx=2.094 gyy=2.031, gzz=2.00, Azetazeta=11 G, Azetazeta=32.5 G, Aetaeta=12.5 G; the Fe-N-O bond angle is about 105 degrees. The paramters for the alpha-nitrosyl hemes are: gxx=2.058, gyy=2.021, gzz=1.977, Azetazeta=24.5 G, Azetazeta less than or equal to 5G, Aetaeta=23 G; the Fe-N-O bond angle is about 167 degrees. Hyperfine splittings of 7 to 8 gauss with 14Nepsilon atom of His(F8) were observed for the beta-nitrosylhemes; none was resolved for the alpha-nitrosylhemes. The results were interpreted to mean that the tension on the iron of the beta subunits is not large in the unliganded state and this tension was not greatly increased by the binding of nitric oxide in the strongly bent configuration. The tension at the iron in the deoxyhemoglobin is dominant at the alpha subunits. Binding of nitric oxide in this case causing either the breaking or great weakening of the Fe-His(F8) bond. The nitrosyl is in a nearly linear configuration. The unpaired electron densities at the nitrogen atom of the bound nitric oxide is about 63% for the beta-nitrosylheme and 37% for the alpha-nitrosylhemes.


Formation of a biologically active, ordered complex from two overlapping fragments of cytochrome c. A noncovalent complex of the apoprotein (1-104) and cyanogen bromide heme fragment containing residues 1 to 65, (1-65) H, has been prepared from horse heart cytochrome c. Conditions under which the redundant portions of the ferrous complex can be removed by limited trypsin digestion have been devised. The complementing fragments have been isolated from the derived complexes and four apofragments and one heme fragment have been identified in the amino acid sequence of cytochrome c. They are (39-104), (40-104), (54-104), (56-104), and (1-53)H. The formation of an ordered ferric complex composed of one heme fragment and one apofragment for the cases (1-53)H (39-104), (1-53)H-(40-104), (1-53)H-(54-104), and (1-53)H-(56-104) has been demonstrated by the quenching of the tryptophan 59 fluorescence and the regain of biological activity in a cytochrome b2 assay. The apparent dissociation constant has been estimated as less than 3 X 10(-7) M in all the aforementioned cases. Thus, the region (between residues 38 and 57) of the amino acid sequence permissible for cleavage without disruption of the ordered structure indicated by the present in vitro experiments corresponds to that (between residues 38 and 57) evolutionally deleted in the three-dimensional structure of Pseudomonas aeruginosa cytochrome c551 discovered by Dickerson et al. (Dickerson, R.E., Timkovich, R., and Almassy, R.J. (1976) J. Mol. Biol. 100, 473-491).


Inactivation of avian myeloblastosis virus DNA polymerase by specific binding of pyridoxal 5'-phosphate to deoxynucleoside triphosphate binding site. Avian myeloblastosis virus (AMV) DNA polymerase is inactivated by preincubation with pyridoxal 5'-phosphate. This inactivation is relatively specific since various pyridoxal-5'-P analogs cause no inactivation. This effect is reversible but can be made irreversible by reduction with sodium borohydride; the reduced pyridoxal-5'-P adduct exhibits a new absorbance maximum at 325 nm and a fluorescence emission at 392 nm when excited at 325 nm. The evidence presented suggests the formation of a Schiff base between pyridoxal-5'-P and a nucleophilic residue of AMV DNA polymerase. The presence of a deoxynucleoside 5'-triphosphate (dTTP) protected the enzyme from inactivation. Reduction of the pyridoxal-5'-P enzyme complex in the presence or absence of a deoxynucleoside 5'-triphosphate showed that the alpha subunit possesses five reactive amino groups, one of which is essential for catalytic activity; the beta subunit has three reactive amino groups which are not involved in the deoxynucleoside binding site.


Concentrations of cyclic AMP-dependent protein kinase subunits in various tissues. The concentrations of the regulatory (R) and catalytic (C) subunits of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase(s) were measured in extracts of skeletal muscle, heart, liver, kidney, and brain. These concentrations were also estimated for the particulate fraction from brain, the only tissue in which a major part of the total activity was not readily extracted in a soluble form. Values for R were determined by measuring the amount of cyclic [3H]amp bound to protein in these tissue fractions under specified conditions; it was assumed that 1 mol of cyclic AMP binds to 1 mol of R. Values for C were determined from measurements of the specific protein kinase activity of the fractions utilizing the turnover number of pure C in the calculations. Turnover numbers for C were found to be identical for this subunit obtained in the pure form from rabbit skeletal muscle, rabbit liver, and beef heart. The methods used for measuring C were evaluated by kinetic studies and through the use of the specific heatstable protein inhibitor of cyclic AMP-dependent protein kinase(s). R and C were found to exist in a 1:1 molar ratio in all of the tissue fractions that were studied. the absolute concentrations of R and C ranged from 0.23 mumol/kg wet weight for liver to 0.78 mumol/kg wet weight for brain. For brain this value was based on the amount of each subunit in the particulate as well as the soluble fraction. For other tissues the values were based solely on the subunit content of the latter fraction. It was noted that the molar concentrations of R are close to those of cyclic AMP under basal conditions in the various tissues.


Regulation of cyclic nucleotide phosphodiesterases in cultured hepatoma cells by dexamethasone and N6,O2'-dibutyryl adenosine 3':k'-monophosphate. DEAE-Bio-Gel chromatography of 100,000 X g supernatant from cultured HTC hepatoma cells separated cyclic nucleotide phosphodiesterase into three forms, numbered E I, E II, and E III in order of elution from the column, E I had a low Km for cyclic guanosine 3':5'-monophosphate (cGMP) and a high Km for cyclic adenosine 3':5'-monophosphate (cAMP), E II exhibited anomalous kinetics. At low substrate concentrations (0.5 muM) cGMP was hydrolyzed more rapidly than cAMP and hydrolysis of 0.5 muM cAMP was stimulated by 1 muM cGMP. E III had a low Km for cAMP. Incubation of cells with 1 muM dexamethasone for 72 h decreased the activity of E I and E II. In cells incubated with N6,O2'-dibutyryl cAMP plus 3-isobutyl-1-methylxanthine for 14 h the activity of E III was increased approximately 100%. Similar activities of calcium-dependent, heat stable phosphodiesterase activator were recovered from supernatants from all cells. These studies have established the presence, in a homogeneous population of hepatoma cells, of at least three forms of cyclic nucleotide phosphodiesterase, the activities of which can be independently regulated.


Role of phosphoprotein phosphatases in reversible deactivation of chicken adipose tissue hormone-sensitive lipase. The reversible deactivation of chicken adipose tissue hormone-sensitive lipase alpha(previously activated with Mg2+ ATP and adenosine 3':5'-monophosphate) required Mg2+ and was inhibited by phosphate. These results are consistent with the assumption that deactivation of the protein kinase-activated enzyme is catalyzed by a lipase phosphatase. Cholesterol ester is catalyzed by a lipase phosphatase. Cholesterol ester hydrolase similarly was activated and reversibly deactivated. The activity of endogenous lipase phosphatase in pH 5.2 precipitate fractions was reduced, and in some cases eliminated, by incubation at 50 degrees for 20 min in buffer containing 20% glycerol. Heating at 50 degrees greatly increased the apparent percentage activation of triglyceride and cholesterol ester hydrolases but this was due to a selective decrease in basal (nonactivated) hydrolase activities. Essentially all endogenous lipase phosphatase could be removed by treatment of the pH 5.2 precipitate fraction with ATP-Sepharose affinity gel. The addition of a partially purified preparation of rat liver phosphorylase phosphatase deactivated triglyceride and cholesterol ester hydrolases. The deactivation process was concentration, 5 mM) and was inhibited by 5 mM phosphate and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipase alpha was also observed with crude prepa- and by phosphorylase alpha. Reversible deactivation of hormone-sensitive lipas alpha was also observed with crude preparations of phosphoprotein phosphatases from rat  and turkey hearts, and from rat epididymal fat pads. Thus, hormone-sensitive lipase is deactivated by a variety of phosphoprotein phosphatases from different tissues and different species, implying a low degree of specificity for the deactivating system.


Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. I. Synthesis of diphtheria toxin fragment A-S-S-human placental lactogen with methyl-5-bromovalerimidate. In order to study the mechanism of entry of plant seed and bacterial toxins into mammalian cells, methods have been developed to synthesize artificial protein hybrid conjugates containing a moiety which binds to a cell membrane receptor and an active fragment of a toxin protein. Utilizing methyl-5-bromovalerimidate, a disulfide cross-linked conjugate of human placental lactogen (hPL) and diphtheria toxin fragment A (toxin A) was synthesized. The reagent was prepared from 5-bromovaleryl nitrile by Pinner synthesis and then used to amidinate hPL. The bromo group thus introduced was converted to S-sulfonate by nucleophilic displacement with 1 M aqueous sodium thiosulfate at room temperature overnight. The S-sulfonated hPL reacted readily with the-SH gorup of reduced toxin A to form a 1 mol/mol of disulfide conjugate in high yield. Thus when reduced toxin A was incubated with a 4-fold excess of the hPL S-sulfonate at 4 degrees and pH 6.5 for 120 h, a conjugate yield of 50% relative to the toxin A input was obtained. Homopolymer formation was negligible and the product was purified by gel filtration on Sephadex G-150. Purity of the conjugate estimated by quantitative analysis of sodium dodecyl sulfate gels was 90%. The toxin A-hPL conjugate retained the activities of both toxin A and hPL, as reported in the accompanying paper. This method of preparing protein hybrid conjugates appeared to have advantages over previous methods utilizing bifunctional reagents with respect to both yield and freedom from homopolymer formation.


Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. II. Biochemical and biologic properties of diphtheria toxin fragment A-S-S-human placental lactogen. The biochemical and biologic properties of a purified disulfide conjugate of diphtheria toxin fragment A and human placental lactogen (toxin A-hPL) have been studied by (a) assaying the ADP-ribosyltransferase activity of the intact conjugate, (b) assaying the binding of the intact conjugate to mammary gland plasma membrane lactogenic receptors, and (c) assaying the effect of the conjugate on the rate of protein synthesis in rabbit mammary gland explants maintained in organ culture. The toxin A-hPL conjugate retains one-third of the NAD+:EF-2 ADP-ribosyltransferase activity of toxin A, and 26% of the hPL-binding activity to lactogenic receptors. Binding activity was demonstrated by radioreceptor assay and by assaying toxin A activity bound to membranes which was competitively displaced by excess hPL. Since the toxin A-hPL conjugate retained activities of its separate subunits, it could be regarded as a structural analogue of nicked diphtheria toxin with replacement of the original membrane-binding chain by another binding chain that is specific for lactogenic receptor. However, the conjugate failed to inhibit protein synthesis in organ-cultured mammary gland explants, although these were sensitive to native diphtheria toxin and could bind hPL. It is concluded from these results that the toxin A-hPL conjugate does not act as a functional analogue of diphtheria toxin with altered receptor specificity, and that the hPL receptor cannot mediate the entry of toxin A or toxin A-hPL from membrane-bound conjugate into the cytosol site of action of toxin A.


Progesterone-stimulated meiotic cell division in Xenopus oocytes. Induction by regulatory subunit and inhibition by catalytic subunit of adenosine 3':5'-monophosphate-dependent protein kinase. Ripe Xenopus oocytes in first meiotic prophase when incubated with progesterone in vitro progress synchronously in 3 to 5 h without interphase to second meiotic metaphase where they remain until fertilization or activation. Using highly purified preparations of regulatory and catalytic subunits of adenosine 3':5'-monophosphate-dependent protein kinase from muscle, this progesterone-stimulated cell division sequence was found to be inhibited by microinjection of the catalytic subunit and induced directly in the absence of progesterone after microinjection of regulatory subunit. Dose-response curves revealed that half-maximal effects of regulatory and catalytic subunits occurred at an internal concentration of approximately 0.1 muM. These results indicate that the catalytic subunit is necessary and sufficient to block progesterone-stimulated meiotic cell division. Other experiments revealed that the catalytic subunit was inhibitory only during the first hour after progesterone exposure, suggesting that initial steps in meiotic cell division are affected. Control experiments demonstrate that the muscle cAMP-dependent protein kinase subunits may interact with the endogenous oocyte protein kinase. The results support a model in which meiotic cell division is regulated by a phosphoprotein subject to control by cAMP-dependent protein kinase.


Turbidimetric ultracentrifugation. Application to the study of human serum very low density lipoprotein distributions. In this communication it is shown that the sedimentation coefficient distribution may be accurately measured for very large particles using turbidimetric techniques and the ultraviolet-scanning analytical ultracentrifuge. A principal advantage is that turbidity is a function of the product of concentration and molecular weight; thus, large particles may be observed even when present in very small amounts. We propose to call this method of analysis "turbidimetric ultracentrifugation." We have used turbidimetric ultracentrifugation ot determine the sedimentation coefficient distribution for a sample of human serum very low density lipoproteins. This distribution is compared to that found with conventional schlieren techniques with good agreement.


Lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat. In the adrenal gland of the rat, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme of cholesterol synthesis, is shown to be regulated by cholesteerol carried in plasma lipoproteins. When plasma cholesterol levels were lowered 90% by administration of the drug 4-aminopyrazolopyrimidine, the cholesteryl ester content of the adrenal gland declined by more than 90% and this was associated with a 150- to 200-fold increase in the activity of adrenal 3-hydroxy-3-methylglutaryl coenzyme A reductase and a 30-fold increase in cholesterol synthesis from [14C]acetate. The subsequent intravenous infusion of cholesterol contained in either rat or human high density or low density lipoproteins restored the adrenal content of cholesteryl esters and reduced the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase to basal levels. The depletion of adrenal cholesteryl esters and the enhancement in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that occurred in the 4-aminopyrazolopyrimidine-treated rat required the action of adrenocorticotropic hormone (ACTH) since neither was observed when ACTH secretion was blocked by administration of dexamethasone. The current data indicate that the low rate of cholesterol synthesis normally observed in the rat adrenal gland is due to a suppression of the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase that is mediated by plasma lipoproteins.


Enzyme and phospholipid asymmetry in liver microsomal membranes. The transverse distribution of enzyme proteins and phospholipids within microsomal membranes was studied by analyzing membrane composition after treatment with proteases and phospholipases. Upon trypsin treatment of closed microsomal vesicles, NADH- and NADPH-cytochrome c reductases as well as cytochrome b5 were solubilized or inactivated, while cytochrome P-450 was partially inactivated. When microsomes were exposed to a concentration of deoxycholate which makes them permeable to macromolecules but does not disrupt the membrane, the detergent alone was sufficient to release four enzymes: nucleoside diphosphatase, esterase, beta-glucuronidase, and a portion of the DT-diaphorase. Introduction of trypsin into the vesicle lumen inactivated glucose-6-phosphatase completely and cytochrome P-450 partially. The rest of this cytochrome, ATPase, AMPase, UDP-glucuronyltransferase, and the remaining 50% of DT-diaphorase activity were not affected by proteolysis from either side of the membrane. Phospholipase A treatment of intact microsomes in the presence of albumin hydrolyzed all of the phosphatidylethanolamine, phosphatidylserine, and 55% of the phosphatidylcholine. From this observation, it was concluded that these lipids are localized in the outer half of the bilayer of the microsomal membrane; Phosphatidylinositol, 45% of the phosphatidylcholine, and sphingomyelin are tentatively assigned to the inner half of this bilayer. It appears that the various enzyme proteins and phospholipids of the microsomal membrane display an asymmetric distribution in the transverse plane.


Ultrastructural changes induced by ACTH in normal adrenocortical cells in culture. The effects of ACTH, its o-nitrophenyl sulfenyl derivative (NPS-ACTH) and dibutyryl cyclic AMP (dbc AMP) on the ultrastructural morphology of adrenocortical cells of adult rats in monolayer culture have been investigated. NPS-ACTH, which has previously been shown to stimulate steroidogenesis but not cAMP synthesis in adrenal cells, induced the same characteristic transformation of mitochondrial architecture as produced by ACTH or high concentrations of dbcAMP. All three agents caused the disappearance of electron-opaque granules present in the mitochondria of unstimulated cells. It was found that these granules could be extracted with EGTA (ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetate). These results are discussed in the light of the known importance of calcium ions in the actions of ACTH.


Delayed malignancy and altered growth properties of somatic cell hybrids between rat hepatoma and mouse L-cells. Somatic cell hybrids of mouse L-cells with rat HTC cells were studied for their growth properties in vitro and tumorigenicity in vivo. Three hybrid clones were selected for detailed study. All were delayed in tumor formation in nude mice compared with the parent lines despite their varied growth properties in vitro. One clone was sensitive to density dependent inhibition of growth (DDIG) and had a relatively low saturation density. A second clone was not sensitive to DDIG and had a higher saturation density. The third clone had atypical morphology, was insensitive to DDIG, and had a relatively high saturation density. All of the clones studied produced colonies suspended in agarose gel which were much smaller than those of the parents incubated for the same period of time. Only the pattern of growth in agarose gel corresponded to the delayed tumor formation in vivo of the hybrids. Sensitivity to DDIG and saturation density were not consistent with tumor growth. The hybrid clone that was sensitive to DDIG was the only one of the three that had a nearly complete set of chromosomes derived from each of the parents. The chromosome numbers of all three clones were unchanged after growth in agarose or as tumors in the nude mice.


Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression. In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.


Quantitative analysis of disulfiram and its metabolites in human blood by gas-liquid chromatography. A simple method is described that permits the direct quantitative determination of carbon disulphide, free diethyldithiocarbamate and disulphides derived from disulfiram in 1 ml of a patient's blood. It is based on a gas chromatographic determination of carbon disulphide produced from diethyldithiocarbamate and disulfiram using the head-space technique and a flame-photometric detector. The method is compared with a recently described spectrophotometric method.